Adaptive associations between total body color dimorphism and climatic stress‐related traits in a stenothermal circumtropical Drosophila species

Abstract Low desiccation resistance of Drosophila ananassae reflects its rarity outside the humid tropics. However, the ability of this sensitive species to evolve under seasonally varying subtropical areas is largely unknown. D. ananassae flies are mostly lighter during the rainy season but darker and lighter flies occur in the autumn season in northern India. We tested the hypothesis whether seasonally varying alternative body color phenotypes of D. ananassae vary in their levels of environmental stress tolerances and mating behavior. Thus, we investigated D. ananassae flies collected during rainy and autumn seasons for changes in body melanization and their genetic basis, desiccation‐related traits, cold tolerance and mating propensity. On the basis of genetic crosses, we found total body color dimorphism consistent with a single gene model in both sexes of D. ananassae. A significant increase in the frequency of the dark morph was observed during the drier autumn season, and body color phenotypes showed significant deviations from Hardy‐Weinberg equilibrium, which suggests climatic selection plays a role. Resistance to desiccation as well as cold stress were two‐ to three‐fold higher in the dark body color strain as compared with the light strain. On the basis of no‐choice mating experiments, we observed significantly higher assortative matings between dark morphs under desiccation or cold stress, and between light morphs under hot or higher humidity conditions. To the best of our knowledge, this is the first report on the ecological significance of seasonally varying total body color dimorphism in a tropical species, D. ananassae.     

Impact of body melanisation on contrasting levels of desiccation resistance in a circumtropical and a generalist <Emphasis Type="Italic">Drosophila</Emphasis> species

We investigated the role of cuticular lipids, body melanisation and body size in conferring contrasting levels of desiccation resistance in latitudinal populations of Drosophila melanogaster and Drosophila ananassae on the Indian subcontinent. Contrary to the well known role of cuticular lipids in water proofing in diverse insect taxa, there is lack of geographical variations in the amount of cuticular lipids per fly in both the species. In D. ananassae, quite low levels of body melanisation are correlated with lower desiccation resistance. By contrast, increased levels of desiccation resistance are correlated with quite high melanisation in D. melanogaster. Thus, species specific cuticular melanisation patterns are significantly correlated with varying levels of desiccation resistance within as well as between populations and across species. Role of body melanisation in desiccation resistance is further supported by the fact that assorted dark and light flies differ significantly in cuticular water loss, hemolymph and dehydration tolerance. However, similar patterns of body size variation do not account for contrasting levels of desiccation resistance in these two Drosophila species. Climatic selection is evidenced by multiple regression analysis with seasonal amplitude of thermal and humidity changes (Tcv and RHcv) along latitude on the Indian subcontinent. Finally, the contrasting levels of species specific distribution patterns are negatively correlated with RHcv of sites of origin of populations i.e. a steeper negative slope for D. ananassae corresponds with its desiccation sensitivity as compared with D. melanogaster. Thus, evolutionary changes in body melanisation impact desiccation resistance potential as well as distribution patterns of these two Drosophila species on the Indian subcontinent.     

Population and behaviour genetics of Drosophila ananassae

Drosophila ananassae is a cosmopolitan and domestic species. It occupies a nuique status among the Drosophila species due to certain peculiarities in its genetic behaviour. The most unusual feature of this species is spontaneous male recombination in appreciable frequency. The present review summarises the work done on population and behaviour genetics of D. ananassae from India. Population dynamics of three cosmopolitan inversions has been studied in Indian population of D. ananassae and it is evident from the results that there is a considerable degree of genetic divergence at the level of inversion polymorphism. In general, the populations from south India show more differentiation than those from the north. These three cosmopolitan inversions, which are coextensive with the species, exhibit heterosis. Interracial hybridization does not lead to beaakdown of heterosis, which suggests that evidence for coadaptation is lacking in geographic populations of D. ananassae. Heterosis appears to be simple luxuriance rather than populational heterosis (coadaptation). Unlinked inversions occur in random associations, indicating no interchromosomal interactions. However, two inversions of the third chromosome often show strong linkage disequilibrium in laboratory populations, which is due to epistatic gene interaction and suppression of crossing-over. Genetic variations for certain allozyme polymorphism and sternoleural bristle phenotypes in Indian populations of D. ananassae have also been observed.A number of investigations have also been carried out on certain aspects of behaviour genetics of Indian D. ananassae. There is evidence for sexual isolation within D. ananassae. Significant variations in mating propensity of several isofemale strains, inversion karyotypes, the diminishing effects of certain mutations on sexual activity of males and positive response to selection for high and low mating propensity provide evidence for genetic control of sexual behaviour in D. ananassae. Males contribute more to variation and thus are more subject to intra-sexual selection than females. Evidence for rare male mating advantage has also been presented. Geographic strains of D. ananassae show variation with respect to oviposition site preference. The results of studies on pupation site preference, which is an important component of larval behaviour, suggest that larval pupation behaviour in D. ananassae is under polygenic control with a substantial amount of additive genetic variation.     

Temperature sensitivity of circadian clocks is conserved across Drosophila species melanogaster,malerkotliana and ananassae

Light and temperature are the major environmental cycles that can synchronize circadian rhythms in a variety of organisms. Previously, we have shown that under light/dark cycles of various photoperiods, the Drosophila species ananassae exhibits unimodal activity pattern with a prominent morning activity peak in contrast with Drosophila melanogaster and Drosophila malerkotliana, which show bimodal activity pattern with morning and evening activity peaks. Here we report that circadian clocks controlling activity/rest rhythm of these two less-studied species D. malerkotliana and D. ananassae can be synchronized by temperature cycles and that even under temperature cycles D. ananassae exhibits only a pronounced morning (thermophase onset) activity peak. Although D. melanogaster and D. ananassae exhibit differences in the phase of activity/rest rhythm under temperature cycles, circadian clocks of both show similar sensitivity to warm temperature pulses. Circadian period of activity/rest rhythm of D. ananassae differs from the other two species at some moderate-range temperatures; however, in conditions that are more extreme, circadian clocks of D. melanogaster, D. malerkotliana and D. ananassae appear to be largely temperature compensated.     

Molecular evolution of sex-biased genes in the Drosophila ananassae subgroup

Background  

Genes with sex-biased expression often show rapid molecular evolution between species. Previous population genetic and comparative genomic studies of Drosophila melanogaster and D. simulans revealed that male-biased genes have especially high rates of adaptive evolution. To test if this is also the case for other lineages within the melanogaster group, we investigated gene expression in D. ananassae, a species that occurs in structured populations in tropical and subtropical regions. We used custom-made microarrays and published microarray data to characterize the sex-biased expression of 129 D. ananassae genes whose D. melanogaster orthologs had been classified previously as male-biased, female-biased, or unbiased in their expression and had been studied extensively at the population-genetic level. For 43 of these genes we surveyed DNA sequence polymorphism in a natural population of D. ananassae and determined divergence to the sister species D. atripex and D. phaeopleura.     

Population genetics of Drosophila ananassae: inversion polymorphism and body size in Indian geographical populations

Twelve Indian natural populations of Drosophila ananassae, a cosmopolitan and domestic species, were sampled and laboratory populations (mass cultures) were established from naturally impregnated females. These populations were maintained in the laboratory for some generations and were analysed chromosomally to know the frequency of different inversions. The chromosomal analysis revealed the presence of three cosmopolitan inversions. The data on the whole show that there are significant differences in the frequencies of different chromosome arrangements in these populations. Body size (wing length and thorax length) was measured in both sexes (50 females and 50 males), in all the 12 geographical populations of D. ananassae. There are statistically significant differences in wing length as well as in thorax length of both sexes among different geographical populations. Five geographical strains were crossed reciprocally and body size (wing length and thorax length) was measured in F₁ and F₂ progeny. The comparison of body size (both traits) between mid‐parent, F₁ and F₂ shows that there is an increase in body size in F₁ and F₂ progeny as compared with parents. Thus, there is no break down of heterosis in F₂, which suggests absence of coadaptation in geographical populations of D. ananassae. Scaling test statistical analysis showed additive, dominance and epistatic effects in certain crosses involving geographical strains of D. ananassae. Correlation between chromosome arrangement frequency and body size has also been tested and significant negative correlation has been found between 2L – ST chromosome arrangement and male thorax.     

Female remating inDrosophila ananassae: shorter duration of copulation during second mating as compared to first mating

Drosophila ananassae, a cosmopolitan and domestic species, belongs to theananassae subgroup of themelanogaster species group. Female remating was observed in ten mass culture stocks of this species, which were initiated from flies collected from different geographic localities. The frequency of female remating ranges from 24% to 56% in different strains. Strains show significant variation in remating latency (days). Significant variation has also been found in all the stocks for duration of copulation between first and second matings. The duration of copulation is shorter in second mating as compared to first mating inD. ananassae.     

Dihydrofolate reductase activity and resistance to aminopterin in various species of Drosophila

Summary The aim of our work was to compare the mechanisms of resistance to aminopterin, inhibitor of the dihydrofolate reductase enzyme, between different Drosophila species and those described for cultured cells. Moreover we compared the systematic species divisions based on morphological traits and those based on a molecular approach. For this purpose, the effect of aminopterin on viability and wing phenotype was studied in different Drosophila species. Dihydrofolate reductase was measured in adult flies. We found an important dihydrofolate reductase activity in the melanogaster sub-group compared to the other species studies. Wing effect was observed only in this sub-group. The effects of aminopterin on the wing phenotype were very similar to the phenotype of rudimentary mutants. Both deplete the pyrimidine pool and it has been shown by the studies of the structural genes of the nucleotide pyrimidine pathway that the wing tissue is very sensitive to every pertubation of this metabolism.The D. ananassae species was found to be fully resistant at the concentrations of the inhibitor tested. No or very little dihydrofolate reductase activity was detected. The binding of the enzyme to the inhibitor was comparable to that found in the Oregon strain of D. melanogaster. The purine and pyrimidine salvage pathways were investigated and the D. ananassae species displayed an important thymidine kinase activity. The D. ananassae flies were sensitive on Sang medium compared to the Oregon flies but were able to use exogenous bases or nucleosides more efficiently. Therefore the mechanism of resistance to aminopterin in Drosophila may be different from those described for methotrexate in mammalian cultured cells, as indicated by the results obtained for D. ananassae.     

The evolutionary history of Drosophila buzzatii XXVII

The correlation between body size and longevity was tested in an Argentinian natural population of Drosophila buzzatii. Mean thorax length of flies newly emerging from rotting cladodes of Opuntia vulgaris was significantly smaller than that of two samples of flies caught at baits. The present results which might be interpreted as directional selection for longevity favoring larger flies are in agreement with previous results achieved in a Spanish natural population of D. buzzatii. Flies emerging from different substrates showed significant differences in thorax length, suggesting that an important fraction of phenotypic variance can be attributed to environmental variability. However, laboratory and field work in different populations of D. buzzatii showed a significant genetic component for thorax length variation.     

Interracial hybridization in Drosophila ananassae

Crosses were made between strains of Drosophila ananassae, homozygous for the ST or AL gene sequences in the second chromosome, and originating from geographically distant localities in India. All interracial crosses were maintained separately in culture bottles for ten generations. After ten generations it was observed that the inversion heterozygotes were heterotic. Thus it was found that interracial hybridization does not lead to breakdown of heterosis. Therefore, it has been suggested that localised coadaptation is absent in the natural populations of Indian D. ananassae. Thus D. ananassae does not show the pattern of some other species.     

Microscale Laser Surgery Demonstrates the Grasping Function of the Male Sex Combs in Drosophila melanogaster and Drosophila bipectinata

Male secondary sexual traits of animals are richly diversified in form and complexity, yet there are many species in which their precise function remains unknown. Within the genus Drosophila, species belonging to the melanogaster and obscura species groups have evolved a remarkable variety of sex combs, male‐limited secondary sexual traits located on the tarsi of both front legs. Information concerning sex comb function is minimal or absent, except for D. melanogaster, where previous studies indicate that the sex combs are used for grasping the female prior to copulation. These studies, however, do not unambiguously demonstrate comb function, because it has not been possible to ascribe observed behavioral outcomes of the various comb manipulations to changes in the combs per se. We used microscale laser surgery to manipulate comb size in D. melanogaster and D. bipectinata, and tested the hypothesis that the sex combs function as grasping devices in courtship, making them essential for copulation to ensue. Results of high‐resolution behavioral analysis in small observation arenas demonstrated that in both species in which sex combs were surgically eliminated, males were unable to grasp, mount or copulate. The combless foretarsi of these altered males slipped off the end (D. melanogaster) and sides (D. bipectinata) of the female abdomen when courting males attempted to grasp. In most cases, males whose sex combs were reduced but not completely removed exhibited similar copulation probabilities as surgical control males, a result we demonstrated in observation chambers as well as under more ecologically realistic conditions inside population cages where males and females interacted on the surface of fruit substrates. Thus, the sex combs in D. melanogaster and D. bipectinata are grasping devices, essential for mounting and copulation.     

Impact of body melanization on mating success in Drosophila melanogaster

Mating speed and copulation duration respond rapidly to laboratory selection in Drosophila melanogaster Meigen (Diptera: Drosophilidae), but there is a lack of data on the evolutionary response to natural selection in the wild. Further, it is not clear whether body melanization and mating behavior are correlated traits. Accordingly, we tested whether variation in body color impacts on mating latency, copulation duration, and fecundity in latitudinal populations of D. melanogaster. We observed geographical variation (cline) for mating propensity, i.e., mating speed as well as copulation duration increased along latitude. Phenotypic plastic responses for body melanization at 17 and 25 °C also showed significant correlations with mating latency and copulation duration. Within‐population analysis based on assorted dark and light flies of five geographical populations showed significant positive correlations of copulation duration and fecundity with body melanization. To assess the role of males and/or females on mating speed and copulation duration, we used atypical body color strains (i.e., dark and light males of D. melanogaster) for no‐choice mating tests. Our data showed a major influence of males for copulation duration and of females for mating speed. Furthermore, a difference in impact of body melanization on mating speed and copulation duration was demonstrated between species, i.e., low melanization in Drosophila ananassae Doleschall is correlated with lower mating speed and shorter copulation duration than in D. melanogaster. Geographical changes in mating propensity were significantly correlated with body melanization at three levels, i.e., within and between populations and between species. Thus, we have shown that a relationship exists between body melanization and mating success. Further, we found seasonal changes in temperature and humidity to confer selection pressures on mating‐related traits.     

Fluorescence patterns of heterochromatin in mitotic and polytene chromosomes in seven members of three sub-groups of the melanogaster species group of Drosophila

A comparative study of fluorescence patterns of heterochromatin in mitotic and polytene chromosomes of seven species belonging to 3 subgroups melanogaster sub-group: D. melanogaster and D. simulans; montium sub-group: D. kikkawai and D. jambulina; ananassae sub-group: D. ananassae, D. malerkotliana and D. bipectinata) of the melanogaster species group of Drosophila (Sophophora) has been made. Hoechst 33258 (H) fluorescence patterns of mitotic chromosomes reveal differences correlated to the taxonomic groupings of these species. The melanogaster sub-group species have H-bright regions on heterochromatin of all chromosomes; the montium subgroup species have H-bright regions mainly on the 4th and Y-chromosomes; in the ananassae sub-group, while D. ananassae chromosomes do not show any H-bright regions, D. malerkotliana and D. bipectinata have small H-bright segments only on their 4th chromosomes. The H-and quinacrine mustard (QM) fluorescence patterns of larval salivary gland polytene chromocentre in these species, however, do not show the same taxonomic correlation. While D. ananassae and D. kikkawai polytene nuclei lack any H-or QMbright region in the chromocentre, the remaining species have prominent H-and/or QM-bright region(s). In D. jambulina, the QM-bright regions are generally bigger than H-bright regions, while in D. malerkotliana and D. bipectinata the situation is reversed. Actinomycin D counterstaining prior to H-staining of polytene preparations of each species confirms that the H-bright region/s in the chromocentre are composed of A-T rich sequences. In vivo labelling of salivary gland polytene nuclei with 5-bromodeoxyuridine for 24 to 48 h and subsequent H-staining reveals that in all the species, the H-bright regions do not replicate in 3rd instar stage and presumably represent the non-replicating alpha heterochromatin. Significantly, in all the species (excepting D. kikkawai and D. ananassae), the size, location and the number of H-and/or QM-bright regions were seen to vary in different polytene nuclei in the same gland. It seems that the organization and the extent of under-replication of alpha heterochromatin varies in different polytene nuclei. Present studies also show that even closely related species differ in the content and organization of H-bright heterochromatin. The 81 F band at the base of 3 R in D. melanogaster, but not in D. simulans, appears to contain non-replicating H-bright sequences in addition to replicating chromatin.     

Recombination between heterozygous inversions in Drosophila ananassae

The rate of recombination between linked inversions of the third chromosome of Drosophila ananassae has been investigated by means of the salivary gland smear technique. Different homozygous strains of the species were utilised during the present investigation. The data show that the crossover rate between heterozygous inversions of the third chromosome of D. ananassae varies among different strains. This suggests that recombination between heterozygous inversions is influenced by genic (strain) factors.     

Accessory gland secretory proteins in relation to fitness parameters of Drosophila ananassae and D. varians

Developmental morphometry, qualitative and quantitative analysis of the accessory gland secretory proteins, fecundity and productivity in relation to protein ejected during subsequent (first to fourth-time) matings have been studied in Drosophila ananassae Doleschall and Drosophila varians Bock. In both species, size and secretion of accessory glands increases from 1 to 8 days and the stored secretion ejected from males to the female genital tract during subsequent mating varies. The maximum number of eggs and flies are produced from the females mated with bachelor males and it is a minimum when virgin females are mated with fourth-time mated males. Sodium dodecylsulfate–polyacrylamide gel electrophoresis analysis of accessory gland secretory protein patterns and their glycosylation differs in both the species. Correlation coefficient analysis between gland size and quantity of secretion, percentage of secretory protein transferred per mating, and eggs and flies that emerged showed a highly significant, positive relationship. Among different matings, the number of eggs laid and flies that emerged per female between subsequent (first to fourth-time) matings of males was found to be highly significant and the difference between fecundity and productivity between the two species was highly significant.     

Effect of Temperature on Longevity and Productivity in Drosophila Ananassae: Evidence for Adaptive Plasticity and Trade-Off Between Longevity and Productivity

The genetic response of body size to temperature in the laboratory provides an interesting example of phenotypic plasticity. We found that females of Drosophila ananassae reared to adulthood at 18°C showed significant increase in body weight as compared to females reared at 25°C. At a given temperature, early productivity and lifetime productivity were the highest when the rearing and test temperature were the same. The effect of test temperature was highly significant for total productivity and early productivity. The interaction between test temperature and development temperature was also highly significant. Effect of development temperature was not significant. The females reared at 18°C showed greater body weight but their productivity was not significantly higher than smaller females reared at 25°C. Thus, the usually close relationship between size and fecundity is lost when the size change is due to rearing temperature. These findings provide evidence for adaptive plasticity in D. ananassae. We also found a negative correlation (trade-off) between longevity and productivity, the first report of such a trade-off between longevity and productivity in D. ananassae.     

High genetic differentiation between an African and a non-African strain of <Emphasis Type="Italic">Drosophila simulans</Emphasis> revealed by segregation distortion and reduced crossover frequency

Drosophila simulans strains originating from Madagascar and nearby islands in the Indian Ocean often differ from those elsewhere in the number of sex comb teeth and the degree of morphological anomaly in hybrids with D. melanogaster. Here, we report a strong segregation distortion in the F1 intercross between two D. simulans strains originating from Madagascar and the US, possibly at both the gametic and zygotic levels. Strong bias against alleles of the Madagascar strain was observed for all ten marker loci distributed over the entire second chromosome in the F1 intercross, but only a few showed a weak distortion in the isogenic backgrounds of either strains. Significant deviations of genotype frequencies from Hardy–Weinberg proportions were consistently observed for the second chromosome. By contrast, the X and third chromosomes did not show any strong segregation distortion. Crossover frequency on the second chromosome was uniformly reduced in isogenic backgrounds whereas the map lengths in the F1 intercross were comparable to or larger than that of the standard D. melanogaster map. We discuss these findings in relation to previous studies on other traits and interspecific differences between D. mauritiana, which is endemic to Mauritius Island, and D. simulans.     

Evolutionary genetics of Drosophila ananassae. I. Effect of selection on body size and inversion frequencies

Adaptive importance of inversion polymorphism has been discussed in Drosophila species at several levels but no study has been carried out demonstrating the individual and combined effects of polymorphic inversions on the fitness of flies through bi‐directional selection. Therefore, artificial bi‐directional selection for thorax length in Drosophila ananassae was carried out for 10 generations. Both, Tukey test for selection difference and regression coefficients of offspring on mid‐parent are highly significant. The realized heritability (h²) in males of both high and low selection lines is more or less similar but is more pronounced in low line females, which suggests the asymmetrical response. This asymmetry in selection is discussed in the light of evidence provided by the study of chromosome inversion frequencies in different selection lines at different generations of selection. Interestingly, chromosome inversion frequency changes towards homozygosity for different gene arrangements in different selection lines. Tests of correlations at G₆ and G₁₀ among different gene arrangements as well as with mean thorax length suggest that 2L‐ST gene arrangement is negatively correlated, while 3L‐ST gene arrangement is positively correlated with thorax length. Furthermore, the present study shows the significant effects of 3L‐ST and 2L + 3L (positive correlation) on thorax length, while 3R‐ST and 2L + 3R show significant effect (negative correlation) on thorax length, which was not evident in the previous study. Present results also suggest how polymorphic inversions and their combinations affect the body size differently in different selection lines. These results suggest that thorax length in D. ananassae is under polygenic control and inversion polymorphism plays crucial role in maintaining body size by modifying genotypic frequency under various selection pressures.     

Shifting clinal patterns of stress resistance traits in Drosophila ananassae

Drosophila ananassae, a desiccation and cold sensitive species, is abundant along the latitudinal gradient of the Indian subcontinent. Analysis of seasonally varying wild-caught flies showed two independent patterns of melanisation: (1) narrow to broad melanic stripes on three anterior abdominal segments only; (2) a novel body color pattern (dark vs. light background). We investigated the degree to which these two melanisation systems vary; first with latitude and secondly among seasons. There is a shallow latitudinal cline for percent striped melanisation as well as for frequency of body color alleles during the rainy season. The frequencies of body color alleles vary significantly across seasons in the northern populations i.e. the light allele occur abundantly (>0.94) during the rainy season while the frequency of the dark allele increases (0.22–0.35) during the dry season causing steeper clines during the dry season. By contrast, the low variations in abdominal stripes showed non-significant changes and the cline was similar across seasons. Furthermore, both types of melanisation patterns showed no plasticity with respect to temperature. The present study also investigated clines related to desiccation, heat and cold stress in D. ananassae females across seasons (rainy and dry) from nine latitudinal populations. The clines for stress related traits changes to steeper and non-linear during the dry season. Thus, latitudinal populations of D. ananassae differ in slope values of clines for stress related traits across seasons. This study reports seasonal changes in latitudinal clines of stress resistance traits as seen in a changing frequency of body color alleles of D. ananassae in northern locality, while in southern localities it remains constant. This is presumably the result of only minor seasonal changes in humidity and temperature in the South, whereas in the North seasonal climatic variability is much higher.