Spiroplasma infection in Drosophila melanogaster: what is the advantage of killing males?

Male-killing bacteria are maternally inherited agents that cause death of sons of infected females. Their transmission rate is commonly high but imperfect and also sensitive to different environmental factors. Therefore, the proportion of infected females should be reduced in each generation. In order to explain male-killers spread and persistence in host population, a mechanism resulting in the relative increase of infected females must outweigh the losses caused by the imperfect transmission. The resource release hypothesis states that the males' death results in increased resources available to sibling females which would otherwise be used by their male siblings. Infected females are then expected: to be larger than uninfected females in natural populations; or to have higher viability; or to have shorter development times; or any combination of these outcomes. Here, we tested the resource release hypothesis by measuring body size of infected and uninfected wild-caught Drosophila melanogaster females and carried out other fitness related measures in the laboratory. Wild-caught infected females produced more daughters than uninfected females in their first days in the laboratory. However, although no significant difference in viability was found in a controlled experiment with infected and uninfected flies from a standard laboratory strain, there was a decrease in development time probably mediated by reduced competition. Fitness effects conditioned by the host genetic background are pointed out as a possible explanation for this difference between wild and laboratory flies. Our findings are discussed in the context of the resource advantage hypothesis.

Wing morphometry as a tool for correct identification of primary and secondary New World screwworm fly.

Cochliomyia hominivorax and Cochliomyia macellaria are endemic Neotropical Calliphoridae species. The former causes severe myiasis in hosts while the latter is Sarcosaprophagous, but commonly found as a second invader in wounds. Due to the morphological similarity between them and the potential losses that C. hominivorax represents for cattle breeders, the rapid and correct identification of these two species is very important. In addition to a correct identification of these species, a good knowledge of C. hominivorax biology can be helpful for designing control programs. We applied geometric morphometric methods to assess wing differences between C. hominivorax and C. macellaria and conduct a preliminary analysis of wing morphological variation in C. hominivorax populations. Canonical variate analysis, using wing shape data, correctly classified 100% of the individuals analyzed according to sex and species. This result demonstrates that wing morphometry is a simple and reliable method for identifying C. hominivorax and C. macellaria samples and can be used to monitor C. hominivorax. Both species show sexual dimorphism, but in C. hominivorax it is magnified. We suggest that this may reflect different histories of selection pressures operating on males and females. Significant differences in wing size and shape were obtained among C. hominivorax populations, with little correlation with latitude. This result suggests that wing variation is also a good morphological marker for studying population variation in C. hominivorax.

Comparative analysis of the chromosomal positions of rDNA genes in species of the tripunctata radiation of Drosophila.

The location of rDNA on chromosomes has been found to be highly variable in different groups of insect families of various orders. Yet, in other insect families the rDNA position is relatively constant. This contrast so far has received limited attention. We investigated the rDNA position on mitotic chromosomes in 18 species of Drosophila, 16 of which are from the tripunctata radiation, subgenus Drosophila, through fluorescent in situ hybridization (FISH). All species showed fluorescent signals only on the sex chromosomes. On the X chromosome a single fluorescent mark, but with variable locations, was found. On the Y, we observed variation both in location and in number of fluorescent marks (from 1 to 5). This constancy of chromosome location, in contrast to the great variability found in other groups, is consistent with the work carried out in other species of Drosophila. This suggests that the presence of a nucleolus organizer region (NOR) on each sex chromosome is probably an ancestral condition in the genus. Moreover, this difference in the variation of rDNA position among groups points out an interesting evolution question, which deserves further study.

Complex patterns of genetic variability in populations of the New World screwworm fly revealed by mitochondrial DNA markers.

Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), the New World screwworm fly, is an important agent of traumatic myiasis, which is endemic in the Neotropical region and which has great economic impact on the development of the livestock industry. International efforts have been aimed at designing programmes to control and eradicate this species from endemic areas. Thorough knowledge of the population genetics of an insect pest is a fundamental component to ensuring the success of a pest management strategy because it enables the determination of an appropriate geographic scale for carrying out effective treatments. This study undertook an analysis of mtDNA polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) in 34 populations of C. hominivorax from 10 countries, encompassing almost all the current distribution of the species. Results showed high levels of mitochondrial DNA variability (pi= 2.9%) and a complex pattern of population genetic structure for this species. Significant population structure (Phi st= 0.5234) and low variability were found in Caribbean populations, suggesting that, in general, islands constitute independent evolutionary entities connected by restricted gene flow. By contrast, high variability and low, but significant, differentiation was found among mainland populations (Phi st= 0.0483), which could not be attributed to geographic distance. Several processes may be acting to maintain the observed patterns, with different implications for establishing control programmes.

Photographic map of the polytene chromosomes of Cochliomyia hominivorax.

Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. A polytene chromosome map is an invaluable tool for the genetic analysis and manipulation of any species because it allows the integration of physical and genetic maps. Cochliomyia hominivorax has a diploid number of 12 chromosomes (2n = 12): five pairs of autosomes and one pair of sex chromosomes (XX/XY), which do not polytenize. We created a new photomap of the polytene chromosomes of C. hominivorax describing its five autosomes (chromosomes 2-6). Pupal trichogen cells, which have chromosomes with a high degree of polytenization, were used to elaborate this map. The photomap was made by comparing 20 different nuclei and choosing, for each chromosome segment, the region with the highest resolution. Thus, we present a new photomap of the five autosomes of this species, with a total resolution of 1450 bands.

Fitness effects of Wolbachia and Spiroplasma in Drosophila melanogaster.

Maternally inherited endosymbionts that manipulate the reproduction of their insect host are very common. Aside from the reproductive manipulation they produce, the fitness of these symbionts depends in part on the direct impact they have on the female host. Although this parameter has commonly been investigated for single infections, it has much more rarely been established in dual infections. We here establish the direct effect of infection with two different symbionts exhibiting different reproductive manipulation phenotypes, both alone and in combination, in the fruit fly Drosophila melanogaster. This species carries a cytoplasmic incompatibility inducing Wolbachia and a male-killing Spiroplasma, occurring as single or double (co-) infections in natural populations. We assessed direct fitness effects of these bacteria on their host, by comparing larval competitiveness and adult fecundity of uninfected, Wolbachia, Spiroplasma and Wolbachia-Spiroplasma co-infected females. We found no effect of infection status on the fitness of females for both estimates, that is, no evidence of any benefits or costs to either single or co-infection. This leads to the conclusion that both bacteria probably have other sources of benefits to persist in D. melanogaster populations, either by means of their reproductive manipulations (fitness compensation from male death in Spiroplasma infection and cytoplasmic incompatibility in Wolbachia infection) or by positive fitness interactions on other fitness components.

Quantitative trait analysis and geographic variability of natural populations of Zaprionus indianus, a recent invader in Brazil.

Five natural samples of a recent South America invader, the drosophilid Zaprionus indianus, were investigated with the isofemale line technique. These samples were compared to five African mainland populations, investigated with the same method. The results were also compared to data obtained on mass cultures of other populations from Africa and India. Three quantitative traits were measured on both sexes, wing and thorax length and sternopleural bristle number. We did not find any latitudinal trend among the American samples, while a significant increase in body size with latitude was observed in the Indian and, to a lesser degree, in the African populations. American populations were also characterized by their bigger size. Genetic variability, estimated by the intraclass correlation among isofemale lines, was similar in American and African populations. The intraline, nongenetic variability was significantly less in the American samples, suggesting a better developmental stability, the origin of which is unclear. A positive relationship was evident between intraline variability of size traits and the wing-thorax length correlation. Altogether, our data suggest that the colonizing propagule introduced to Brazil had a fairly large size, preventing any bottleneck effect being detected. The big body size of American flies suggests that they came from a high-latitude African country. The lack of a latitudinal dine in America seems to be related to the short time elapsed since introduction. The very rapid spread of Z. indianus all over South America suggests that it might rapidly invade North America.

Male-killing Spiroplasma naturally infecting Drosophila melanogaster.

Elucidation of the mechanism of action of selfish genetic elements is difficult outside species with well-defined genetics. Male-killing, the phenomenon whereby inherited bacteria kill male hosts during embryogenesis, is thus uncharacterized in mechanistic terms despite being common and important in insects. We characterized the prevalence, identity and source of the male-killing infection recently discovered in Drosophila melanogaster in Brazil. Male-killing was found to be present in 2.3% of flies from Recife, Brazil, and was uniquely associated with the presence of Spiroplasma infection. The identity of sequences across part of the 16S and across the 16S-23S ITS region indicated that the male-killing infection of D. melanogaster was very closely related to S. poulsonii, the source of the male-killing infection in willistoni group flies also found in South America. The sequences of two further protein-coding genes indicated the D. melanogaster infection to be most closely related to that found in D. nebulosa, from the willistoni group. Our data suggest that the establishment of D. melanogaster in South America was associated with the movement of male-killing bacteria between species.

Colour polymorphism in Drosophila mediopunctata: genetic (chromosomal) analysis and nonrandom association with chromosome inversions.

The presence of three dark spots on the abdomen is typical of the tripunctata group of Drosophila, which is the second largest Neotropical group, with 56 species. In some species, such as D. mediopunctata, the colour pattern varies considerably: ranging from flies showing no spots up to flies with three dark spots. In this paper, we present a genetic (chromosomal) analysis of this character showing that this colour polymorphism is genetically determined mainly by the second chromosome. Since this chromosome is the most polymorphic for inversions in this species, we also examined the influence of the inversions on this character. We used strains in which different second chromosomes were placed on the same genetic background and the offspring between them. We found a nonrandom association between the number of spots and the inversions PA0 and PC0. Thus, our results are consistent with the idea that the factors or genes determining a conspicuous polymorphism are likely to be associated, forming a supergene, and this association would be most efficiently accomplished through a chromosome inversion. Moreover, this is the first time that an association between a conspicuous morphological polymorphism and chromosome inversions has been described.

Low temperature cure of a male killing agent in Drosophila melanogaster.

Environmental factors can affect transmission or phenotype expression of selfish cytoplasmic endosymbionts such as embryonic male killers. Temperature is one factor that usually affects the transmission rate of selfish cytoplasmic endosymbionts. Heat cures have been described for several host-parasite systems, cold cures, however, are rare. We report a temperature cure of the Drosophila melanogaster male-killing agent, which occurs when flies are raised at 16.5 degrees C. Flies grown at 20, 24, and 28 degrees C maintained an extremely female biased sexual proportion.

Male-killing selfish cytoplasmic element causes sex-ratio distortion in Drosophila melanogaster.

Sex ratio distortion induced by a male-killing agent has been found to affect Drosophila melanogaster. The trait was discovered accidentally in a collection of flies from markets in Campinas, São Paulo State, Brazil. Repeated crosses with Canton-S males (for 15 generations to date) and successful transmission using the injection of macerates of sex ratio flies, have shown that the trait is inherited maternally, is cytoplasmic and is infectious. Crosses with strains marked with the visible mutation white and viability experiments at pre-adult stages of development, indicate that the skewed sex ratio results from male mortality before hatching. Males do not transmit the trait to their progeny.

Plasticity of Drosophila melanogaster wing morphology: effects of sex, temperature and density.

In this paper we use an adjusted ellipse to the contour of the wings of Drosophila as an experimental model to study phenotypic plasticity. The geometric properties of the ellipse describe the wing morphology. Size is the geometric mean of its two radii; shape is the ratio between them; and, the positions of the apexes of the longitudinal veins are determined by their angular distances to the major axis of the ellipse. Flies of an inbred laboratory strain of Drosophila melanogaster raised at two temperatures (16.5 degrees C and 25 degrees C) and two densities (10 and 100 larvae per vial) were used. One wing of at least 40 animals of each sex and environmental condition were analyzed (total = 380), a measurement of thorax length was also taken. Wing size variation could be approximately divided into two components: one related to shape variation and the other shape independent. The latter was influenced primarily by temperature, while the former was related to sex and density. A general pattern could be identified for the shape dependent variation: when wings become larger they become longer and the second, fourth and fifth longitudinal veins get closer to the tip of the wing.

Size and shape heritability in natural populations of Drosophila mediopunctata: temporal and microgeographical variation.

'Traditional morphometrics' allows us to decompose morphological variation into its major independent sources, identifying them usually as size and shape. To compare and investigate the properties of size and shape in natural populations of Drosophila mediopunctata, estimating their heritabilities and analysing their temporal and microgeographic changes, we carried out collections on seven occasions in Parque Nacional do Itatiaia, Brazil. In one of these collections, we took samples from five different altitudes. Measurements were taken from wild caught inseminated females and up to three of their laboratory-reared daughters. Through a principal component analysis, three major sources of variation were identified as due to size (the first one) and shape (the remaining two). The overall amount of variation among laboratory flies was about half of that observed among wild flies and this reduction was primarily due to size. Shape variation was about the same under natural and artificial conditions. A genetic altitudinal cline was detected for size and shape, although altitude explained only a small part of their variation. Differences among collections were detected both for size and shape in wild and laboratory flies, but no simple pattern emerged. Shape variation had high heritability in nature, close to or above 40% and did not vary significantly temporally. Although on the overall size heritability (18 +/- 6%) was significant its estimates were not consistent along months--they were non-significant in all but one month, when it reached a value of 51 +/- 11%. Overall, this suggests that size and shape have different genetic properties.

Heritability, phenotypic and genetic correlations of size and shape of Drosophila mediopunctata wings.

We have studied the morphology of wings of Drosophila mediopunctata employing the ellipse method, a procedure that allows precise descriptions of wing size (SI), wing shape outline (SH), and placement of longitudinal wing veins. We have found that the SH and the points which determine the position of the apices of the third, fourth and fifth longitudinal wing veins show high heritability in nature (the lower bound for the natural heritability is above 0.25). The values found are similar to those obtained for the broad-sense heritabilities (H2) in the laboratory. However, SI and the point which determines the apex of the second longitudinal wing vein showed small lower bounds for heritability in nature, 0.05 and 0.07, respectively, in spite of the high estimates of H2 in the laboratory. These results suggest that size and shape have different genetic properties. We observed a high positive phenotypic correlation between the SH, the fourth and the fifth longitudinal wing veins, which contrasts with a negative correlation between these traits and the second longitudinal vein. That is, as the SH gets longer, the apices of the second and fifth veins become closer to each other. Positive genetic correlations in the field were detected between SH, the fourth and the fifth longitudinal veins and also between the third and the fourth veins.

Variation and heritability of aristal morphology in a natural population of Drosophila mediopunctata.

We studied the major sources influencing the variation of the number of aristal branches in a natural population of Drosophila mediopunctata. Flies were collected on six occasions at different altitudes in Parque Nacional do Itatiaia (Brazil). The progenies of these flies were reared in the laboratory at 16.5 degrees C. The number of aristal branches ranges from 11 to 15 and is influenced by sex. Estimates of the natural heritability showed that at least 20% of the total phenotypic variation is due to additive genetic variation. Although the heritability of this trait estimate in the laboratory was larger (42%), the difference between the two estimates is not statistically significant. Thus, for the number of aristal branches, laboratory estimates of heritability provide reasonable estimations of both the magnitude and significance of heritabilities in nature. The mean numbers of aristal branches in the wild-caught flies from different altitudes or months are homogeneous. The same was observed for the means of its progeny kept in the laboratory under controlled conditions. On the other hand, wild-caught females have significantly fewer aristal branches than their laboratory-raised daughters, which suggests that an environmental factor or factors may have an important influence on this trait.

An experimental demonstration of Fisher's principle: evolution of sexual proportion by natural selection.

Most sexually reproducing species have sexual proportions around 1:1. This major biological phenomenon remained unexplained until 1930, when FISHER proposed that it results from a mechanism of natural selection. Here we report the first experimental test of his model that obeys all its assumptions. We used a naturally occurring X-Y meiotic drive system--the sex-ratio trait of Drosophila mediopunctat--to generate female-biased experimental populations. As predicted by FISHER, these populations evolved toward equal sex proportions due to natural selection, by accumulation of autosomal alleles that direct the parental reproductive effort toward the rare sex. Classical Fisherian evolution is a rather slow mechanism: despite a very large amount of genetic variability, the experimental populations evolved from 16% of males to 32% of males in 49 generations and would take 330 generations (29 years) to reach 49%. This slowness has important implications for species potentially endangered by skewed sexual proportions, such as reptiles with temperature sex determination.

Polymorphism for Y-linked suppressors of sex-ratio in two natural populations of Drosophila mediopunctata.

In several Drosophila species there is a trait known as "sex-ratio": males carrying certain X chromosomes (called "SR") produce female biased progenies due to X-Y meiotic drive. In Drosophila mediopunctata this trait has a variable expression due to Y-linked suppressors of sex-ratio expression, among other factors. There are tow types of Y chromosomes (suppressor and nonsuppressor) and two types of SR chromosomes (suppressible and unsuppressible). Sex-ratio expression is suppressed in males with the SRsuppressible/Ysuppressor genotype, whereas the remaining three genotypes produce female biased progenies. Now we have found that approximately 10-20% of the Y chromosomes from two natural populations 1500 km apart are suppressors of sex-ratio expression. Preliminary estimates indicate that Ysuppressor has a meiotic drive advantage of 6% over Ynonsuppressor. This Y polymorphism for a nonneutral trait is unexpected under current population genetics theory. We propose that this polymorphism is stabilized by an equilibrium between meiotic drive and natural selection, resulting from interactions in the population dynamics of X and Y alleles. Numerical simulations showed that this mechanism may stabilize nonneutral Y polymorphisms such as we have found in D. mediopunctata.

Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp.

The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.

Morphological variation in a natural population of Drosophila mediopunctata: altitudinal cline, temporal changes and influence of chromosome inversions.

To characterize the morphological variation in a natural population of Drosophila mediopunctata, males were collected on three occasions at a single locality. From each wild-caught male 14 body measures were taken and the karyotype for inversions on chromosomes X and II was determined. Through a principal components analysis, two sources of variation, identified as size and shape, accounted for approximately 80 and 6 per cent of the total morphological variability, respectively. The shape component was determined primarily by variations in the position of the wing second longitudinal vein. Differences between collections were detected both for size and shape. An altitudinal cline was observed in respect of wing shape, although altitude explained only a small part of the shape variation. Size and shape were affected by chromosome II inversions. However, in respect of size, no direct differences were detected between karyotypes but a significant interaction between collecting date and karyotype was found. This suggests that karyotypes might differ in their norms of reaction in the field.

Y-linked suppressors of the sex-ratio trait in Drosophila mediopunctata.

X-linked meiotic drive causing female-biased progenies is known to occur in nine Drosophila species and is called 'sex-ratio'. In D. mediopunctata this trait is associated with the X:21 chromosome inversion and has variable expression. We describe here a powerful Y-linked suppressor system of sex-ratio expression in this species. There are two types of Y chromosomes (suppressor and nonsuppressor) and two types of X:21 chromosomes (suppressible and unsuppressible). Sex-ratio expression is suppressed in males with the 21suppressible/Ysuppressor genotype, whereas the remaining three genotypes produce female-biased progenies.