Cassandra G. Extavour

Horizontal gene transfer (HGT) in animals is thought to be rare and largely restricted to transfers from non-animal sources. In contrast to this expectation, we discovered that 9.2% of the protein-coding genes in the genome of the dark-winged fungus gnat Bradysia coprophila (order: Diptera, family: Sciaridae) are of potential HGT origin. Most of these genes have phylogenetic affinity with sequences from other distantly related arthropods that inhabit a shared soil-dwelling niche with B. coprophila. We show that these findings are inconsistent with genomic contamination or widespread gene loss and instead suggest a level of inter-animal HGT that is, to our knowledge, unprecedented. Consistent with their potential functional importance, ~75% of putative B. coprophila horizontally transferred genes (HTGs) are transcribed, and nearly all are shared across multiple sciarid fungus gnat species that diverged tens of millions of years ago. Further, domain annotations suggest that HTGs participate in diverse processes potentially important to B. coprophila ecology. We tested the functions of eight putative B. coprophila HTGs through transgenic expression in the fly Drosophila melanogaster and the yeast Saccharomyces cerevisiae, and found evidence that HGT has likely endowed B. coprophila with the novel capability to biosynthesize NAD+, as well as enhanced capacity to withstand stressors imposed by arsenic, cyanide, alkylating agents, and reactive oxygen species. Our findings reveal a previously underappreciated contribution of inter-animal genetic exchange to the evolution of genomic, physiological, and ecological novelty.

Phenotypic traits can vary based on genes or environment. Nevertheless, some quantitative traits remain invariant. Understanding the developmental mechanisms that generate such invariance can reveal how phenotypic robustness evolves. Insect reproductive capacity is largely determined by the number of gamete-producing subunits within the gonad, providing a tractable system to investigate these mechanisms as they operate on a quantitative trait that impacts fitness. In most insects, gonad subunit number varies within and between species. Here, we investigate gonad development in the milkweed bug Oncopeltus fasciatus , wherein subunit number is invariant. We show that subunit number is established during embryogenesis, when some abdominal segments, but not others, specify somatic gonad precursors. The position and number of these precursors depend on segment identity, and experimentally modified segment number or Hox-mediated segment identity changes the number of gonad subunits. Thus, O. fasciatus establishes invariant subunit number by coupling gonad development to segmental patterning machinery. We propose that co-option of the Hox code provides a developmental mechanism for generating robustness in gonad subunit number and may thereby contribute to the evolution of reproductive capacity in other insects.

Many studies investigating the proximate causes of evolutionary adaptations dissect potentially adaptive phenotypes under laboratory conditions where organisms are more easily experimentally manipulated. For such models, it remains unclear if organisms maintained long-term under laboratory conditions are representative of relatives in their natural environment. Drosophila sechellia, a fly endemic to the Seychelles that uses the toxic Morinda citrifolia noni fruit as a sole niche, has emerged as a powerful (neuro)genetic model for studying the molecular basis of ecological adaptations. Many studies have investigated the genetic and cellular basis of this species’ specialization in a laboratory setting using inbred strains that were collected many decades ago and have been maintained under conditions very different from those in nature. Here, we compare the phenotypes of these strains with recently collected wild D. sechellia to ask if long-term laboratory maintenance has led to a loss or degradation of phenotypes associated with their specialization. We show that newly derived D. sechellia lines display highly similar anatomical and behavioural phenotypes to long-term laboratory strains, including noni attraction and olfactory sensory neuron numbers, toxin resistance, egg size and circadian plasticity. These results support the use of inbred, genetically manipulatable, strains to study ecological adaptation.

Insects show extreme diversity and have long intrigued biologists. Although gene editing and transgenesis should enable the use of almost any insect species for biological research, species-specific challenges remain and conditions must be optimized carefully. Here, using the milkweed bug Oncopeltus fasciatus , we first generate a useful eye- and body-color mutant strain by CRISPR/Cas9-mediated genome editing. Using this strain, we develop an efficient piggyBac -mediated transgenesis system with a nuclear targeting approach. Incorporating 1× and 3× nuclear localization signals into piggyBac mRNA substantially enhances overall transformation efficiency in O. fasciatus . Taking advantage of the mutant strain and the efficient transgenesis system, we integrated multiple expression cassettes ranging from 1.8 to 7.2 kb, including attP strains for phiC31-mediated integration and histone-labelled strains for live imaging. We further show that the Q system, a binary expression system, is functional in this species, paving the way for sophisticated genetic manipulations. Together, our results expand the genetic toolkit of O. fasciatus as a comparative model insect and provide a practical framework for developing efficient transgenesis in other non-traditional model organisms. Summary statement: We developed an efficient piggyBac transgenesis system in the milkweed bug with a nuclear targeting approach, expanding genetic tools for non-model insects.

Novel genes arise through multiple mechanisms, including gene duplication, gene fusion, and horizontal gene transfer (HGT). While HGT has increasingly been documented in animals, the posttransfer evolutionary fate of horizontally acquired genes is less well understood. We hypothesized that fusion with endogenous sequences in animal genomes might generate what we call “HGT-chimeras”: genes with regions of nonmetazoan and metazoan descent in the same open reading frame. To test this hypothesis, we developed a molecular phylogenetics pipeline that enables the identification of HGT-chimeras. We applied our pipeline to 319 high-quality annotated arthropod genomes and uncovered a high-confidence set of 274 HGT-chimeras corresponding to 104 independent origination events across diverse arthropods. HGT-chimeras contain intervals acquired from across the tree of life, and many likely originated via a gene duplication-based mechanism. To assess whether HGT-chimeras might be functionally important, we performed RT-PCR and Sanger sequencing of tissues from 20 arthropod species predicted to harbor HGT-chimeras in their genome. We found evidence for the expression of contiguous chimeric messenger RNA transcripts (mRNAs) for 36 of 41 tested HGT-chimeras across 18 of 20 different tested species. We also found evidence that HGT-chimeras evolve under purifying selection and have acquired potentially functional domain architectures, consistent with the hypothesis that these genes are in active use and may participate in diverse biological processes. These results illuminate an underappreciated combinatorial mechanism underlying the origin of novel genes across the largest animal phylum, and suggest that interdomain sequence fusion can play important roles in animal biology and evolution.

Antonio García-Bellido was a pioneer of developmental biology in Spain who had the rare ability to see beyond the trees and grasp not just the forest, but entire landscapes. Intellectually and personally, he was indefatigable, uncompromising, and wholly committed to science. He led his trainees by example in his unflagging demands on honoring the data above all else. Antonio discovered the existence of “developmental compartments” in the wing of Drosophila and proposed that these territories corresponded to the realm of action of homeotic genes. He was a fierce defender of hypotheses that he thought best explained available data but did not cling blindly to ideas when they no longer served to understand the phenomena at hand. With his distinctive style he founded a successful school of Drosophila developmental geneticists that will sorely miss his unparalleled passion for science.

Cell migration across epithelial barriers occurs in diverse developmental, immunological, and pathological contexts. Here, we investigate the contribution of heterotypic adhesion between migrating cells and epithelial “substrate” cells to transepithelial migration. Using an in silico model inspired by the migration of primordial germ cells across the midgut epithelium in the Drosophila embryo, we show that heterotypic adhesion modulates migration efficiency in a nonmonotonic manner, revealing the existence of an optimal adhesion regime. Consistent with this prediction, in vivo overexpression of E-cadherin in germ cells accelerated their exit from the midgut relative to controls. Beyond providing experimentally testable predictions, our model integrates and explains previous observations on the role of heterotypic adhesion in cell-on-cell migration, offering a framework for understanding transepithelial migration across biological contexts.

Biases in synonymous codon use occur in many unicellular and multicellular organisms. Optimal codons, defined as those most commonly used in highly transcribed genes, are thought to arise from selection for cost-efficient translation, which would favor codons with abundant matching tRNAs. Such presumed selection is described as optimal codon choice. Non-optimal codons, defined as those least commonly used in highly transcribed genes, may in principle also play important roles, but the dynamics of their use remain understudied. Here, we examine non-optimal codon use using sex-biased genes expressed in the gonads of Drosophila melanogaster as a case study. We show that genes with sex-biased expression exhibit a preference for non-optimal codon use, especially testis-biased genes. Further, we show that the use of non-optimal codons is not random. Instead, specific non-optimal codons are favored, again especially in testis-biased genes. Non-optimal codon use is positively linked to elevated disorder of the encoded proteins. Remarkably, all 18 degenerate amino acids were associated with higher disorder when encoded by the identified primary non-optimal codon, than when encoded by its sister optimal codon. We hypothesize that selection may have promoted non-optimal codon choice for a subset of favored non-optimal codons to regulate translation. We discuss the putative roles of tRNA gene copy numbers, pleiotropy, and sex-biased expression in the evolution of this level of gene regulation.

Germ cells enable the reproduction of an organism and the continuity of its lineage. Across animals, these crucial cells are segregated from the soma at different times and places and via distinct mechanisms. Understanding the evolution of germ cell specification across animals is complicated by the difficulty of making meaningful comparisons of embryonic development between diverse animal species. Here, we characterize germ cell specification in Panarthropoda, an ancient clade that encompasses massive animal biodiversity, within which we can conduct meaningful comparative embryology. We amass data from centuries of studies describing the timing and mechanisms of germ cell formation, and apply ancestral state reconstruction to these data to propose hypotheses about the trajectory of evolution in this process. Furthermore, we speculate about the mechanisms underlying these evolutionary dynamics by considering the relationships among germ cell specification, concurrent developmental processes and the germ line gene network. Collectively, this Review derives new insights from a rich historical database of embryological observations, offering broad implications for understanding the evolution of metazoan germ cells.

Recent advances in adult injection-based insect genome editing have enabled genetic manipulation of a wide range of insect species, including those previously considered difficult or even impervious to genetic modification. However, achieving efficient knock-in remains a significant challenge with this approach. Here, we demonstrate that fusing a HUH endonuclease tag to Cas9 significantly enhances both non-homologous end joining (NHEJ)-mediated knockout and homology-directed repair (HDR)-mediated knock-in via adult injection. This fusion increased knockout efficiency by up to fivefold in the beetle Tribolium castaneum through adult injection, likely due to its previously unrecognized nuclear localization activity. It also improved single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in efficiency, which we attribute to its characteristic ssDNA-tethering activity. To evaluate its versatility, we tested the HUH-tagged Cas9 in conventional embryo injection, which significantly enhanced HDR-mediated knock-in of an epitope tag in cricket and milkweed bug embryos. Our findings establish the HUH-tag as a versatile platform for improving both NHEJ- and HDR-based genome editing, providing a robust framework to advance genetic engineering across a broad spectrum of arthropods.