Jonchee A. Kao

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.

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.

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.

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 novel 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.

During development, sexually reproducing animals must specify and maintain the germ line, the lineage of cells that gives rise to the next generation of animals. In the fruit fly Drosophila melanogaster , germ cell-less ( gcl ) is required for the formation of primordial germ cells in the form of cells that cellularize at the posterior pole of the embryo, called pole cells. Forming pole cells is a mechanism of germ cell formation unique to a subset of insects. Even though most animals do not form pole cells as primordial germ cells, gcl is conserved across Metazoa, raising the question of how this conserved gene acquired its central role in the evolutionarily derived process of pole cell formation. Here, we examine the functions of gcl in two other insects with different modes of germ cell specification: the milkweed bug Oncopeltus fasciatus and the cricket Gryllus bimaculatus . We found that gcl is involved in germ cell development, but not strictly required for germ cell specification, in O. fasciatus , although it appears to function through a mechanism different from that in D. melanogaster . In contrast, we could not detect any impact on the embryonic germ line upon gcl knockdown in G. bimaculatus . This work serves as a case study into how the roles of genes in the process of germ line development can change over evolutionary time across animals. Highlights: germ cell-less knockout reduces germ cell number in Oncopeltus fasciatus . germ cell-less knockdown does not affect germ cell number in Gryllus bimaculatus . The genetic mechanisms through which gcl affects germ cell number have diverged among insects. Graphical

Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into preexisting regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene oskar, was first identified based on its role in establishing the Drosophila melanogaster germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a neural role for oskar . We show that oskar is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket Gryllus bimaculatus . In these stem cells, called neuroblasts, oskar is required together with the ancient animal transcription factor Creb to regulate long-term (but not short-term) olfactory memory. We provide evidence that oskar positively regulates Creb , which plays a conserved role in long-term memory across animals, and that oskar in turn may be a direct target of Creb. Together with previous reports of a role for oskar in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that oskar ’s original somatic role may have been in the insect nervous system. Moreover, its colocalization and functional cooperation with the conserved pluripotency gene piwi in the nervous system may have facilitated oskar ’s later co-option to the germ line in holometabolous insects.

Novel genes have the potential to drive the evolution of new biological mechanisms, or to integrate into pre-existing regulatory circuits and contribute to the regulation of older, conserved biological functions. One such gene, the novel insect-specific gene oskar , was first identified based on its role in establishing the Drosophila melanogaster germ line. We previously showed that this gene likely arose through an unusual domain transfer event involving bacterial endosymbionts, and played a somatic role before evolving its well-known germ line function. Here, we provide empirical support for this hypothesis in the form of evidence for a novel neural role for oskar . We show that oskar is expressed in the adult neural stem cells of a hemimetabolous insect, the cricket Gryllus bimaculatus . In these stem cells, called neuroblasts, oskar is required together with the ancient animal transcription factor Creb to regulate long-term (but not short-term) olfactory memory. We provide evidence that oskar positively regulates Creb , which plays a conserved role in long-term memory across animals, and that oskar in turn may be a direct target of Creb. Together with previous reports of a role for oskar in nervous system development and function in crickets and flies, our results are consistent with the hypothesis that oskar ’s original somatic role may have been in the insect nervous system. Moreover, its co-localization and functional cooperation with the conserved pluripotency gene piwi in the nervous system may have facilitated oskar ’s later co-option to the germ line in holometabolous insects.

Liquid–liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using Drosophila melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.