Evolution of larval foraging behaviour in Drosophila and its effects on growth and metabolic rates

Abstract.  The evolution of foraging in Drosophila melanogaster (Meigen) is studied using outbred populations that had been differentiated using laboratory selection. The foraging behaviour of Drosophila larvae is measured using the foraging path length of 72-h-old larvae. The foraging path length is the distance travelled by foraging larvae over 5 min. Populations of Drosophila selected for rapid development show significantly greater path lengths than their controls. Populations of Drosophila selected for resistance to ammonia and urea in their larval food have shorter path lengths than their controls. Individuals in the ammonia-resistant populations are smaller than those in the control populations, but the size-adjusted metabolic rates are not significantly different. A simple model is proposed suggesting that changes in larval foraging behaviour may be a means for Drosophila larvae to adapt to new environments that require additional maintenance energy. In the ammonia-selected populations, crucial tests of these ideas will have to be conducted in environments with ammonia.     

Foraging behaviour in Drosophila larvae: mushroom body ablation

Drosophila larvae and adults exhibit a naturally occurring genetically based behavioural polymorphism in locomotor activity while foraging. Larvae of the rover morph exhibit longer foraging trails than sitters and forage between food patches, while sitters have shorter foraging trails and forage within patches. This behaviour is influenced by levels of cGMP-dependent protein kinase (PGK) encoded by the foraging (for) gene. Rover larvae have higher expression levels and higher PGK activities than do sitters. Here we discuss the importance of the for gene for studies of the mechanistic and evolutionary significance of individual differences in behaviour. We also show how structure-function analysis can be used to investigate a role for mushroom bodies in larval behaviour both in the presence and in the absence of food. Hydroxyurea fed to newly hatched larvae prevents the development of all post-embryonically derived mushroom body (MB) neuropil. This method was used to ablate MBs in rover and sitter genetic variants of foraging to test whether these structures mediate expression of the foraging behavioural polymorphism. We found that locomotor activity levels during foraging of both the rover and sitter larval morphs were not significantly influenced by MB ablation. Alternative hypotheses that may explain how variation in foraging behaviour is generated are discussed.     

Rover/sitterDrosophila melanogaster larval foraging polymorphism as a function of larval development,food-patch quality,and starvation

The genetically based rover/sitter behavioral difference in Drosophila melanogasterlarval foraging is expressed throughout most of the larval instars when larvae forage on food patches of differing food quality. The amount of locomotor behavior decreases when third-instar larvae of both rover and sitter strains are starved just prior to the behavioral test. Such strain differences in locomotor behavior are maintained despite the starvation-induced decrease in locomotion found in both strains. Measurements of larval body length and width, taken at 24, 48, 72, and 96 h posthatching, reveal that rover and sitter larval growth rates do not differ. The finding that rover/sitter differences are expressed in a variety of environments and throughout the majority of the larval instars should aid in attempts to uncover selection pressures which may differentially affect the two morphs in environmentally heterogeneous natural populations.     

The foraging locus: behavioral tests for normal muscle movement in rover and sitter Drosophila melanogaster larvae

We used Drosophila melanogaster larvae with different alleles at the foraging (for) locus in a variety of behavioral tests to evaluate normal muscle usage of rover and sitter phenotypes. The results show that sitter and lethal sitter alleles of for do not affect larval behavior through a mutation which affects larval muscle usage. In general the behavior of rovers and sitters differed on food but not on non-nutritive substrates. Rovers and sitters moved equally well on non-nutritive substrates, and measures such as the time to roll over and length of forward stride showed no significant strain differences. Larvae with different alleles at for did not differ in body length. Rovers took more strides, not longer ones, than sitters while on foraging substrates. We conclude that differences in larval locomotion during foraging found in larvae with different alleles at for can not be explained on the basis of muscle usage alone. It is more likely that for affects larval ability to perceive or respond to the foraging environment.     

Natural selection in the laboratory for a change in resistance byDrosophila melanogaster to the parasitoid waspAsobara tabida

The selection response of the polymorphic hostD. melanogaster (Meigen) to the braconid waspA. tabida (Nees) is addressed. Cages of flies with and without wasps were initiated with a population ofD. melanogaster that exhibited variation both in larval foraging behavior and in encapsulation ability. Encapsulation ability was measured as the proportion of parasitized larvae that produce a hardened capsule which encapsulates the wasp egg and ultimately kills the wasp larva. We determined whether the host population changed its encapsulation ability and/or its foraging behavior in response to the wasp. Both species were collected from a local orchard whereA. tabida is the only wasp known to parasitizeD. melanogaster larvae. The naturally occurring genetic polymorphism for rover and sitter larval foraging behavior inD. melanogaster is also found in this field population.A. tabida's vibrotactic search behavior enables it to detect rover more frequently than sitter larvae. Rover larvae move significantly more while feeding than do sitter larvae. In this field population, rover larvae also show higher encapsulation abilities than do sitter larvae. Six cage populations, three without wasps and three with wasps, each containing an equal mixture of rover and sitter flies, were established in the laboratory and maintained for 19 fly generations. Selection pressure in the laboratory was similar to that found in the field population from which the flies and wasps were derived. We found that larvae from cages with wasps developed a significantly higher frequency of encapsulation than those reared without wasps. We were, however, unable to detect a change in larval movement (rover or sitter behavior) in larvae from cages subject to selection from wasps compared to larvae from cages containing no wasps. This may have resulted from a balance between two selective forces, selection against rovers by the wasps' use of vibrotaxis, and selection for rovers resulting from their increased encapsulation abilities     

Social Environment Influences Performance in a Cognitive Task in Natural Variants of the Foraging Gene

In Drosophila melanogaster, natural genetic variation in the foraging gene affects the foraging behaviour of larval and adult flies, larval reward learning, adult visual learning, and adult aversive training tasks. Sitters (for ^s) are more sedentary and aggregate within food patches whereas rovers (for^R) have greater movement within and between food patches, suggesting that these natural variants are likely to experience different social environments. We hypothesized that social context would differentially influence rover and sitter behaviour in a cognitive task. We measured adult rover and sitter performance in a classical olfactory training test in groups and alone. All flies were reared in groups, but fly training and testing were done alone and in groups. Sitters trained and tested in a group had significantly higher learning performances compared to sitters trained and tested alone. Rovers performed similarly when trained and tested alone and in a group. In other words, rovers learning ability is independent of group training and testing. This suggests that sitters may be more sensitive to the social context than rovers. These differences in learning performance can be altered by pharmacological manipulations of PKG activity levels, the foraging (for) gene''s gene product. Learning and memory is also affected by the type of social interaction (being in a group of the same strain or in a group of a different strain) in rovers, but not in sitters. These results suggest that for mediates social learning and memory in D. melanogaster.     

Polymorphic Foraging Behavior Among Caenorhabditis elegans: Frequency- and Density-Dependent Selection

Strains of Caenorhabditis elegans obtained from their natural soil environment exhibit one of two forms of foraging behavior. Some strains forage solitarily and disperse evenly on a bacterial lawn. Other strains move rapidly until they encounter groups of conspecifics, and then slow their movement and join the group. Strains expressing these behaviors are globally widespread and have been isolated from the same location, suggesting a foraging polymorphism. We hypothesized that density-dependent selection maintains both foraging alleles in populations. Alternatively, both foraging alleles could be retained in populations through frequency-dependent selection. We tested both of these hypotheses by manipulating strain density and frequency, and observing changes in population density over time. Our results indicated that neither density- nor frequency-dependent selection appears to be responsible for the observed polymorphism. The clumping strain consistently out-competed the solitary strain over all treatment levels. We suggest other potential factors that may maintain both alleles in populations.     

LIFE-HISTORY CONSEQUENCES OF ADAPTATION TO LARVAL NUTRITIONAL STRESS IN DROSOPHILA

Many animal species face periods of chronic nutritional stress during which the individuals must continue to develop, grow, and/or reproduce despite low quantity or quality of food. Here, we use experimental evolution to study adaptation to such chronic nutritional stress in six replicate Drosophila melanogaster populations selected for the ability to survive and develop within a limited time on a very poor larval food. In unselected control populations, this poor food resulted in 20% lower egg‐to‐adult viability, 70% longer egg‐to‐adult development, and 50% lower adult body weight (compared to the standard food on which the flies were normally maintained). The evolutionary changes associated with adaptation to the poor food were assayed by comparing the selected and control lines in a common environment for different traits after 29–64 generations of selection. The selected populations evolved improved egg‐to‐adult viability and faster development on poor food. Even though the adult dry weight of selected flies when raised on the poor food was lower than that of controls, their average larval growth rate was higher. No differences in proportional pupal lipid content were observed. When raised on the standard food, the selected flies showed the same egg‐to‐adult viability and the same resistance to larval heat and cold shock as the controls and a slightly shorter developmental time. However, despite only 4% shorter development time, the adults of selected populations raised on the standard food were 13% smaller and showed 20% lower early‐life fecundity than the controls, with no differences in life span. The selected flies also turned out less tolerant to adult malnutrition. Thus, fruit flies have the genetic potential to adapt to poor larval food, with no detectable loss of larval performance on the standard food. However, adaptation to larval nutritional stress is associated with trade‐offs with adult fitness components, including adult tolerance to nutritional stress.     

Plastic and evolutionary responses of cell size and number to larval malnutrition in Drosophila melanogaster

Both development and evolution under chronic malnutrition lead to reduced adult size in Drosophila. We studied the contribution of changes in size vs. number of epidermal cells to plastic and evolutionary reduction of wing size in response to poor larval food. We used flies from six populations selected for tolerance to larval malnutrition and from six unselected control populations, raised either under standard conditions or under larval malnutrition. In the control populations, phenotypic plasticity of wing size was mediated by both cell size and cell number. In contrast, evolutionary change in wing size, which was only observed as a correlated response expressed on standard food, was mediated entirely by reduction in cell number. Plasticity of cell number had been lost in the selected populations, and cell number did not differ between the sexes despite males having smaller wings. Results of this and other experimental evolution studies are consistent with the hypothesis that alleles which increase body size through prolonged growth affect wing size mostly via cell number, whereas alleles which increase size through higher growth rate do so via cell size.     

Chronic malnutrition favours smaller critical size for metamorphosis initiation in Drosophila melanogaster

Critical size at which metamorphosis is initiated represents an important checkpoint in insect development. Here, we use experimental evolution in Drosophila melanogaster to test the long-standing hypothesis that larval malnutrition should favour a smaller critical size. We report that six fly populations subject to 112 generations of laboratory natural selection on an extremely poor larval food evolved an 18% smaller critical size (compared to six unselected control populations). Thus, even though critical size is not plastic with respect to nutrition, smaller critical size can evolve as an adaptation to nutritional stress. We also demonstrate that this reduction in critical size (rather than differences in growth rate) mediates a trade-off in body weight that the selected populations experience on standard food, on which they show a 15-17% smaller adult body weight. This illustrates how developmental mechanisms that control life history may shape constraints and trade-offs in life history evolution.     

The Genomic Architecture of Adaptation to Larval Malnutrition Points to a Trade-off with Adult Starvation Resistance in Drosophila

Periods of nutrient shortage impose strong selection on animal populations. Experimental studies of genetic adaptation to nutrient shortage largely focus on resistance to acute starvation at adult stage; it is not clear how conclusions drawn from these studies extrapolate to other forms of nutritional stress. We studied the genomic signature of adaptation to chronic juvenile malnutrition in six populations of Drosophila melanogaster evolved for 150 generations on an extremely nutrient-poor larval diet. Comparison with control populations evolved on standard food revealed repeatable genomic differentiation between the two set of population, involving >3,000 candidate SNPs forming >100 independently evolving clusters. The candidate genomic regions were enriched in genes implicated in hormone, carbohydrate, and lipid metabolism, including some with known effects on fitness-related life-history traits. Rather than being close to fixation, a substantial fraction of candidate SNPs segregated at intermediate allele frequencies in all malnutrition-adapted populations. This, together with patterns of among-population variation in allele frequencies and estimates of Tajima’s D, suggests that the poor diet results in balancing selection on some genomic regions. Our candidate genes for tolerance to larval malnutrition showed a high overlap with genes previously implicated in acute starvation resistance. However, adaptation to larval malnutrition in our study was associated with reduced tolerance to acute adult starvation. Thus, rather than reflecting synergy, the shared genomic architecture appears to mediate an evolutionary trade-off between tolerances to these two forms of nutritional stress.     

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 Localization of Foraging (For): A Major Gene for Larval Behavior in Drosophila Melanogaster

Localizing genes for quantitative traits by conventional recombination mapping is a formidable challenge because environmental variation, minor genes, and genetic markers have modifying effects on continuously varying phenotypes. We describe "lethal tagging," a method used in conjunction with deficiency mapping for localizing major genes associated with quantitative traits. Rover/sitter is a naturally occurring larval foraging polymorphism in Drosophila melanogaster which has a polygenic pattern of inheritance comprised of a single major gene (foraging) and minor modifier genes. We have successfully localized the lethal tagged foraging (for, 2-10) gene by deficiency mapping to 24A3-C5 on the polytene chromosome map.     

The evolution of larval foraging behaviour in response to host plant variation in a leaf beetle

The evolutionary causes of variation in host specialization among phytophagous insects are still not well understood and identifying them is a central task in insect–host plant biology. Here we examine host utilization of the chrysomelid beetle Oreina elongata that shows interpopulation variation in the degree of specialization. We focus on larval behaviour and on what selection pressures may favour the use of two different larval host plants ( Adenostyles alliariae and Cirsium spinosissimum ) in one population as opposed to specialization onto one of them as is seen in other populations. The results suggest that the degree of exploratory foraging behaviour is higher in larvae from the two-host population than in single host populations, and a field survey of the two-host population also indicated that larvae do move between host species. A field experiment indicated that predation rates on O. elongata larvae in the two-host population are higher on one of the host species, A. alliariae , than on the alternative C. spinosissimum . In combination with earlier results this finding suggest that larvae move between hosts to obtain better food on one host, and to get better protection from predators on the other. It appears that in this two-host situation a single plant species does not provide the most beneficial conditions in all parts of O. elongata life cycle and individuals may obtain different plant-specific benefits by moving between host species. This heterogeneous host situation appears to have selected for the explorative larval foraging strategy seen in the in the two-host population. In general, the results support the notion that to understand patterns of host plant use in insects it is often vital to consider a range of host related selection pressures whose relative importance may vary between life stages of the insect.     

Chaser (Csr), a New Gene Affecting Larval Foraging Behavior in Drosophila Melanogaster

Chaser (Csr) was uncovered in a gamma mutagenesis screen to identify genes that modify the larval foraging behavior of sitters to rovers. Rover larvae have significantly longer path lenghts than sitters while foraging on a yeast and water paste. This difference is influenced by one major gene, foraging (for), which has two naturally occurring alleles, for(R) (rover) and for(s) (sitter). In a mutagenesis screen for modifiers of for, we identified three lines with viable mutations on chromosome 3 that alter foraging behavior. Each of these mutations increased larval path lengths in for(s)/for(s) larvae in a dominant fashion, and were not separable by recombination. These mutations are therefore probably allelic and define a new gene that we have called Csr. Csr was genetically localized using the lethal-tagging technique. This technique resulted in seven lines with a significant decrease in larval path-length and recessive lethal mutations on chromosome 3. We refer to these as reverted Csr (Csr(rv)) lines. Deficiencies that uncovered cytologically visible chromosome rearrangements in three of the seven reverted lines were used in a complementation analysis. In this way we mapped the lethal mutations in the Csr(rv) lines to cytological region 95F7-96A1 on the right arm of chromosome 3.     

Idiosyncratic evolution of maternal effects in response to juvenile malnutrition in Drosophila

Maternal effects often affect fitness traits, but there is little experimental evidence pertaining to their contribution to response to selection imposed by novel environments. We studied the evolution of maternal effects in Drosophila populations selected for tolerance to chronic larval malnutrition. To this end, we performed pairwise reciprocal F₁ crosses between six selected (malnutrition tolerant) populations and six unselected control populations and assessed the effect of cross direction on larval growth and developmental rate, adult weight and egg‐to‐adult viability expressed under the malnutrition regime. Each pair of reciprocal crosses revealed large maternal effects (possibly including cytoplasmic genetic effects) on at least one trait, but the magnitude, sign and which traits were affected varied among populations. Thus, maternal effects contributed significantly to the response to selection imposed by the malnutrition regime, but these changes were idiosyncratic, suggesting a rugged adaptive landscape. Furthermore, although the selected populations evolved both faster growth and higher viability, the maternal effects on growth rate and viability were negatively correlated across populations. Thus, genes mediating maternal effects can evolve to partially counteract the response to selection mediated by the effects of alleles on their own carriers’ phenotype, and maternal effects may contribute to evolutionary trade‐offs between components of offspring fitness.     

Activity Variants of Acid Phosphatase-3 among Chromosome 3 Inversions of DROSOPHILA PSEUDOOBSCURA

Sexual selection is measured between two strains of Drosophila melanogaster: a wild strain and a strain mutant at the sepia locus. Frequency-dependent male mating was found to be successful, whereas the female genotype exerted no influence. The rarer the male genotype becomes, the greater is its mating success. A selection model is built for this behavior characteristic in which selection operates differently in the two sexes. The genetic consequencies of this model upon the maintenance of genetic polymorphism at the sepia locus are compared to experimental data from previous population cage studies. The fit obtained with this sexual selection model is compared to that of the larvel selection model previously investigated. A model composed of both sexual and larval components of fitness is presented. The role that each major selection component is expected to play in experimental populations as the gene frequency changes is discussed. Sexual selection leads to an equilibrium level higher than larval selection, and the combined model is very close to the experimental values.     

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.     

Alleles underlying larval foraging behaviour influence adult dispersal in nature

The dispersal and migration of organisms have resulted in the colonisation of nearly every possible habitat and ultimately the extraordinary diversity of life. Animal dispersal tendencies are commonly heterogeneous (e.g. long vs. short) and non‐random suggesting that phenotypic and genotypic variability between individuals can contribute to population‐level heterogeneity in dispersal. Using laboratory and field experiments, we demonstrate that natural allelic variation in a gene underlying a foraging polymorphism in larval fruit flies (for), also influences their dispersal tendencies as adults. Rover flies (for^R; higher foraging activity) have consistently greater dispersal tendencies and are more likely to disperse longer distances than sitter flies (for^s; lower foraging activity). Increasing for expression in the brain and nervous system increases dispersal in sitter flies. Our study supports the notion that variation in dispersal can be driven by intrinsic variation in food‐dependent search behaviours and confirms that single gene pleiotropic effects can contribute to population‐level heterogeneity in dispersal.