Many genomes contain rapidly evolving and highly divergent genes whose homology to genes of known function often cannot be determined from sequence similarity alone. However, coding sequence-independent features of genes, such as intron-exon boundaries, often evolve more slowly than coding sequences and can provide complementary evidence for homology. We found that a linear logistic regression classifier using only structural features of rapidly evolving bicycle aphid effector genes identified many putative bicycle homologs in aphids, phylloxerids, and scale insects, whereas sequence similarity search methods yielded few homologs in most aphids and no homologs in phylloxerids and scale insects. Subsequent examination of sequence features and intron locations supported homology assignments. Differential expression studies of newly-identified bicycle homologs, together with prior proteomic studies, support the hypothesis that BICYCLE proteins act as plant effector proteins in many aphid species and perhaps also in phylloxerids and scale insects.

Motor systems flexibly implement diverse motor programs to pattern behavioral sequences, yet their neural underpinnings remain unclear. Here, we investigated the neural circuit mechanisms of flexible courtship behavior in Drosophila. Courting males alternately produce two types of courtship song. By recording calcium signals in the ventral nerve cord (VNC) in behaving flies, we found that different songs are produced by activating overlapping neural populations with distinct motor functions in a combinatorial manner. Recordings from the brain suggest that song is driven by two descending pathways – one defines when to sing and the other specifies what song to sing. Connectomic analysis reveals that these “when” and “what” descending pathways provide structured input to VNC neurons with different motor functions. These results suggest that dynamic changes in the activation patterns of descending pathways drive different combinations of motor modules, thereby flexibly switching between different motor actions.

The pea aphid, Acyrthosiphon pisum, exhibits several environmentally cued polyphenisms, in which discrete, alternative phenotypes are produced. At low-density, parthenogenetic females produce unwinged female progeny, but at high-density females produce progeny that develop with wings. These alternative phenotypes represent a solution to the competing demands of dispersal and reproduction. Males also develop as either winged or unwinged, but these alternatives are determined by a genetic polymorphism. Winged and unwinged males are morphologically less distinct from each other than winged and unwinged females, possibly because males experience fewer trade-offs between dispersal and reproduction. To assess whether shared physiological differences mirror the shared morphological differences that characterize the wing polyphenism and polymorphism, we used a cDNA microarray representing an estimated 10% of the coding genome (1734 genes) to examine differential transcript accumulation between winged and unwinged females and males. We identified several transcripts that differentially accumulate between winged and unwinged morphs in both sexes, the majority of which are involved in energy production. Unexpectedly, the extent of differential transcript accumulation between winged and unwinged morphs was greater for adult males than for adult females. Together, these results suggest not only that similar physiological differences underlie the polyphenism and polymorphism, but that male morphs, like females, are subject to trade-offs between reproduction and dispersal that are reflected in levels of transcript accumulation and possibly genome-wide patterns of gene regulation. These data also provide a baseline for future studies of the molecular and physiological basis of life-history trade-offs.

Interspecific comparisons of protein sequences can reveal regions of evolutionary conservation that are under purifying selection because of functional constraints. Interpreting these constraints requires combining evolutionary information with structural, biochemical, and physiological data to understand the biological function of conserved regions. We take this integrative approach to investigate the evolution and function of the nuclear-encoded subunits of cytochrome c oxidase (COX). We find that the nuclear-encoded subunits evolved subsequent to the origin of mitochondria and the subunit composition of the holoenzyme varies across diverse taxa that include animals, yeasts, and plants. By mapping conserved amino acids onto the crystal structure of bovine COX, we show that conserved residues are structurally organized into functional domains. These domains correspond to some known functional sites as well as to other uncharacterized regions. We find that amino acids that are important for structural stability are conserved at frequencies higher than expected within each taxon, and groups of conserved residues cluster together at distances of less than 5 A more frequently than do randomly selected residues. We, therefore, suggest that selection is acting to maintain the structural foundation of COX across taxa, whereas active sites vary or coevolve within lineages.

Developmental genes can have complex cis-regulatory regions with multiple enhancers. Early work revealed remarkable modularity of enhancers, whereby distinct DNA regions drive gene expression in defined spatiotemporal domains. Nevertheless, a few reports have shown that enhancers function in multiple developmental stages, implying that enhancers can be pleiotropic. Here, we have studied the activity of the enhancers of the shavenbaby gene throughout D. melanogaster development. We found that all seven shavenbaby enhancers drive expression in multiple tissues and developmental stages. We explored how enhancer pleiotropy is encoded in two of these enhancers. In one enhancer, the same transcription factor binding sites contribute to embryonic and pupal expression, revealing site pleiotropy, whereas for a second enhancer, these roles are encoded by distinct sites. Enhancer pleiotropy may be a common feature of cis-regulatory regions of developmental genes, and site pleiotropy may constrain enhancer evolution in some cases.

Aphids exhibit unique attributes, such as polyphenisms and specialized cells to house endosymbionts, that make them an interesting system for studies at the interface of ecology, evolution and development. Here we present a comprehensive characterization of the developmental genes in the pea aphid, Acyrthosiphon pisum, and compare our results to other sequenced insects. We investigated genes involved in fundamental developmental processes such as establishment of the body plan and organogenesis, focusing on transcription factors and components of signalling pathways. We found that most developmental genes were well conserved in the pea aphid, although many lineage-specific gene duplications and gene losses have occurred in several gene families. In particular, genetic components of transforming growth factor beta (TGFbeta) Wnt, JAK/STAT (Janus kinase/signal transducer and activator of transcription) and EGF (Epidermal Growth Factor) pathways appear to have been significantly modified in the pea aphid.

Diverse traits often covary between species [1-3]. The possibility that a single mutation could contribute to the evolution of several characters between species [3] is rarely investigated as relatively few cases are dissected at the nucleotide level. Drosophila santomea has evolved additional sex comb sensory teeth on its legs and has lost two sensory bristles on its genitalia. We present evidence that a single nucleotide substitution in an enhancer of the scute gene contributes to both changes. The mutation alters a binding site for the Hox protein Abdominal-B in the developing genitalia, leading to bristle loss, and for another factor in the developing leg, leading to bristle gain. Our study suggests that morphological evolution between species can occur through a single nucleotide change affecting several sexually dimorphic traits. VIDEO ABSTRACT.

The courtship song of Drosophila melanogaster has long served as excellent model system for studies of animal communication and differences in courtship song have been demonstrated among populations and between species. Here, we report that flies of African and European origin, which diverged approximately 13,000 years ago, show significant genetic differentiation in the use of slow versus fast pulse song. Using a combination of quantitative trait mapping and population genetic analysis we detected a single strong QTL underlying this trait and we identified candidate genes that may contribute to the evolution of this trait. Song trait variation between parental strains of our recombinant inbred panel enabled detection of genomic intervals associated with six additional song traits, some of which include known courtship-related genes. These findings improve the prospects for further genetic insights into the evolution of reproductive behavior and the biology underlying courtship song.

Changes in gene regulation underlie much of phenotypic evolution. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations. Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

Aphid taxonomy is often frustrated by the host alternation and extensive polyphenism displayed by many species. Here we examine the utility of using molecular data to assist in life cycle and taxonomic determination. We found that a relatively small amount of DNA sequence data can greatly assist in these tasks. Molecular data have identified the synonymy of five species: Tuberaphis plicator (Noordam) is a junior synonym of T.takenouchii (Takahashi), T.taiwana (Takahashi) is a junior synonym of T.coreana Takahashi, Hamiltonaphis styraci (Matsumura) is transferred to Tuberaphis Takahashi, Astegopteryx roepkei Hille Ris Lambers is transferred to Ceratoglyphina van der Goot, and A.vandermeermohri Hille Ris Lambers is transferred to Cerataphis Lichtenstein. We have elucidated the complete life cycles of five species: A.basalis (van der Goot) alternates between Styrax benzoin and bamboos, Ceratoglyphina bambusae van der Goot alternates between S.benzoin and bamboos, Pseudoregma sundanica (van der Goot) alternates between S.paralleloneura and Zingiberaceae, T.coreana alternates between S.formosana and Loranthaceae, and T.takenouchii alternates between S.japonica and Loranthaceae. In all cases the molecular data agreed with available morphological data. This analysis demonstrates the utility of DNA sequence comparisons for elucidating complex life cycles and the taxonomy of difficult insect groups.