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.     

Natural Conditions Override Differences in Emergence Rhythm among Closely Related Drosophilids

Previous studies on adult emergence rhythm of Drosophila melanogaster (DM) done under semi-natural conditions have shown that emergence is correlated to daily changes in temperature, humidity and light at dawn. Recently we showed that under laboratory conditions D. ananassae (DA), a closely related species of DM exhibits patterns in its activity/rest rhythm distinct from the latter. Here, we report the results of a study aimed at examining whether this difference in activity/rest rhythm among species extends to other circadian behaviours such as the adult emergence rhythm under a more natural environment with multiple cyclic time cues. We monitored the adult emergence rhythm of recently wild-caught DM and DA populations in parallel with those of a related species D. malerkotliana (DK), both in the laboratory and under semi-natural conditions. We find that although DM, DK and DA showed marked difference from one another under laboratory conditions, such differences were not detectable in the emergence behaviour of these three species under semi-natural conditions, and that they respond very similarly to seasonal changes in the environment. The results suggest that seasonal changes in temperature and humidity contribute largely to the variation in adult emergence waveform in terms of gate width, phase and amplitude of the peak and day-to-day variance in the timing of the emergence peak. In all three species, seasons with cooler and wetter conditions make the rhythm less tightly gated, with low amplitude peak and high day-to-day variation in timing of the peak of emergence. We show that in nature the emergence rhythm of DM, DK and DA is strongly influenced by environmental factors such that in a given season all of them exhibit similar time course and waveform and that with the changing season, they all modify their emergence patterns in a similar manner.     

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.     

Larval pupation site preference on fruit in different species of Drosophila

Larval pupation site preference (PSP) of different species of Drosophila was analyzed on fruit in the laboratory. The larvae of D. melanogaster, D. ananassae, D. virilis, D. novamexicana and D. hydei pupated on the surface of glass vials; D. simulans, D. yakuba, D. mauritiana and D. malerkotliana pupated in/on fruit; and D. rajasekari pupated on cotton plugs in all experiments. D. bipectinata larvae changed their preference from fruit in the control to glass surface for all of the fruits tested. The statistical analysis of PSP (glass and fruit) found a significant result in that compared to other species, D. mauritiana and D. ananassae preferred to pupate on cotton compared to the control.     

Evolution of increased larval competitive ability in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> without increased larval feeding rate

Multiple experimental evolution studies on Drosophila melanogaster in the 1980s and 1990s indicated that enhanced competitive ability evolved primarily through increased larval tolerance to nitrogenous wastes and increased larval feeding and foraging rate, at the cost of efficiency of food conversion to biomass, and this became the widely accepted view of how adaptation to larval crowding evolves in fruitflies. We recently showed that populations of D. ananassae and D. n. nasuta subjected to extreme larval crowding evolved greater competitive ability without evolving higher feeding rates, primarily through a combination of reduced larval duration, faster attainment of minimum critical size for pupation, greater efficiency of food conversion to biomass, increased pupation height and, perhaps, greater urea/ammonia tolerance. This was a very different suite of traits than that seen to evolve under similar selection in D. melanogaster and was closer to the expectations from the theory of K-selection. At that time, we suggested two possible reasons for the differences in the phenotypic correlates of greater competitive ability seen in the studies with D. melanogaster and the other two species. First, that D. ananassae and D. n. nasuta had a very different genetic architecture of traits affecting competitive ability compared to the long-term laboratory populations of D. melanogaster used in the earlier studies, either because the populations of the former two species were relatively recently wild-caught, or by virtue of being different species. Second, that the different evolutionary trajectories in D. ananassae and D. n. nasuta versus D. melanogaster were a reflection of differences in the manner in which larval crowding was imposed in the two sets of selection experiments. The D. melanogaster studies used a higher absolute density of eggs per unit volume of food, and a substantially larger total volume of food, than the studies on D. ananassae and D. n. nasuta. Here, we show that long-term laboratory populations of D. melanogaster, descended from some of the populations used in the earlier studies, evolve essentially the same set of traits as the D. ananassae and D. n. nasuta crowding-adapted populations when subjected to a similar larval density at low absolute volumes of food. As in the case of D. ananassae and D. n. nasuta, and in stark contrast to earlier studies with D. melanogaster, these crowding-adapted populations of D. melanogaster did not evolve greater larval feeding rates as a correlate of increased competitive ability. The present results clearly suggest that the suite of phenotypes through which the evolution of greater competitive ability is achieved in fruitflies depends critically not just on larval density per unit volume of food, but also on the total amount of food available in the culture vials. We discuss these results in the context of an hypothesis about how larval density and the height of the food column in culture vials might interact to alter the fitness costs and benefits of increased larval feeding rates, thus resulting in different routes to the evolution of greater competitive ability, depending on the details of exactly how the larval crowding was implemented.     

Stability of Adult Emergence and Activity/Rest Rhythms in Fruit Flies Drosophila melanogaster under Semi-Natural Condition

Here we report the results of a study aimed at examining stability of adult emergence and activity/rest rhythms under semi-natural conditions (henceforth SN), in four large outbred fruit fly Drosophila melanogaster populations, selected for emergence in a narrow window of time under laboratory (henceforth LAB) light/dark (LD) cycles. When assessed under LAB, selected flies display enhanced stability in terms of higher amplitude, synchrony and accuracy in emergence and activity rhythms compared to controls. The present study was conducted to assess whether such differences in stability between selected and control populations, persist under SN where several gradually changing time-cues are present in their strongest form. The study revealed that under SN, emergence waveform of selected flies was modified, with even more enhanced peak and narrower gate-width compared to those observed in the LAB and compared to control populations in SN. Furthermore, flies from selected populations continued to exhibit enhanced synchrony and accuracy in their emergence and activity rhythms under SN compared to controls. Further analysis of zeitgeber effects revealed that enhanced stability in the rhythmicity of selected flies under SN was primarily due to increased sensitivity to light because emergence and activity rhythms of selected flies were as stable as controls under temperature cycles. These results thus suggest that stability of circadian rhythms in fruit flies D. melanogaster, which evolved as a consequence of selection for emergence in a narrow window of time under weak zeitgeber condition of LAB, persists robustly in the face of day-to-day variations in cycling environmental factors of nature.     

Status of research on Drosophila ananassae at global level

Drosophila, a dipteran insect, has been found to be the best biological model for different kinds of studies. D melanogaster was first described by Meigen in 1830, is most extensively studied species of the genus Drosophila and a number of investigations employing this species have been documented in areas such as genetics, behaviour, evolution, development, molecular biology, ecology, population biology, etc. Besides D. melanogaster, a number of other species of the genus Drosophila have also been used for different kinds of investigations. Among these, D. ananassae, a cosmopolitan and domestic species endowed with several unusual genetic features, is noteworthy. Described for the first time from Indonesia (Doleschall 1858), this species is commonly distributed in India. Extensive research work on D. ananassae has been done by numerous researchers pertaining to cytology, genetics, mutagenesis, gene mapping, crossing-over in both sexes, population and evolutionary genetics, behaviour genetics, ecological genetics, sexual isolation, fluctuating asymmetry, trade-offs etc. Genome of D. ananassae has also been sequenced. The status of research on D. ananassae at global level is briefly described in this review. Bibliography on this species from different countries worldwide reveals that maximum contribution is from India.     

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.     

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.     

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.     

The <Emphasis Type="Italic">Drosophila bipectinata</Emphasis> species complex: phylogenetic relationship among different members based on chromosomal variations

Making interspecific hybridizations, where possible remains an unparalleled option for studying the intricacies of speciation. In the Drosophila bipectinata species complex comprising of four species, namely D. bipectinata, D. parabipectinata, D. malerkotliana and D. pseudoananassae, interspecific hybrids can be obtained in the laboratory, thus bequeathing an ideal opportunity for studying speciation and phylogeny. With the view of investigating the degree of divergence between each species pair, we planned to study the polytene chromosomes of the F ₁ hybrids, as it would mirror the level of compatibility between the genomes of the parental species. Two sets of crosses were made, one involving homozygous strains of all four species from India and the other including homozygous strains from different places across the globe. Polytene chromosomes of F ₁ larvae from both sets of crosses had similar configurations. In F ₁ larvae from crosses involving D. bipectinata, D. parabipectinata and D. malerkotliana, complex configurations (depicting overlapping inversions) could be detected in different arms. However, they were fairly synapsed, indicating that the differences are only at the level of gene arrangements. The polytene chromosomes of larvae obtained by crossing D. pseudoananassae with the other three species were very thin with gross asynapsis in all the arms, demonstrating that the genome of D. pseudoananassae is widely diverged from rest of the species. The overlapping inversions (reflected in complex configuration), are inferred in the light of earlier chromosomal studies performed in this complex.     

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.     

The influence of species frequency,temperature regime and previous development condition on relative competitive ability between Drosophila melanogaster and Drosophila simulans

Some fitness components of Drosophila melanogaster and D. simulans were measured in control and inter-specific competition tests. The effects derived from different relative frequencies of the competitors were examined under a factorial scheme with two temperatures, 21 °C or room temperature, and with adults developed in mixed- or pure-species cultures. D. melanogaster appeared as a strong competitor and outnumbered D. simulans in all the culture conditions. This was because intraspecific competition was stronger than inter-specific competition for D. melanogaster whereas the reverse occurred for D. simulans. In competition, the productivity of both species generally appeared as frequency-dependent, although density-dependent productivity seems to be a more accurate explanation. D. simulans was very sensitive to variations of laboratory conditions. Room temperature and previous development with D. melanogaster were more favorable for D. simulans than 21 °C and previous development in pure cultures. These factors did not substantially affect D. melanogaster, which showed a greater ability of adaptation to laboratory conditions than its sibling D. simulans.     

Coexistence of three differentDrosophila species by rescheduling their life history traits in a natural population

We present evidence for coexistence of three differentDrosophila species by rescheduling their life history traits in a natural population using the same resource, at the same time and same place.D. ananassae has faster larval development time (DT) and faster DT(egg-fly) than other two species thus utilizing the resources at maximum at both larval and adult stages respectively. Therefore,D. ananassae skips the interspecific competition at preadult stage but suffers more from intraspecific competition. However,D. melanogaster andD. biarmipes have rescheduled their various life history traits to avoid interspecific competition. Differences of ranks tests for various life history traits suggest that except for DT(egg-pupa), the difference of ranks is highest for the combination ofD. melanogaster andD. ananassae for all other life history traits. This difference is maintained by tradeoffs between larval development time and pupal period and between pupal period and DT(egg-pupa) 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.     

DESICCATION AND STARVATION TOLERANCE OF ADULT DROSOPHILA: OPPOSITE LATITUDINAL CLINES IN NATURAL POPULATIONS OF THREE DIFFERENT SPECIES

Desiccation and starvation tolerance were measured along latitudinal transects in three Drosophilid species (Drosophila ananassae, D. melanogaster, and Zaprionus indianus) of the Indian subcontinent. In each case, significant latitudinal clines were observed; desiccation tolerance increased with latitude while starvation tolerance decreased. Such field observations suggest that desiccation and starvation tolerance are fitness related traits that are independently selected in nature and genetically independent. It was, however, difficult to relate these genetic changes with precise climatic variables, except winter temperature. The overall negative correlation between the two traits, which was evidenced in natural populations, contrasts with a positive correlation generally observed in various laboratory selection experiments and that also seems to exist between different species. These observations point to the difficulty of interpreting correlations among fitness-related traits when different evolutionary levels are compared, and also different sets of data, that is, field versus laboratory studies.     

Oviposition pattern of several Drosophila species under various light environments

The oviposition pattern over two continuous days under constant light (LL) and light-and-dark (LD) conditions was studied by means of a special apparatus; four Drosophila species (D. lutescens, D. melanogaster, D. hydei, and D. virilis) were used. The results showed that: (1) Under the LL condition, the oviposition pattern was characteristic for each species. The number of eggs laid by D. lutescens was smaller than that laid by the other species, and the oviposition pattern of this species was rhythmical. Females of D. melanogaster laid eggs continuously, but at a low rate. In contrast, D. hydei and D. virilis laid eggs in clusters, the total numbers of eggs being larger than those of D. lutescens and D. melanogaster. (2) Females of three species (D. lutescens, D. melanogaster, and D. virilis) laid more eggs in the light phase than in the dark phase under the LD condition. However, no consistent trend was obtained with D. hydei, suggesting that the oviposition pattern of this species is indifferent to the light condition (i.e., it is independent of it). (3) No effects of pre-experimental light condition on the oviposition were found for any of the species. The light-dependency of the oviposition pattern is discussed from the viewpoint of adaptation.     

Evolutionary History and Mode of the <Emphasis Type="Italic">amylase</Emphasis> Multigene Family in <Emphasis Type="Italic">Drosophila</Emphasis>

Previous studies indicate that the tandemly repeated members of the amylase (Amy) gene family evolved in a concerted manner in the melanogaster subgroup and in some other species. In this paper, we analyzed all of the 49 active and complete Amy gene sequences in Drosophila, mostly from subgenus Sophophora. Phylogenetic analysis indicated that the two types of diverged Amy genes in the Drosophila montium subgroup and Drosophila ananassae, which are located in distant chromosomal regions from each other, originated independently in different evolutionary lineages of the melanogaster group after the split of the obscura and melanogaster groups. One of the two clusters was lost after duplication in the melanogaster subgroup. Given the time, 24.9 mya, of divergence between the obscura and the melanogaster groups (Russo et al. 1995), the two duplication events were estimated to occur at about 13.96 ± 1.93 and 12.38 ± 1.76 mya in the montium subgroup and D. ananassae, respectively. An accelerated rate of amino acid changes was not observed in either lineage after these gene duplications. However, the G+C contents at the third codon positions (GC3) decreased significantly along one of the two Amy clusters both in the montium subgroup and in D. ananassae right after gene duplication. Furthermore, one of the two types of the Amy genes with a lower GC3 content has lost a specific regulatory element within the montium subgroup species and D. ananassae. While the tandemly repeated members evolved in a concerted manner, the two types of diverged Amy genes in Drosophila experienced frequent gene duplication, gene loss, and divergent evolution following the model of a birth-and-death process.     

Female Remating in Drosophila ananassae: Evidence for the Effect of Density on Female Remating Frequency

Drosophila ananassae, a cosmopolitan and domestic species, is largely circumtropical in distribution and belongs to the ananassae species complex of the ananassae subgroup of the melanogaster species group. In the present study, experiments were conducted to investigate the effect of density on female remating frequency by employing different wild-type and mutant strains of D. ananassae. Two experimental designs, i.e., 2-h daily observation and continuous confinement, were used. The results show that there is significant dependence of remating frequency on density in all strains tested under both experimental designs except in a wild-type strain (Bhutan), which shows no dependence of remating frequency on density under 2-h daily observation design. This finding provides evidence that density may increase the frequency of female remating in D. ananassae.