The Loci of Behavioral Evolution: Evidence That Fas2 and tilB Underlie Differences in Pupation Site Choice Behavior between Drosophila melanogaster and D. simulans

The behaviors of closely related species can be remarkably different, and these differences have important ecological and evolutionary consequences. Although the recent boom in genotype–phenotype studies has led to a greater understanding of the genetic architecture and evolution of a variety of traits, studies identifying the genetic basis of behaviors are, comparatively, still lacking. This is likely because they are complex and environmentally sensitive phenotypes, making them difficult to measure reliably for association studies. The Drosophila species complex holds promise for addressing these challenges, as the behaviors of closely related species can be readily assayed in a common environment. Here, we investigate the genetic basis of an evolved behavioral difference, pupation site choice, between Drosophila melanogaster and D. simulans. In this study, we demonstrate a significant contribution of the X chromosome to the difference in pupation site choice behavior between these species. Using a panel of X-chromosome deficiencies, we screened the majority of the X chromosome for causal loci and identified two regions associated with this X-effect. We then collect gene disruption and RNAi data supporting a single gene that affects pupation behavior within each region: Fas2 and tilB. Finally, we show that differences in tilB expression correlate with the differences in pupation site choice behavior between species. This evidence associating two genes with differences in a complex, environmentally sensitive behavior represents the first step toward a functional and evolutionary understanding of this behavioral divergence.

Testing implications of the polygenic model for the genetic analysis of loci identified through genome-wide association

It has been proposed that many loci with no significant association in GWA studies can nonetheless contribute to the phenotype through modifier interactions with the core genes, implying a polygenic determination of quantitative traits. We have tested this hypothesis by using Drosophila pupal phenotypes. We identified candidate genes for pupal length determination in a GWA and show for disrupted versions of the genes that most are indeed involved in the phenotype, presumably forming a core pathway. We then randomly chose genes below the GWA threshold and found that three quarters of them had also an effect on the trait. We further tested the effects of these knockout lines on an independent behavioral pupal trait (pupation site choice) and found that a similar, but non-correlated fraction of them had a significant effect as well. Our data thus confirm the prediction that a large number of genes can influence independent quantitative traits.Impact statementQuantitative traits are similarly likely influenced by randomly picked loci as by loci identified in a genome-wide association study.

The loci of behavioral evolution: Fas2 and tilB underlie differences in pupation site choice behavior between Drosophila melanogaster and D. simulans

The recent boom in genotype-phenotype studies has led to a greater understanding of the genetic architecture of a variety of traits. Among these traits, however, behaviors are still lacking, perhaps because they are complex and environmentally sensitive phenotypes, making them difficult to measure reliably for association studies. Here, we aim to fill this gap in knowledge with the results of a genetic screen for a complex behavioral difference, pupation site choice, between Drosophila melanogaster and D. simulans. In this study, we demonstrate a significant contribution of the X chromosome to the difference in pupation site choice behavior between these species. Using a panel of X-chromosome deletions, we screened the majority of the X chromosome for causal loci, and identified two regions that explain a large proportion of the X-effect. We then used gene disruptions and RNAi to demonstrate the substantial effects of a single gene within each region: Fas2 and tilB. Finally, we show that differences in tilB expression underlie species differences in pupation site choice behavior, and that generally, pupation site choice behavior appears to be correlated with relative expression of this gene. Our results suggest that even complex, environmentally sensitive behaviors may evolve through changes to loci with large phenotypic effects.Author summaryBehaviors are complex traits that involve sensory detection, higher level processing, and a coordinated output by the nervous system. This level of processing is highly susceptible to environmentally induced variation. Because of their complexity and sensitivity, behaviors are difficult to study; as a result, we have very little understanding of the genes involved in behavioral variation. In this study, we use common laboratory fruit fly model, Drosophila, to address this gap and dissect the genetic underpinnings of an environmentally sensitive behavior that differs between species. We find that a significant amount of the phenotypic difference between species is explained by a single chromosome. We further show that just two genes on this chromosome account for a large majority of its effect, suggesting that the genetic basis of complex behavioral evolution may be simpler than anticipated. For one of these genes, we show that a species-level difference in gene expression is associated with the difference in behavior. Our results contribute to a growing number of studies identifying the genetic components of behavior. Ultimately, we hope to use these data to better predict the number, types, and effects of genetic mutations necessary for complex behaviors to evolve.

Pupation site preference selection in Drosophila jambulina

Larvae of Drosophila jambulina belonging to montium subgroup were tested for pupation site preference in relation to temperature. At higher temperature (30 °C), larvae preferred to pupate on food whereas at lower temperature (21 °C) pupation occurred on the cotton. Genetic basis of larval pupation behavior was studied by conducting reciprocal crosses for 30 generations on food-selected and on cotton-selected larvae. Results from genetic analysis between food-selected and cotton-selected strains suggested a single gene responsible for the pupation site preference, with F1 progeny pupated on cotton and F2 (F1×;F1) larvae pupated on both food as well on cotton. Although we found no change in morphological traits in food vs. cotton selected population, significantly different growth rate (body weight) between the two strains was observed. These results suggest that pupation site preferences can affect life-history traits in D. jambulina.

Hormonal signaling cascades required for phototaxis switch in wandering Leptinotarsa decemlineata larvae

Many animals exploit several niches sequentially during their life cycles, a fitness referred to as ontogenetic niche shift (ONS). To successfully accomplish ONS, transition between development stages is often coupled with changes in one or more primitive, instinctive behaviors. Yet, the underlining molecular mechanisms remain elusive. We show here that Leptinotarsa decemlineata larvae finish their ONS at the wandering stage by leaving the plant and pupating in soil. At middle wandering phase, larvae also switch their phototactic behavior, from photophilic at foraging period to photophobic. We find that enhancement of juvenile hormone (JH) signal delays the phototactic switch, and vise verse. Moreover, RNA interference (RNAi)-aided knockdown of LdPTTH (prothoracicotropic hormone gene) or LdTorso (PTTH receptor gene) impairs avoidance response to light, a phenotype nonrescuable by 20-hydroxyecdysone. Consequently, the RNAi beetles pupate at the soil surface or in shallow layer of soil, with most of them failing to construct pupation chambers. Furthermore, a combination of depletion of LdPTTH/LdTorso and disturbance of JH signal causes no additional effects on light avoidance response and pupation site. Finally, we establish that TrpA1 (transient receptor potential (TRP) cation channel) is necessary for light avoidance behavior, acting downstream of PTTH. We conclude that JH/PTTH cascade concomitantly regulates metamorphosis and the phototaxis switch, to drive ONS of the wandering beetles from plant into soil to start the immobile pupal stage.Author summaryMany animals occupy distinct niches and utilize diverse resources at different development stages in order to meet stage-dependent requirements and overcome stage-specific limitations. This fitness is referred to as ontogenetic niche shift (ONS). During the preparation for ONS, animals often change one or more primitive, instinctive behaviors. Holometabolous insects, with four discrete developmental periods usually in different niches, are a suitable animal group to explore the molecular modes of these behavioral switches. Here we find that Leptinotarsa decemlineata larvae, an insect defoliator of potatoes, switch their phototactic behavior, from photophilic at feeding period to photophobic during the larval-pupal transition (wandering stage). This phototactic switch facilitates the wandering larvae to accomplish the ONS from potato plant to their pupation site below ground. We show that JH/PTTH cascade controls the phototaxis switch, through a step in photo transduction between the photoreceptor molecule and the transient receptor potential cation channel.