The courtship song of Drosophila melanogaster has long served as an 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.

bioRxiv Preprint: https://www.biorxiv.org/content/early/2024/05/17/2024.05.14.594231

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.

Symbiotic relationships between bacteria and insect hosts are common. Although the bacterial endosymbionts have been subjected to intense investigation, little is known of the host cells in which they reside, the bacteriocytes. We have studied the development and evolution of aphid bacteriocytes, the host cells that contain the endosymbiotic bacteria Buchnera aphidicola. We show that bacteriocytes of Acyrthosiphon pisum express several gene products (or their paralogues): Distal-less, Ultrabithorax/Abdominal-A, and Engrailed. Using these markers, we find that a subpopulation of the bacteriocytes is specified prior to the transmission of maternal bacteria to the embryo. In addition, we discovered that a second population of cells is recruited to the bacteriocyte fate later in development. We experimentally demonstrate that bacteriocyte induction and proliferation occur independently of B. aphidicola. Major features of bacteriocyte development, including the two-step recruitment of bacteriocytes, have been conserved in aphids for 80-150 million years. Furthermore, we have investigated two cases of evolutionary loss of bacterial symbionts: in one case, where novel extracellular, eukaryotic symbionts replaced the bacteria, the bacteriocyte is maintained; in another case, where symbionts are absent, the bacteriocytes are initiated but not maintained. The bacteriocyte represents an evolutionarily novel cell fate, which is developmentally determined independently of the bacteria. Three of five transcription factors we examined show novel expression patterns in bacteriocytes, suggesting that bacteriocytes may have evolved to express many additional transcription factors. The evolutionary transition to a symbiosis in which bacteria and an aphid cell form a functional unit, similar to the origin of plastids, has apparently involved extensive molecular adaptations on the part of the host cell.

BACKGROUND: Many insects undergo a period of arrested development, called diapause, to avoid seasonally recurring adverse conditions. Whilst the phenology and endocrinology of insect diapause have been well studied, there has been comparatively little research into the developmental details of diapause. We investigated developmental aspects of diapause in sexually-produced embryos of the pea aphid, Acyrthosiphon pisum.

RESULTS: We found that early stages of embryogenesis progressed at a temperature-independent rate, characteristic of diapause, whereas later stages of embryogenesis progressed at a temperature-dependent rate. However, embryos maintained at very high temperatures during the temperature-independent stage showed severe developmental abnormalities. Under no temperature regime did embryos display a distinct resting stage. Rather, morphological development progressed slowly but continuously throughout embryogenesis.

CONCLUSION: Diapause in the pea aphid, and perhaps in many other insects, is a temperature-independent slowing but not a cessation of morphological development. This suggests that the mechanisms limiting developmental rate during diapause may be the same as those controlling developmental rate at other stages of growth.

Pigmentation divergence between Drosophila species has emerged as a model trait for studying the genetic basis of phenotypic evolution, with genetic changes contributing to pigmentation differences often mapping to genes in the pigment synthesis pathway and their regulators. These studies of Drosophila pigmentation have tended to focus on pigmentation changes in one body part for a particular pair of species, but changes in pigmentation are often observed in multiple body parts between the same pair of species. The similarities and differences of genetic changes responsible for divergent pigmentation in different body parts of the same species thus remain largely unknown. Here we compare the genetic basis of pigmentation divergence between Drosophila elegans and D. gunungcola in the wing, legs, and thorax. Prior work has shown that regions of the genome containing the pigmentation genes yellow and ebony influence the size of divergent male-specific wing spots between these two species. We find that these same two regions of the genome underlie differences in leg and thorax pigmentation; however, divergent alleles in these regions show differences in allelic dominance and epistasis among the three body parts. These complex patterns of inheritance can be explained by a model of evolution involving tissue-specific changes in the expression of Yellow and Ebony between D. elegans and D. gunungcola.

We report an extreme morphological difference between Drosophila sechellia and related species of the pattern of hairs on first-instar larvae. On the dorsum of most species, the posterior region of the anterior compartment of most segments is covered by a carpet of fine hairs. In D. sechellia, these hairs have been lost and replaced with naked cuticle. Genetic mapping experiments and interspecific complementation tests indicate that this difference is caused, in its entirety, by evolution at the ovo/shaven-baby locus. The pattern of expression of the ovo/shaven-baby transcript is correlated with this morphological change. The altered dorsal cuticle pattern is probably caused by evolution of the cis-regulatory region of ovo/shaven-baby in the D. sechellia lineage.

It is unclear how regulatory genes establish neural circuits that compose sex-specific behaviors. The Drosophila melanogaster male courtship song provides a powerful model to study this problem. Courting males vibrate a wing to sing bouts of pulses and hums, called pulse and sine song, respectively. We report the discovery of male-specific thoracic interneurons—the TN1A neurons—that are required specifically for sine song. The TN1A neurons can drive the activity of a sex-non-specific wing motoneuron, hg1, which is also required for sine song. The male-specific connection between the TN1A neurons and the hg1 motoneuron is regulated by the sexual differentiation gene doublesex. We find that doublesex is required in the TN1A neurons during development to increase the density of the TN1A arbors that interact with dendrites of the hg1motoneuron. Our findings demonstrate how a sexual differentiation gene can build a sex-specific circuit motif by modulating neuronal arborization.

•Doublesex-expressing TN1 neurons are necessary and sufficient for the male sine song•A subclass of TN1 neurons, TN1A, contributes to the sine song•TN1A neurons are functionally coupled to a sine song motoneuron, hg1•Doublesex regulates the connectivity between the TN1A and hg1 neurons

It is unclear how developmental regulatory genes specify sex-specific behaviors. Shirangi et al. demonstrate that the Drosophila sexual differentiation gene doublesex encodes a sex-specific behavior—male song—by promoting the connectivity between the male-specific TN1A neurons and the sex-non-specific hg1 neurons, which are required for production of the song.

Aphids are sap-feeding insects that host a range of bacterial endosymbionts including the obligate, nutritional mutualist Buchnera plus several bacteria that are not required for host survival. Among the latter, ’Candidatus Regiella insecticola’ and ’Candidatus Hamiltonella defensa’ are found in pea aphids and other hosts and have been shown to protect aphids from natural enemies. We have sequenced almost the entire genome of R. insecticola (2.07 Mbp) and compared it with the recently published genome of H. defensa (2.11 Mbp). Despite being sister species the two genomes are highly rearranged and the genomes only have \~{}55% of genes in common. The functions encoded by the shared genes imply that the bacteria have similar metabolic capabilities, including only two essential amino acid biosynthetic pathways and active uptake mechanisms for the remaining eight, and similar capacities for host cell toxicity and invasion (type 3 secretion systems and RTX toxins). These observations, combined with high sequence divergence of orthologues, strongly suggest an ancient divergence after establishment of a symbiotic lifestyle. The divergence in gene sets and in genome architecture implies a history of rampant recombination and gene inactivation and the ongoing integration of mobile DNA (insertion sequence elements, prophage and plasmids).