Genetic architecture of variation in sex-comb tooth number in Drosophila simulans

The sex comb on the forelegs of Drosophila males is a secondary sexual trait, and the number of teeth on these combs varies greatly within and between species. To understand the relationship between the intra- and interspecific variation, we performed quantitative trait locus (QTL) analyses of the intraspecific variation in sex-comb tooth number. We used five mapping populations derived from two inbred Drosophila simulans strains that were divergent in the number of sex-comb teeth. Although no QTLs were detected on the X chromosome, we identified four QTLs on the second chromosome and three QTLs on the third chromosome. While identification and estimated effects of the second-chromosome QTLs depend on genetic backgrounds, significant and consistent effects of the two third-chromosome QTLs were found in two genetic backgrounds. There were significant epistatic interactions between a second-chromosome QTL and a third-chromosome QTL, as well as between two second-chromosome QTLs. The third-chromosome QTLs are concordant with the locations of the QTLs responsible for the previously observed differences in sex-comb tooth number between D. simulans and D. mauritiana.     

Correlation of diversity of leg morphology in Gryllus bimaculatus (cricket) with divergence in dpp expression pattern during leg development

Insects can be grouped into mainly two categories, holometabolous and hemimetabolous, according to the extent of their morphological change during metamorphosis. The three thoracic legs, for example, are known to develop through two overtly different pathways: holometabolous insects make legs through their imaginal discs, while hemimetabolous legs develop from their leg buds. Thus, how the molecular mechanisms of leg development differ from each other is an intriguing question. In the holometabolous long-germ insect, these mechanisms have been extensively studied using Drosophila melanogaster. However, little is known about the mechanism in the hemimetabolous insect. Thus, we studied leg development of the hemimetabolous short-germ insect, Gryllus bimaculatus (cricket), focusing on expression patterns of the three key signaling molecules, hedgehog (hh), wingless (wg) and decapentaplegic (dpp), which are essential during leg development in Drosophila. In Gryllus embryos, expression of hh is restricted in the posterior half of each leg bud, while dpp and wg are expressed in the dorsal and ventral sides of its anteroposterior (A/P) boundary, respectively. Their expression patterns are essentially comparable with those of the three genes in Drosophila leg imaginal discs, suggesting the existence of the common mechanism for leg pattern formation. However, we found that expression pattern of dpp was significantly divergent among Gryllus, Schistocerca (grasshopper) and Drosophila embryos, while expression patterns of hh and wg are conserved. Furthermore, the divergence was found between the pro/mesothoracic and metathoracic Gryllus leg buds. These observations imply that the divergence in the dpp expression pattern may correlate with diversity of leg morphology.     

Kinematics and motor activity during tethered walking and turning in the cockroach, <Emphasis Type="Italic">Blaberus discoidalis</Emphasis>

When insects turn from walking straight, their legs have to follow different motor patterns. In order to examine such pattern change precisely, we stimulated single antenna of an insect, thereby initiating its turning behavior, tethered over a lightly oiled glass plate. The resulting behavior included asymmetrical movements of prothoracic and mesothoracic legs. The mesothoracic leg on the inside of the turn (in the apparent direction of turning) extended the coxa-trochanter and femur-tibia joints during swing rather than during stance as in walking, while the outside mesothoracic leg kept a slow walking pattern. Electromyograms in mesothoracic legs revealed consistent changes in the motor neuron activity controlling extension of the coxa-trochanter and femur-tibia joints. In tethered walking, depressor trochanter activity consistently preceded slow extensor tibia activity. This pattern was reversed in the inside mesothoracic leg during turning. Also for turning, extensor and depressor motor neurons of the inside legs were activated in swing phase instead of stance. Turning was also examined in free ranging animals. Although more variable, some trials resembled the pattern generated by tethered animals. The distinct inter-joint and inter-leg coordination between tethered turning and walking, therefore, provides a good model to further study the neural control of changing locomotion patterns.     

The genetics of mating recognition between Drosophila simulans and D. sechellia

During courtship, visual and chemical signals are often exchanged between the sexes. The proper exchange of such signals ensures intraspecific recognition. We have examined the genetic basis of interspecific differences in male mating behaviour and pheromone concentration between Drosophila simulans and D. sechellia by using Drosophila simulans/D. sechellia introgression lines. Our results show a majority of quantitative trait loci (QTLs) explaining variation in both male mating behaviour and pheromone concentration to be located on the third chromosome. One QTL found on the third chromosome explains variation in time needed to start courtship and copulation as well as time spent courting. The position of such QTL (approximately 84A-88B) with effects on courtship and copulation aspects of mating includes the candidate sex determination gene doublesex (84E5-6) and Voila (86E1-2), a gene that affects male courtship in D. melanogaster. One additional third chromosome QTL explained variation in 7-tricosene pheromone concentrations among males. The interval mapping position of this QTL (approximately 68E-76E) did not overlap with the position detected for differences in mating behaviour and the intervals did not include candidate genes previously identified as having an effect on D. melanogaster cuticular hydrocarbon production. We did not detect any directionality of the effect of Drosophila sechellia allele introgressions in male mating recognition.     

Ultrabithorax gene expression in Drosophila imaginal discs and larval nervous system

Using a monoclonal antibody and image-processing procedures, the patterns of expression of the Ultrabithorax (Ubx) gene product have been characterized in Drosophila larvae. As reported previously, the metathoracic imaginal discs stain most intensely with anti-Ubx, with some mesothoracic and no prothoracic expression detectable. In the metathoracic discs, the greatest modulation in anti-Ubx staining is along the proximodistal axis. Ubx is generally expressed at higher levels in the posterior regions of metathoracic discs, although relatively high anterior expression is found in some areas. Expression in the mature wing disc is confined to the squamous peripodial membrane cells; in younger wings, Ubx expression fills the posterior half of the peripodial side of the disc. The mesothoracic leg stains with a pattern that is qualitatively similar (but not identical) to that of the metathoracic leg; Ubx is expressed in some anterior regions of the mesothoracic leg, in parasegment 4. Double staining with anti-Ubx and anti-engrailed reveals that discontinuities in Ubx expression that have been suggested to correspond to compartment borders do not coincide with the compartment boundaries in some cases. In the larval ventral ganglion, Ubx expression is greatest in parasegments 5 and 6, as in the embryonic nervous system.     

Comparative morphology of the prothoracic leg in heliconian butterflies: Tracing size allometry,podite fusions and losses in ontogeny and phylogeny

Prothoracic legs of heliconian butterflies (Nymphalidae, Heliconiinae, Heliconiini) are reduced in size compared to mesothoracic and metathoracic legs. They have no apparent function in males, but are used by females for drumming on host plants, a behavior related to oviposition site selection. Here, taking into account all recognized lineages of heliconian butterflies, we described their tarsi using optical and scanning electron microscopy and searched for podite fusions and losses, and analyzed allometry at the static, ontogenetic and phylogenetic levels. Female tarsi were similar, club-shaped, showing from four to five tarsomeres, each bearing sensilla chaetica and trichodea. Male tarsi were cylindrical, formed from five (early diverging lineages) to one (descendant lineages) either partially or totally fused tarsomeres, all deprived of sensilla. Pretarsi were reduced in both sexes, in some species being either vestigial or absent. Tarsal lengths were smaller for males in almost all species. An abrupt decrease in size was detected for the prothoracic legs during molting to the last larval instar at both histological and morphometric levels. In both sexes, most allometric coefficients found at the population level for the prothoracic legs were negative compared to the mesothoracic leg and also to wings. Prothoracic tarsi decreased proportionally in size over evolutionary time; the largest and smallest values being found for nodes of the oldest and youngest lineages, respectively. Our results demonstrate that evolution of the prothoracic leg in heliconian butterflies has been based on losses and fusions of podites, in association with negative size allometry at static, ontogenetic and phylogenetic levels. These processes have been more pronounced in males. Our study provided further support to the hypothesis that evolution of these leg structures is driven by females, by changing their use from walking to drumming during oviposition site selection. In males the leg would have been selected against due to absence of function and thus progressively reduced in size, in association with podites fusions and lost.     

Genetic analysis of extra sex combs in the hybrids between Drosophila subobscura and D. madeirensis

Drosophila subobscura and D. madeirensis are closely related species, the first distributed over a large area and the latter restricted to the island of Madeira. These species can hybridize in laboratory conditions, yielding fertile females and sterile males. Hybrid offspring show several phenotypic anomalies, including sex combs on the second and third pairs of legs in males. The extra sex comb trait is a homeotic transformation of the mesothoracic and metathoracic legs into prothoracic legs. This anomaly is observed almost exclusively in F₁ males with D. madeirensis mothers. Analysis of backcross males shows that D. subobscura and D. madeirensis have diverged at a minimum of four autosomal loci affecting the extra sex comb anomaly. In addition, some incompatibility involving the X chromosome and/or a maternal effect is also implicated.     

Quantitative trait loci for thermotolerance phenotypes in Drosophila melanogaster

For insects, temperature is a major environmental variable that can influence an individual's behavioral activities and fitness. Drosophila melanogaster is a cosmopolitan species that has had great success in adapting to and colonizing diverse thermal niches. This adaptation and colonization has resulted in complex patterns of genetic variation in thermotolerance phenotypes in nature. Although extensive work has been conducted documenting patterns of genetic variation, substantially less is known about the genomic regions or genes that underlie this ecologically and evolutionarily important genetic variation. To begin to understand and identify the genes controlling thermotolerance phenotypes, we have used a mapping population of recombinant inbred (RI) lines to map quantitative trait loci (QTL) that affect variation in both heat- and cold-stress resistance. The mapping population was derived from a cross between two lines of D. melanogaster (Oregon-R and 2b) that were not selected for thermotolerance phenotypes, but exhibit significant genetic divergence for both phenotypes. Using a design in which each RI line was backcrossed to both parental lines, we mapped seven QTL affecting thermotolerance on the second and third chromosomes. Three of the QTL influence cold-stress resistance and four affect heat-stress resistance. Most of the QTL were trait or sex specific, suggesting that overlapping but generally unique genetic architectures underlie resistance to low- and high-temperature extremes. Each QTL explained between 5 and 14% of the genetic variance among lines, and degrees of dominance ranged from completely additive to partial dominance. Potential thermotolerance candidate loci contained within our QTL regions are identified and discussed.     

A quantitative genetic analysis of male sexual traits distinguishing the sibling species Drosophila simulans and D. sechellia

A quantitative trait locus (QTL) genetic analysis of morphological and reproductive traits distinguishing the sibling species Drosophila simulans and D. sechellia was carried out in a backcross design, using 38 markers with an average spacing of 8.4 cM. The direction of QTL effects for the size of the posterior lobe was consistent across the identified QTL, indicating directional selection for this trait. Directional selection also appears to have acted on testis length, indicating that sexual selection may have influenced many reproductive traits, although other forms of directional selection cannot be ruled out. Sex comb tooth number exhibited high levels of variation both within and among isofemale lines and showed no evidence for directional selection and, therefore, may not have been involved in the early speciation process. A database search for genes associated with significant QTL revealed a set of candidate loci for posterior lobe shape and size, sex comb tooth number, testis length, tibia length, and hybrid male fertility. In particular, decapentaplegic (dpp), a gene known to influence the genital arch, was found to be associated with the largest LOD peak for posterior lobe shape and size.     

In vitro culture of Drosophila imaginal disc cells: aggregation, sorting out, and differentiative abilities

This paper describes the aggregation in vitro of cells dissociated from imaginal discs and demonstrates the sorting out of undifferentiated cells from different imaginal discs and from differently determined regions of the same imaginal disc, as well as the abilities of such cells to undergo pattern reconstruction when injected into larvae. Dissociated cells begin to aggregate by 1.5 hr of rotation. By 5 hr of rotation, large aggregates of loosely associated cells appear. By 18 hr the aggregates have condensed and taken on a characteristic epithelial structure. To study sorting out in undifferentiated cells, we combined a histochemical stain for acid phosphatase with the use of the acid phosphatase null mutant acphn-11. We performed cell mixing experiments with 0-2 (prospective notum) and 2-8 (prospective wing) fragments, with the A and P (prospective anterior and posterior) fragments of the dorsal mesothoracic disc and with mixtures of cells from ventral prothoracic and dorsal mesothoracic discs. We found that prospective anterior and posterior dorsal mesothoracic cells do not sort out, but that prospective notum and wing and leg and wing cells do. The results from differentiated implants are consistent with those from undifferentiated mixes.     

Molecular population genetics of male accessory gland proteins in Drosophila

Drosophila seminal proteins have an unusually high rate of molecular sequence evolution, suggesting either a high rate of neutral substitution or rapid adaptive evolution. To further quantify patterns of polymorphism and divergence in genes encoding seminal proteins, also called accessory gland proteins (Acp's), we conducted a sequencing survey of 10 Acp genes in samples of Drosophila melanogaster and D. simulans (Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB). Mean heterozygosity at replacement sites in D. simulans was 0.0074 for Acp genes and 0.0013 for a set of 19 non-Acp genes, and mean melanogaster-simulans divergence at replacement sites was 0.0497 for Acp genes and 0.0107 at non-Acp genes. The elevated divergence of Acp genes is thus accompanied by elevated within-species polymorphism. In addition to the already-reported departures of Acp26A, Acp29AB, and Acp70A from neutrality, our data reject neutrality at Acp29AB and Acp36DE in the direction of excess replacements in interspecific comparisons.     

Genetic studies of two sister species in the Drosophila melanogaster subgroup, D. yakuba and D. santomea

We performed genetic analysis of hybrid sterility and of one morphological difference (sex-comb tooth number) on D. yakuba and D. santomea, the former species widespread in Africa and the latter endemic to the oceanic island of S?o Tomé, on which there is a hybrid zone. The sterility of hybrid males is due to at least three genes on the X chromosome and at least one on the Y, with the cytoplasm and large sections of the autosomes having no effect. F1 hybrid females carrying two X chromosomes from either species are perfectly fertile despite their genetic similarity to completely sterile F1 hybrid males. This implies that the appearance of Haldane's rule in this cross is at least partially due to the faster accumulation of genes causing male than female sterility. The larger effects of the X and Y chromosomes than of the autosomes, however, also suggest that the genes causing male sterility are recessive in hybrids. Some female sterility is also seen in interspecific crosses, but this does not occur between all strains. This is seen in pure-species females inseminated by heterospecific males (probably reflecting incompatibility between the sperm of one species and the female reproductive tract of the other) as well as in inseminated F1 and backcross females, probably reflecting genetically based incompatibilities in hybrids that affect the reproductive system. The latter 'innate' sterility appears to involve deleterious interactions between D. santomea chromosomes and D. yakuba cytoplasm. The difference in male sex-comb tooth number appears to involve fairly large effects of the X chromosome. We discuss the striking evolutionary parallels in the genetic basis of sterility, in the nature of sexual isolation, and in morphological differences between the D. santomea/D. yakuba divergence and two other speciation events in the D. melanogaster subgroup involving island colonization.     

Quantitative trait loci affecting a courtship signal in Drosophila melanogaster

Courtship plays a major role in the sexual isolation of species, yet the genetics underlying courtship behaviour are poorly understood. Here we analyse quantitative trait loci (QTL) for a major component of courtship song in recombinant inbred lines derived from two laboratory strains of Drosophila melanogaster. The total variance among lines exceeds that between parental strains, and is broadly similar to that seen among geographic strains of the Cosmopolitan form of this species. Previous studies of the quantitative genetics of fly song have implied a polygenic additive inheritance with numerous genes spread throughout the genome. We find evidence for only three significant QTLs explaining 54% of the genetic variance in total. Thus there is evidence for a few large effect genes contributing to the genetic variance among lines. Interestingly, almost all of the candidate song genes previously described for D. melanogaster do not coincide with our QTLs.     

Leg development in flies versus grasshoppers: differences in dpp expression do not lead to differences in the expression of downstream components of the leg patterning pathway

All insect legs are structurally similar, characterized by five primary segments. However, this final form is achieved in different ways. Primitively, the legs developed as direct outgrowths of the body wall, a condition retained in most insect species. In some groups, including the lineage containing the genus Drosophila, legs develop indirectly from imaginal discs. Our understanding of the molecular mechanisms regulating leg development is based largely on analysis of this derived mode of leg development in the species D. melanogaster. The current model for Drosophila leg development is divided into two phases, embryonic allocation and imaginal disc patterning, which are distinguished by interactions among the genes wingless (wg), decapentaplegic (dpp) and distalless (dll). In the allocation phase, dll is activated by wg but repressed by dpp. During imaginal disc patterning, dpp and wg cooperatively activate dll and also indirectly inhibit the nuclear localization of Extradenticle (Exd), which divide the leg into distal and proximal domains. In the grasshopper Schistocerca americana, the early expression pattern of dpp differs radically from the Drosophila pattern, suggesting that the genetic interactions that allocate the leg differ between the two species. Despite early differences in dpp expression, wg, Dll and Exd are expressed in similar patterns throughout the development of grasshopper and fly legs, suggesting that some aspects of proximodistal (P/D) patterning are evolutionarily conserved. We also detect differences in later dpp expression, which suggests that dpp likely plays a role in limb segmentation in Schistocerca, but not in Drosophila. The divergence in dpp expression is surprising given that all other comparative data on gene expression during insect leg development indicate that the molecular pathways regulating this process are conserved. However, it is consistent with the early divergence in developmental mode between fly and grasshopper limbs.     

Pattern formation in imaginal discs of Drosophila melanogaster after irradiation of embryos and young larvae

Embryos and first instar larvae of Drosophila melanogaster were X-irradiated in order to study pattern formation in discs damaged at early stages. After treatment, appendages were found in which some pattern elements were duplicated and others were absent. In some strains legs were preferentially duplicated, and in others, antennae were preferentially duplicated. Duplicated appendages were mirror images and resulted most frequently when animals were irradiated during late embryonic or early larval stages. Appendages varied from those which showed complete duplication of only the distal parts (claws or aristae), to those which showed duplication of almost the entire appendage. Examination of the cuticular patterns in duplicated mesothoracic legs showed that in legs with complete duplication solely in distal regions, only extreme lateral leg parts were duplicated, and medial leg parts were absent. In legs with duplication extending into proximal regions, much of the lateral side was duplicated, and only extreme medial parts were missing. The situation for partially duplicated antennae was similar. Prothoracic legs were found fused in some X-rayed flies. The cuticular patterns were almost perfect mirror images, although the amount of fusion varied widely between different individuals. Apparently the pattern forming processes in the right and left first leg discs are coordinated in X-rayed animals. The results were consistent with a model embracing a gradient of developmental capacity in the early disc similar to that postulated to exist in the late third instar leg disc. This model is also consistent with results of various surgical experiments reported in the literature. Several predictions of the model are explained including the possible mode of action of a specific class of mutants which may affect pattern formation by altering a morphogenetic gradient.     

Quantitative trait loci and interaction effects responsible for variation in female postmating mortality in Drosophila simulans and D. sechellia introgression lines

Mating appears to inflict a cost to Drosophila females, resulting in a reduction of their lifespan shortly after mating. Males from different chromosome extracted lines differ significantly in their detrimental effects on postmating female survival, and seminal fluid proteins produced in the male accessory glands are at least partially responsible for the effect. This suggests that there is a genetic basis underlying the male inflicted effect on female's postmating mortality. However, the genes responsible for this effect remain elusive. Using males from introgression lines between D. simulans and D. sechellia genomes and a quantitative trait locus (QTL) mapping approach, we identified chromosomal regions that affect postmating mortality of females. We found a second chromosome QTL with an effect on average female lifespan after mating and a third chromosome QTL with an effect on postmating female mortality rate. Under the general observation of a faster divergence of sex-related genes among closely related species, it is predicted that genes for reproductive traits other than hybrid sterility will show evidence of epistatic effects when brought into a heterospecific background. We detected a significant epistatic genetic effect on postmating female mortality rate that supports this prediction.