Copulatory courtship behavior and sine song as a mate recognition cue in Drosophila lini and its sibling species.

Most Drosophila species sing species-specific pulse songs during their "precopulatory courtship." Three sibling species of the Drosophila montium species subgroup performed "copulatory courtship": males generated courtship songs by vibrating either wing only after mounting and during copulation. In these three species, strong sexual isolation was detected between D. ohnishii and D. lini and between D. ohnishii and D. ogumai, but not between D. lini and D. ogumai. Female showed strong repelling behavior when they were mounted by a heterospecific male in the species combinations including D. ohnishii, resulting in failure of the copulation attempt of the male. Acoustic analyses of courtship songs revealed that the pulse song was irregular, without any species-specific parameters, but that the frequency of the sine song was different among the three species in accordance with the modes of sexual isolation between them; it was significantly lower in D. ohnishii (mean ± SE = 193.0 ± 1.7 Hz) but higher in D. lini (253.4 ± 2.7 Hz) and D. ogumai (246.7 ± 5.3 Hz). We suggest that this difference in the sine song frequency is a sexual signal in the Specific Mate Recognition System (SMRS) among these three Drosophila species.

Evolutionary relationships in the Drosophila ananassae species cluster based on introns of multiple nuclear loci.

The Drosophila ananassae species cluster includes D. ananassae, D. pallidosa, D. parapallidosa, and the cryptic species "pallidosa-like", "pallidosa-like Wau" and "papuensis-like" Some of the taxa are sympatric in the South Pacific, Papua New Guinea, and Southeast Asia, and gene flow between different taxa has been suspected for a handful of genes. In the present analysis, we examined DNA sequences of introns in four loci: alpha actinin (Actn) on XL, white (w) on XR, CG7785 on 2L, and zinc ion transmembrane transporter 63C (ZnT63C) on 2R. Phylogenetic trees (neighbor-joining and haplotype network) were inconsistent among these loci. Some haplotypes shared between taxa were found for w, CG7785, and ZnT63C, suggesting recent gene flow. However, no haplotypes were shared, for example, between D. ananassae and D. pallidosa for CG7785, which is close to the proximal breakpoint of In(2L)D. This suggests that taxon-specific inversions prevent gene flow, as predicted by the chromosomal speciation hypothesis.

Potential gene flow in natural populations of the Drosophila ananassae species cluster inferred from a nuclear mitochondrial pseudogene.

A pseudogene with 94% similarity to mitochondrial cytochrome c oxidase subunit I (COI) was identified and localized to chromosome 4 of Drosophila ananassae. Because this chromosome is believed to have reduced recombination, its history can be traced using the pseudo-COI sequence. Pseudo-COI sequences were obtained from 27 iso-female lines of six taxa belonging to the D. ananassae species cluster in which reproductive isolation is incomplete. The phylogenetic network constructed from seven recognized haplotypes (#0-#6) indicated that different taxa inhabiting the same geographic area share the haplotypes: #1 from Papua New Guinean populations of D. ananassae and pallidosa-like-Wau; #2 from Papua New Guinean populations of D. ananassae, pallidosa-like, and papuensis-like; and #4 from South Pacific populations of D. ananassae and D. pallidosa. Taxon-K has a unique haplotype (#6), and 18 mutation steps separate it from the closest haplotype, #2. We discuss the possibility of chromosome 4 introgression beyond taxon boundaries.

Genetic analysis of female mating recognition between Drosophila ananassae and Drosophila pallidosa: application of interspecific mosaic genome lines.

Drosophila ananassae and Drosophila pallidosa are closely related species that can produce viable and fertile hybrids of both sexes, although strong sexual isolation exists between the two species. Females are thought to discriminate conspecific from heterospecific males based on their courtship songs. The genetic basis of female discrimination behavior was analyzed using isogenic females from interspecific mosaic genome lines that carry homozygous recombinant chromosomes. Multiple regression analysis indicated a highly significant effect of the left arm of chromosome 2 (2L) on the willingness of females to mate with D. ananassae males. Not only 2L but also the left arm of chromosome X (XL) and the right arm of chromosome 3 (3R) had significant effects on the females' willingness to mate with D. pallidosa males. All regions with strong effects on mate choice have chromosome arrangements characterized by species-specific inversions. Heterospecific combinations of 2L and 3R have previously been suggested to cause postzygotic reproductive isolation. Thus, genes involved in premating as well as postmating isolation are located in or near chromosomal inversions. This conclusion is consistent with the recently proposed hypothesis that "speciation genes" accumulate at a higher rate in non-recombining genome regions when species divergence occurs in the presence of gene flow.

Seasonal fluctuation in susceptibility to insecticides within natural populations of Drosophila melanogaster. II. Features of genetic variation in susceptibility to organophosphate insecticides within natural populations of D. melanogaster.

To elucidate genetic variation in susceptibility to organophosphate insecticides within natural populations of Drosophila melanogaster, we conducted an analysis of variance for mortality data sets of isofemale lines (10-286 lines) used in the previous studies. Susceptibility of isofemale lines to the three organophosphate insecticides was continuously distributed within each natural population, ranging from susceptible to resistant. Analysis of variance showed highly significant variation among isofemale lines in susceptibility to each insecticide for each natural population. Significant genetic variances in susceptibility to the three chemicals were estimated for the Katsunuma population; 0.0529-0.2722 for malathion, 0.0492-0.1603 for prothiophos, and 0.0469-0.1696 for fenitrothion. Contrary to the consistent seasonal tendency towards an increase in mean susceptibility in the fall, reported in the previous study, genetic variances in susceptibility to the three organophosphates did not change significantly in 1997 but tended to increase by 2- to 5-times in 1998. We tested whether both the observed situations, maintenance and increase in genetic variance in organophosphate resistance, can be generated under circumstances in which the levels of resistance to the three organophosphates tended to decrease, by conducting a simulation analysis, based on the hypothesis that resistant genotypes have lower fitnesses than susceptible ones under the density-independent condition. The simulation analysis generally explained the pattern in the mean susceptibility and genetic variances in susceptibility to the three organophosphates, observed in the Katsunuma population of D. melanogaster. It was suggested that the differences in the frequencies of resistance genes in the summer population could affect the patterns in genetic variance in organophosphate resistance in the fall population.

Establishing interspecific mosaic genome lines between Drosophila ananassae and Drosophila pallidosa by means of parthenogenesis.

Strong sexual isolation exists between the closely related species Drosophila ananassae and D. pallidosa, but there is no obvious post-mating isolation; both sexes of the hybrids and their descendants appear to be completely viable and fertile. Strains exhibiting parthenogenesis have been derived from wild populations of both species. We intercrossed such strains and established iso-female lines after the second generation of parthenogenesis. These lines are clones, carrying homozygous chromosomes that are interspecific recombinants. We established 266 such isogenic lines and determined their genetic constitution by using chromosomal and molecular markers. Strong pseudo-linkage was seen between loci on the left arm of chromosome 2 and on the right arm of chromosome 3; the frequency of inheriting the two chromosome regions from the same species was significantly larger than expected. One possible cause of pseudo-linkage is female meiotic bias, so that chromosomes of the same species origin tend to be distributed to the same gamete. But this possibility is ruled out; backcross analysis indicated that the two chromosome regions segregated independently in female hybrids. The remaining possibility is elimination of low-fitness flies carrying the two chromosome regions from different species. Thus, genetic incompatibility was detected in the species pair for which no hybrid breakdown had previously been indicated. The 'interspecific mosaic genome' lines reported here will be useful for future research to identify genes involved in speciation and phenotypic evolution.

Simple synthesis of the major sex pheromone components of Drosophila ananassae and D. pallidosa.

The major sex pheromone components of Drosophila ananassae and D. pallidosa, (Z,Z)-5,25-hentriacontadiene and (Z,Z)-5,27-tritriacontadiene, respectively, were synthesized by using the Wittig olefination and sulfone coupling reactions as the C-C bond-forming steps.

Female mate recognition and sexual isolation depending on courtship song in Drosophila sechellia and its siblings.

In Drosophila sechellia, females accept males that sing heterospecific songs less than those that do not sing, whereas in D. melanogaster and D. simulans, females accept males that sing heterospecific song more than those that do not sing. Here we studied the sexual isolation of D. sechellia and its siblings using interspecific hybrids to reveal the mechanisms underlying female mate recognition. The females of hybrids mated more with winged males of the parent species than with wingless ones, suggesting that the discrimination against heterospecific songs by D. sechellia females is recessive. Female preference for courtship songs seems to be inherited additively or semidominantly. In addition, we examined female receptivity without the stimuli of courtship songs by comparing the mating frequencies between the crosses using wingless males and found that it is also inherited additively or semidominantly.

The comparison of intrinsic rates of increase among chromosome-substituted lines resistant and susceptible to organophosphate insecticides in Drosophila melanogaster.

To investigate the genetic basis of the seasonal fluctuations in resistance to three organophosphates, observed within a natural population of Drosophila melanogaster (Meigen), we compared the intrinsic rate of increase, generation time and net reproduction rate among chromosome substitution lines derived from a resistant and a susceptible line, obtained from this natural population. There was significant variation among substituted lines; lines possessing the third chromosome from the resistant line, which confers resistance to the three organophosphates, generally showed lower mean values of these fitness measures. Chromosomal analyses also indicated significant negative contributions of the third chromosome from the resistant line. However, significant positive contributions of the interactions among chromosomes from the resistant line to these fitness measures were also detected. We further conducted a local stability analysis, in which each chromosome-substituted line was assumed to be introduced at a low frequency into the initial susceptible population. It was demonstrated that the resistance factor(s) on the third chromosome tend to decrease in their frequency under both density-independent and juvenile density-regulated conditions. Based on these results, a possible explanation for the seasonal fluctuations in resistance to the three organophosphates observed in the natural population was proposed.

Genetic basis of cross-resistance to three organophosphate insecticides in Drosophila melanogaster (Diptera: Drosophilidae).

To investigate the genetic basis of cross-resistance to insecticides, we conducted genetic analyses of resistance to three organophosphate insecticides, malathion, prothiophos, and fenitrothion. After isofemale lines resistant and susceptible to all of the three organophosphates had been screened from natural populations of Drosophila melanogaster (Meigen), chromosomal analyses were performed by using chromosome-substituted lines between the resistant and the susceptible lines. The chromosomal analyses revealed that both the second and the third chromosomes contributed to resistance to the organophosphates, suggesting that this resistant line possessed at least two factors for organophosphate resistance. However, the relative contribution of each chromosome was different in resistance to different organophosphates. We further carried out genetic mapping of a resistance factor for each organophosphate on each of the two chromosomes. Each resistance factor was mapped to the position of each chromosome, about II-62 and III-50. Results of the chromosomal analyses and the genetic mapping revealed that at least two resistance factors exhibiting different patterns of cross-resistance to the organophosphates existed within a natural population of D. melanogaster. Based on this research, genetic variation in insecticide resistance within natural populations and complex as well as simple aspects of the mechanism of cross-resistance are discussed.

Light wavelength dependency of mating activity in the Drosophila melanogaster species subgroup.

The action spectra of mating activity among the six species of the Drosophila melanogaster species subgroup were compared to understand how light wavelength affects mating activity. The species fell into three groups with respect to the action spectrum of mating activity. We chose one representative species from each of the three types for detailed study: D. melanogaster, D. sechellia and D. yakuba. The mating activities were investigated under three different light intensities of three monochromatic lights stimulus. Each species showed a unique spectral and intensity response. To know the evolutionary meaning of the light wavelength dependency of mating activity, we superimposed the type of action spectrum of mating activity in these six species on a cladogram. Mating inhibition under UV was conserved in evolution among these species. Furthermore we clarified that D. melanogaster showed low mating activity under UV because males courted less under UV.

Search for species-specific mating signal in courtship songs of sympatric sibling species, Drosophila ananassae and D. pallidosa.

Sexual isolation is one of the most important mechanisms that may lead to speciation. Drosophila ananassae and D. pallidosa are useful for the study of sexual isolation, because of their sympatric distribution and no postmating isolation between them. Courtship songs are considered to play a crucial role in sexual isolation between D. ananassae and D. pallidosa. We recorded and analyzed male courtship songs of D. ananassae and D. pallidosa for eight and four geographical strains, respectively. Courtship behaviors of the two species were consistent with those previously described, however, male's middle leg shaking, which had not before been described, was observed in both species. Males sing by wing vibration only during courtship. Their song oscillograms were distinct between species, but those of conspecific strains were very similar, in spite of their different geographical derivation. We found species-specificity in burst length, pulse length, cycle number in a pulse, and frequency spectra of bursts; these results suggest that these song parameters may play a role in mate recognition that enforces their sexual isolation. The specific values of interpulse interval, cycle number in a pulse and intrapulse frequency were involved with the determination of specificity in frequency spectra of bursts. We discussed the possibility that the specific frequency spectra of bursts are recognized by females as the species-specific signal rather than each parameter individually.

Genetics of sexual isolation based on courtship song between two sympatric species: Drosophila ananassae and D. pallidosa.

Sexual isolation has been considered one of the primary causes of speciation and its genetic study has the potential to reveal the genetics of speciation. In Drosophila, the importance of courtship songs in sexual isolation between closely related species has been well investigated, but studies analysing the genetic basis of the difference in the courtship songs associated with sexual isolation are less well documented. Drosophila ananassae and Drosophila pallidosa are useful for studies of sexual isolation, because of their sympatric distribution and absence of postmating isolation. Courtship songs are known to play a crucial role in sexual isolation between these two species, and the female discrimination behaviour against the courting male has been revealed to be controlled by a very narrow region on the second chromosome. In this study we investigated the genetic basis controlling the song differences associated with their sexual isolation, using intact and wingless males with chromosomes substituted between species. The results obtained from F1 hybrid males between these species indicate the dominance of the song characters favoured by D. pallidosa females. In addition, the results obtained from backcross F2 males indicate that chromosome 2 had a major effect on the control of the song characters associated with sexual isolation.

Negative correlations between resistance to three organophosphate insecticides and productivity within a natural population of Drosophila melanogaster (Diptera: Drosophilidae).

To investigate the relationship between resistance to organophosphate insecticides and fitness components, we first measured resistance to three organophosphates, malathion, prothiophos, and fenitrothion, and productivity, a measure of fitness components, for each of the isofemale lines from the same natural population of Drosophila melanogaster (Meigen). Pearson correlation coefficients indicated that positive correlations among resistance to the organophosphates and negative correlations between resistance to each of the organophosphates and the productivity existed within the natural population. We further investigated the genetic basis of the correlations among resistance to the organophosphates and the productivity, by using chromosome-substituted lines between a resistant and a susceptible inbred line established from the same natural population. Chromosomal analyses indicated that the third chromosome from the resistant line exhibited not only significant, positive effects on resistance to all of the organophosphates tested but also a significant negative effect on the productivity, suggesting positive genetic correlations between resistance to each organophosphate and negative genetic correlations between resistance to each organophosphate and the productivity. In addition, a significant negative effect on the productivity was also detected from the second chromosome, which did not exhibit significant major effects on resistance to the organophosphates. This suggests that fitness components of resistant lines could be also affected by factors independent of insecticide resistance. The dynamics of genetic variation in resistance to the organophosphates within the natural population of D. melanogaster are discussed from the standpoint of negative genetic correlations between resistance to the organophosphates and the productivity.