Circadian clocks and life-history related traits: Is pupation height affected by circadian organization in<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>?

InD. melanogaster, the observation of greater pupation height under constant darkness than under constant light has been explained by the hypothesis that light has an inhibitory effect on larval wandering behaviour, preventing larvae from crawling higher up the walls of culture vials prior to pupation. If this is the only role of light in affecting pupation height, then various light : dark regimes would be predicted to yield pupation heights intermediate between those seen in constant light and constant darkness. We tested this hypothesis by measuring pupation height under various light : dark regimes in four laboratory populations ofDrosophila melanogaster. Pupation height was the greatest in constant darkness, intermediate in constant light, and the least in a light/dark regime of LD 14:14 h. The results clearly suggest that there is more to light regime effects on pupation height than mere behavioural inhibition of wandering larvae, and that circadian organization may play some role in determining pupation height, although the details of this role are not yet clear. We briefly discuss these results in the context of the possible involvement of circadian clocks in life-history evolution.     

Dynamics of Social Behavior in Fruit Fly Larvae

We quantified the extent and dynamics of social interactions among fruit fly larvae over time. Both a wild-type laboratory population and a recently-caught strain of larvae spontaneously formed social foraging groups. Levels of aggregation initially increased during larval development and then declined with the wandering stage before pupation. We show that larvae aggregated more on hard than soft food, and more at sites where we had previously broken the surface of the food. Groups of larvae initiated burrowing sooner than solitary individuals, indicating that one potential benefit of larval aggregations is an improved ability to dig and burrow into the food substrate. We also show that two closely related species, D. melanogaster and D. simulans, differ in their tendency to aggregate, which may reflect different evolutionary histories. Our protocol for quantifying social behavior in larvae uncovered robust social aggregations in this simple model, which is highly amenable to neurogenetic analyses, and can serve for future research into the mechanisms and evolution of social behavior.     

EXPERIMENTAL EVOLUTION OF ACCELERATED DEVELOPMENT IN DROSOPHILA. 1. DEVELOPMENTAL SPEED AND LARVAL SURVIVAL

Developmental time is a trait of great relevance to fitness in all organisms. In holometabolous species that occupy ephemeral habitat, like Drosophila melanogaster, the impact of developmental time upon fitness is further exaggerated. We explored the trade-offs surrounding developmental time by selecting 10 independent populations from two distantly related selection treatments (CB_1-5 and CO_1-5) for faster development. After 125 generations, the resulting accelerated populations (ACB_1-5 and ACO_1-5) displayed net selection responses for development time of -33.4 hours (or 15%) for ACB and -38.6 hours (or 17%) for ACO. Since most of the change in egg-to-adult developmental time was accounted for by changes in larval duration, the “accelerated” larvae were estimated to develop 25-30% faster than their control/ancestor populations. The responses of ACB and ACO lines were remarkably parallel, despite being founded from populations evolved independently for more than 300 generations. On average, these “A” populations developed from egg to adult in less than eight days and produced fertile eggs less than 24 hours after emerging. Accelerated populations showed no change in larval feeding rate, but a reduction in pupation height, the latter being a trait relating to larval energetic expenditure in wandering prior to pupation. This experiment demonstrates the existence of a negative evolutionary correlation between preadult developmental time and viability, as accelerated populations experienced a severe cost in preadult survivorship. In the final assay generation, viability of accelerated treatments had declined by more than 10%, on average. A diallel cross demonstrated that the loss of viability in the ACO lines was not due to inbreeding depression. These results suggest the existence of a rapid development syndrome, in which the fitness benefits of fast development are balanced by fitness costs resulting from reduced preadult survivorship, marginal larval storage of metabolites, and reduced adult size.     

Genetics and ecology of Drosophila melanogaster larval foraging and pupation behaviour

In this paper we show that, (1) Drosophila melanogaster larvae utilize a variety of pupal microhabitats in an orchard, (2) variation in larval foraging path length, pupation distance from the food and pupal microhabitat preference (on or off the fruit) is genetically based and, (3) variation in these behaviours can be maintained in a spatially heterogenous environment since there is a reversal in pupation site suitability in wet and dry pupal microhabitats. Differences in path length in both laboratory and natural populations can be attributed to genes on the second pair of chromosomes and is under simple genetic control, whereas differences in pupal height are polygenically inherited (the second pair of chromosomes influences pupal height three times more than the third pair). Pupae collected from on-fruit sites had shorter foraging path lengths and lower pupal heights than off-fruit populations. Populations from the orchard maintained their field pupal microhabitat preferences even after 1 year of rearing them in the laboratory. Larvae with the sitter larval phenotype (short path lengths and low pupal heights tended to pupate more on-fruit than those with the rover phenotype (long path lengths and high pupal heights). To determined if these genetically based differences in microhabitat preference contributed to fitness, larval pupation behaviour was studied in a “field assay” (dish with fruit on soil) with soil water content varied. At low soil water contents, pupal survivorship was significantly better on the fruit whereas, at high soil water contents, survivorship was better in the soil. There was a reversal in which microhabitat (dry or wet) was a better site for pupation. In the field environment where soil water content fluctuates in space and time, such a reversal would explain the maintenance of genetic variation for these larval behaviours. Another selective agent acting on D. melanogaster larvae in our orchard is parasitization by Asobara tabida. This parasitoid parasitizes larvae with high locomotory scores (e.g. rovers) significantly more than those with low scores (sitters). This study relates laboratory phenotypes to field phenotypes thereby linking the ecological, behavioural and genetic components of larval habitat selection in D. melanogaster.     

Genetic and behavioral analysis of natural variation in Drosophila melanogaster pupation position

Drosophila melanogaster pupae are exposed to many biotic and abiotic dangers while immobilized during several days of metamorphosis. As a passive defense mechanism, appropriate pupation site selection represents an important mitigation of these threats. Pupation site selection is sensitive to genetic and environmental influences, but the specific mechanisms of the behavior are largely unknown. Using a set of 76 recombinant inbred strains we identify a single quantitative trait locus, at polytene position 56A01-C11, associated with pupation site variation. We furthermore present a detailed investigation into the wandering behaviors of two strains expressing different pupation position tendencies, and identify behavioral differences. Larvae from a strain that tends to pupate relatively far from the food also tend to travel significantly farther from the media during wandering. We did not observe consistent differences in either the number or duration of wandering forays made by near or far pupating strains. The ability of larvae to integrate several internal and external environmental cues while choosing a contextually appropriate pupation site, and specifically, the variation in this ability, presents a very interesting behavioral phenotype in this highly tractable genetic model organism.     

DIRECTIONAL AND STABILIZING DENSITY-DEPENDENT NATURAL SELECTION FOR PUPATION HEIGHT IN DROSOPHILA MELANOGASTER

Six populations of Drosophila melanogaster have been kept at extreme population densities, three high and three low, for 175 generations. Larvae from the high density populations pupate 50%-100% higher than larvae from the low density populations. At high larval test densities there is both a directional and a stabilizing component to selection, with viabilities ranging from 0.14 to 0.992, depending on the choice of pupation site. The directional component is stronger on the populations which have evolved at low densities, while the stabilizing component is stronger on the populations which have evolved at high densities. There is no indication that the evolution of this trait, in response to density, has altered its phenotypic plasticity.     

Host plant exodus and larval wandering behaviour in a butterfly: diapause generation larvae wander for longer periods than do non‐diapause generation larvae

1. Prior to pupation, lepidopteran larvae enter a wandering phase lasting up to 30 h before choosing a pupation site. Because stillness is important for concealment, this behaviour calls for an adaptive explanation. 2. The explanation most likely relates to the need to find a suitable pupation substrate, especially in terms of shelter from predation, and given that many predators and parasitoids use host plants as prey‐location cues, mortality probably decreases with distance from the host plant. Hence, remaining on the host includes a long‐term risk, while moving away from the host introduces an increased risk during locomotion. 3. Bivoltine species that overwinter in the pupal stage produce two kinds of pupae; non‐diapausing pupae from which adults emerge after 1–2 weeks, or diapausing pupae that overwinter with adults emerging after 8–10 months. 4. Given the hypothesis of distance‐from‐host‐plant‐related predation, this should select for phenotypic plasticity with larvae in the diapausing generation having a longer wandering phase than larvae under direct development, if there is a trade‐off between mortality during the wandering phase and accumulated mortality during winter. 5. Here this prediction is tested by studying the duration of the wandering period in larvae of the partially bivoltine swallowtail butterfly, Papilio machaon, under both developmental pathways. 6. The results are in agreement with the predictions and show that the larval wandering phase is approximately twice as long under diapause development. The authors suggest that the longer duration of the wandering phase in the diapause generation is a general phenomenon in Lepidoptera.     

Study of Natural Genetic Variation in Early Fitness Traits Reveals Decoupling Between Larval and Pupal Developmental Time in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Characterizing the relationships between genotype and phenotype for developmental adaptive traits is essential to understand the evolutionary dynamics underlying biodiversity. In holometabolous insects, the time to reach the reproductive stage and pupation site preference are two such traits. Here we characterize aspects of the genetic architecture for Developmental Time (decomposed in Larval and Pupal components) and Pupation Height using lines derived from three natural populations of Drosophila melanogaster raised at two temperatures. For all traits, phenotypic differences and variation in plasticity between populations suggest adaptation to the original thermal regimes. However, high variability within populations shows that selection does not exhaust genetic variance for these traits. This could be partly explained by local adaptation, environmental heterogeneity and modifications in the genetic architecture of traits according to environment and ontogenetic stage. Indeed, our results show that the genetic factors affecting Developmental Time and Pupation Height are temperature-specific. Varying relationships between Larval and Pupal Developmental Time between and within populations also suggest stage-specific modifications of genetic architecture for this trait. This flexibility would allow for a somewhat independent evolution of adaptive traits at different environments and life stages, favoring the maintenance of genetic variability and thus sustaining the traits’ evolvabilities.     

Correlated changes in circadian clocks in response to selection for faster pre-adult development in fruit flies Drosophila melanogaster

Although, circadian clocks are believed to be involved in the regulation of life-history traits such as pre-adult development time and lifespan in fruit flies Drosophila melanogaster, there is very little unequivocal evidence either to support or refute this. Here we report the results of a long-term study aimed at examining the role of circadian clocks in the temporal regulation of pre-adult development in D. melanogaster. We employed laboratory selection protocol for faster pre-adult development on four large, outbred, random mating populations of Drosophila. We assayed pre-adult development time and circadian period of locomotor activity rhythm of these flies at regular intervals of 5–10 generations. After 50 generations of selection, the overall egg-to-adult duration in the selected stocks was reduced by ~29 h (~12.5 %) relative to controls, with the selected populations showing a concurrent reduction in time taken to hatching, pupation and wing pigmentation, by ~2, ~16, and ~25.2 h, respectively. Furthermore, selected populations showed a concomitant reduction in the circadian period of locomotor activity rhythm, implying that circadian clocks and development time are correlated. Thus, our study provides the first ever unequivocal evidence for the evolution of circadian clocks as a correlated response to selection for faster pre-adult development, suggesting that circadian clocks and development are linked in fruit flies D. melanogaster.     

Drosophila larval foraging behaviour: Developmental stages

Two distinct developmentally related behaviour patterns can be identified in third-instar larvae of Drosophila melanogaster, ‘foraging’ behaviour and ‘wandering’, a pre-pupation behaviour. An age-related change in behaviour from foraging to wandering is quantified by measuring larval locomotion at the early, middle and late third instar in an environment where food is distributed in patches. Strain, moisture, food and inhomogeneities in the texture of the surface of the medium significantly influence larval locomotory behaviour.     

A genetic locus affecting the developmental expression of an enzyme in Drosophila melanogaster

We have discovered and characterized strains of Drosophila melanogaster showing a genetically controlled modification of the developmental program for accumulation of aldehyde oxidase. Most strains show a sharp increase in specific activity just before pupation. The variant class, represented by several strains, does not show the increase at this stage even though changes in specific activity at other stages are comparable in the two classes. This developmental difference is controlled by a single gene or a small chromosome segment closely linked to the structural gene and apparently exercising cis-dominant control over its expression. It is hoped that this and other similar mutations currently under study will provide some insight into the organization of regulatory loci in the eukaryotic genome.     

Experimental evolution of post-ingestive nutritional compensation in response to a nutrient-poor diet

The geometric framework of nutrition predicts that populations restricted to a single imbalanced diet should evolve post-ingestive nutritional compensation mechanisms bringing the blend of assimilated nutrients closer to physiological optimum. The evolution of such nutritional compensation is thought to be mainly driven by the ratios of major nutrients rather than overall nutritional content of the diet. We report experimental evolution of divergence in post-ingestive nutritional compensation in populations of Drosophila melanogaster adapted to diets that contained identical imbalanced nutrient ratios but differed in total nutrient concentration. Larvae from ‘Selected’ populations maintained for over 200 generations on a nutrient-poor diet with a 1 : 13.5 protein : carbohydrate ratio showed enhanced assimilation of nitrogen from yeasts and reduced assimilation of carbon from sucrose than ‘Control’ populations evolved on a diet with the same nutrient ratio but fourfold greater nutrient concentration. Compared to the Controls, the Selected larvae also accumulated less triglycerides relative to protein. This implies that the Selected populations evolved a higher assimilation rate of amino acids from the poor imbalanced diet and a lower assimilation of carbohydrates than Controls. Thus, the evolution of nutritional compensation may be driven by changes in total nutrient abundance, even if the ratios of different nutrients remain unchanged.     

Pleiotropic Effects of a Mitochondrial–Nuclear Incompatibility Depend upon the Accelerating Effect of Temperature in Drosophila

Interactions between mitochondrial and nuclear gene products that underlie eukaryotic energy metabolism can cause the fitness effects of mutations in one genome to be conditional on variation in the other genome. In ectotherms, the effects of these interactions are likely to depend upon the thermal environment, because increasing temperature accelerates molecular rates. We find that temperature strongly modifies the pleiotropic phenotypic effects of an incompatible interaction between a Drosophila melanogaster polymorphism in the nuclear-encoded, mitochondrial tyrosyl-transfer (t)RNA synthetase and a D. simulans polymorphism in the mitochondrially encoded tRNA^Tyr. The incompatible mitochondrial–nuclear genotype extends development time, decreases larval survivorship, and reduces pupation height, indicative of decreased energetic performance. These deleterious effects are ameliorated when larvae develop at 16° and exacerbated at warmer temperatures, leading to complete sterility in both sexes at 28°. The incompatible genotype has a normal metabolic rate at 16° but a significantly elevated rate at 25°, consistent with the hypothesis that inefficient energy metabolism extends development in this genotype at warmer temperatures. Furthermore, the incompatibility decreases metabolic plasticity of larvae developed at 16°, indicating that cooler development temperatures do not completely mitigate the deleterious effects of this genetic interaction. Our results suggest that the epistatic fitness effects of metabolic mutations may generally be conditional on the thermal environment. The expression of epistatic interactions in some environments, but not others, weakens the efficacy of selection in removing deleterious epistatic variants from populations and may promote the accumulation of incompatibilities whose fitness effects will depend upon the environment in which hybrids occur.     

The contribution of ancestry,chance, and past and ongoing selection to adaptive evolution

The relative contributions of ancestry, chance, and past and ongoing election to variation in one adaptive (larval feeding rate) and one seemingly nonadaptive (pupation height) trait were determined in populations ofDrosophila melanogaster adapting to either low or high larval densities in the laboratory. Larval feeding rates increased rapidly in response to high density, and the effects of ancestry, past selection and chance were ameliorated by ongoing selection within 15–20 generations. Similarly, in populations previously kept at high larval density, and then switched to low larval density, the decline of larval feeding rate to ancestral levels was rapid (15-20 generations) and complete, providing support for a previously stated hypothesis regarding the costs of faster feeding inDrosophila larvae. Variation among individuals was the major contributor to variation in pupation height, a trait that would superficially appear to be nonadaptive in the environmental context of the populations used in this study because it did not diverge between sets of populations kept at low versus high larval density for many generations. However, the degree of divergence among populations (FST) for pupation height was significantly less than expected for a selectively neutral trait, and we integrate results from previous studies to suggest that the variation for pupation height among populations is constrained by stabilizing selection, with a flat, plateau-like fitness function that, consequently, allows for substantial phenotypic variation within populations. Our results support the view that the genetic imprints of history (ancestry and past selection) in outbreeding sexual populations are typically likely to be transient in the face of ongoing selection and recombination. The results also illustrate the heuristic point that different forms of selection-for example directional versus stabilizing selection—acting on a trait in different populations may often not be due to differently shaped fitness functions, but rather due to differences in how the fitness function maps onto the actual distribution of phenotypes in a given population. We discuss these results in the light of previous work on reverse evolution, and the role of ancestry, chance, and past and ongoing selection in adaptive evolution.     

The effects of adaptation to urea on feeding rates and growth in Drosophila larvae

A collection of forty populations were used to study the phenotypic adaptation of Drosophila melanogaster larvae to urea‐laced food. A long‐term goal of this research is to map genes responsible for these phenotypes. This mapping requires large numbers of populations. Thus, we studied fifteen populations subjected to direct selection for urea tolerance and five controls. In addition, we studied another twenty populations which had not been exposed to urea but were subjected to stress or demographic selection. In this study, we describe the differentiation in these population for six phenotypes: (1) larval feeding rates, (2) larval viability in urea‐laced food, (3) larval development time in urea‐laced food, (4) adult starvation times, (5) adult desiccation times, and (6) larval growth rates. No significant differences were observed for desiccation resistance. The demographically/stress‐selected populations had longer times to starvation than urea‐selected populations. The urea‐adapted populations showed elevated survival and reduced development time in urea‐laced food relative to the control and nonadapted populations. The urea‐adapted populations also showed reduced larval feeding rates relative to controls. We show that there is a strong linear relationship between feeding rates and growth rates at the same larval ages feeding rates were measured. This suggests that feeding rates are correlated with food intake and growth. This relationship between larval feeding rates, food consumption, and efficiency has been postulated to involve important trade‐offs that govern larval evolution in stressful environments. Our results support the idea that energy allocation is a central organizing theme in adaptive evolution.     

Stage-Specific Effects of Candidate Heterochronic Genes on Variation in Developmental Time along an Altitudinal Cline of Drosophila melanogaster

Background

Previously, we have shown there is clinal variation for egg-to-adult developmental time along geographic gradients in Drosophila melanogaster. Further, we also have identified mutations in genes involved in metabolic and neurogenic pathways that affect development time (heterochronic genes). However, we do not know whether these loci affect variation in developmental time in natural populations.

Methodology/Principal Findings

Here, we constructed second chromosome substitution lines from natural populations of Drosophila melanogaster from an altitudinal cline, and measured egg-adult development time for each line. We found not only a large amount of genetic variation for developmental time, but also positive associations of the development time with thermal amplitude and altitude. We performed genetic complementation tests using substitution lines with the longest and shortest developmental times and heterochronic mutations. We identified segregating variation for neurogenic and metabolic genes that largely affected the duration of the larval stages but had no impact on the timing of metamorphosis.

Conclusions/Significance

Altitudinal clinal variation in developmental time for natural chromosome substitution lines provides a unique opportunity to dissect the response of heterochronic genes to environmental gradients. Ontogenetic stage-specific variation in invected, mastermind, cricklet and CG14591 may affect natural variation in development time and thermal evolution.     

Nonrandom Association between Structural Amy and Regulatory map Variants in DROSOPHILA MELANOGASTER

Populations of Drosophila melanogaster were investigated for variation in structural Amy genes, coding for different electrophoretic variants, and regulatory genes that determine the tissue-specific production patterns of α-amylase in the midguts of adults and larvae. Analysis of strains homozygous for second chromosomes extracted from three cage populations of different geographical origin revealed a consistent nonrandom association between Amy and midgut activity pattern (map) variants of α-amylase in adults and third-instar larvae. The origin and maintenance of the linkage disequilibrium between Amy and map genes are discussed.     

Identification of a gene, Desiccate, contributing to desiccation resistance in Drosophila melanogaster

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress.     

Tissue Damage Disrupts Developmental Progression and Ecdysteroid Biosynthesis in Drosophila

In humans, chronic inflammation, severe injury, infection and disease can result in changes in steroid hormone titers and delayed onset of puberty; however the pathway by which this occurs remains largely unknown. Similarly, in insects injury to specific tissues can result in a global developmental delay (e.g. prolonged larval/pupal stages) often associated with decreased levels of ecdysone – a steroid hormone that regulates developmental transitions in insects. We use Drosophila melanogaster as a model to examine the pathway by which tissue injury disrupts developmental progression. Imaginal disc damage inflicted early in larval development triggers developmental delays while the effects are minimized in older larvae. We find that the switch in injury response (e.g. delay/no delay) is coincident with the mid-3rd instar transition – a developmental time-point that is characterized by widespread changes in gene expression and marks the initial steps of metamorphosis. Finally, we show that developmental delays induced by tissue damage are associated with decreased expression of genes involved in ecdysteroid synthesis and signaling.     

Drosophila hemolymph proteins: Purification,characterization, and genetic mapping of larval serum protein 2 in D. melanogaster

Three of the major protein species present in the hemolymph of Drosophila melanogaster larvae just prior to pupation are absent from second instar larvae but accumulate rapidly during the third instar. This article describes the purification and characterization of one of these, larval serum protein (LSP) 2, using an immunological assay. It is a homohexamer of molecular weight about 450,000, with a polypeptide molecular weight of 78,000–83,000. Fast and slow electrophoretic variants of this protein map between the markers vin and gs, at 36–37 on chromosome 3.This work was partially supported by M.R.C. Research Studentships to J.W. and M.E.A.