Germline mutagenesis of<i>Nasonia vitripennis</i>through ovarian delivery of CRISPR-Cas9 ribonucleoprotein

Chaverra-Rodríguez, Duverney; Benetta, Elena Dalla; Heu, Chan C.; Rasgon, Jason L.; Ferree, Patrick M.; Akbari, Omar S.

doi:10.1101/2020.05.10.087494

Cited by 6 publications

(4 citation statements)

References 45 publications

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“…Indeed, tools in parasitic hymenopterans or other haplodiploid arthropods are quite sparse. Even in the most wellstudied species, Nasonia vitripenis, CRISPR has only recently been used for gene-editing (Li, et al 2017;Chaverra-Rodriguez, et al 2020), but as of yet no one has achieved integration of a transgene in any Hymenopteran. Ongoing investigation into the mechanisms of symbiontmediated PI and deciphering the involvement of PifA and PifB will thus require a stronger focus on biochemical approaches and methods development for cell biology and genetics in these difficult to work with insects.…”

Section: Discussionmentioning

confidence: 99%

Identification of parthenogenesis-inducing effector proteins inWolbachia

Fricke,

Lindsey

2023

Preprint

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Bacteria in the genusWolbachiahave evolved numerous strategies to manipulate arthropod sex, including the conversion of would-be male offspring to asexually reproducing females. This so-called “parthenogenesis-induction” phenotype can be found in a number ofWolbachiastrains that infect arthropods with haplodiploid sex determination systems, including parasitoid wasps. Despite the discovery of microbe-mediated parthenogenesis more than 30 years ago, the underlying genetic mechanisms have remained elusive. We used a suite of genomic, computational, and molecular tools to identify and characterize two proteins that are uniquely found in parthenogenesis-inducingWolbachiaand have strong signatures of host-associated bacterial effector proteins. These putative parthenogenesis-inducing proteins have structural homology to eukaryotic protein domains including nucleoporins, the key insect sex-determining factor Transformer, and a eukaryotic-like serine-threonine kinase with leucine rich repeats. Furthermore, these proteins significantly impact eukaryotic cell biology in the model,Saccharomyces cerevisiae. We suggest these proteins are parthenogenesis-inducing factors and our results indicate this would be made possible by a novel mechanism of bacterial-host interaction.

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Section: Discussionmentioning

confidence: 99%

Identification of parthenogenesis-inducing effector proteins inWolbachia

Fricke,

Lindsey

2023

Preprint

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“…Additionally, better understanding of the reproductive system of B. stephanieae will in turn enhance our ability to introduce genetic material in the Berghia system. For example, powerful new techniques such as ReMOT Control, which introduce transgenic components into adult animals, targeting the ovaries directly (Chaverra-Rodriguez et al 2018, 2020), will require detailed understanding of reproductive processes in order to be effective. The increasing availability of genetic tools opens new avenues for exploring the diversity of sexual systems.…”

Section: Discussionmentioning

confidence: 99%

Precocious sperm exchange in the simultaneously hermaphroditic nudibranch,Berghia stephanieae

Taraporevala

Lesoway

Goodheart

et al. 2022

Preprint

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Sexual systems vary greatly across molluscs. This diversity includes simultaneous hermaphroditism, with both sexes functional at the same time. Most nudibranch molluscs are thought to be simultaneous hermaphrodites, but detailed studies of reproductive development and timing remain rare as most species cannot be cultured in the lab. The aeolid nudibranch, Berghia stephanieae, is one such species that can be cultured through multiple generations on the benchtop. We studied B. stephanieae reproductive timing to establish when animals first exchange sperm and how long sperm can be stored. We isolated age- and size-matched individuals at sequential timepoints to learn how early individuals exchange sperm. Individuals isolated at 13 weeks post laying (wpl) can produce fertilized eggs. This is 6 weeks before animals first lay egg masses, indicating that sperm exchange occurs well before individuals are capable of laying eggs. Our results indicate that male gonads become functional for animals between 6 mm (~9 wpl) and 9 mm (~15 wpl) in length. That is much smaller (and sooner) than the size (and age) of individuals at first laying (12-19 mm; ~19 wpl), indicating that male and female functions do not develop simultaneously. We also tracked the number of fertilized eggs in each egg mass, which remained steady for the first 10-15 egg masses, followed by a decline to near-to-no fertilization. This large, novel dataset provides insights into precise timing of the onset of functionality of the male and female reproductive systems in B. stephanieae. These data contribute to a broader understanding of reproductive development and the potential for understanding evolution of diverse sexual systems in molluscs.

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“…There is a need to apply genetics and functional genomics in the mosquito to better understand the small RNA pathway features. New genetic tools for mosquitoes are still emerging, such as germline editing using CRISPR in mosquitoes [ 288 , 289 ] and novel Cas9 delivery via ReMOT [ 294 , 295 , 296 ] that may enable creating Piwi pathway mosquito mutants. These mutants could inform on whether infertility results from loss of mosquito Piwi genes, but the redundancy of Piwi genes in mosquitoes may also obscure an overt phenotype.…”

Section: Final Thoughts: Strengthening the Fly-mosquito Partnershmentioning

confidence: 99%

Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways

Gamez

Srivastav

Akbari

et al. 2020

Cells

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Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.

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Germline mutagenesis ofNasonia vitripennisthrough ovarian delivery of CRISPR-Cas9 ribonucleoprotein

Cited by 6 publications

References 45 publications

Identification of parthenogenesis-inducing effector proteins inWolbachia

Identification of parthenogenesis-inducing effector proteins inWolbachia

Precocious sperm exchange in the simultaneously hermaphroditic nudibranch,Berghia stephanieae

Diverse Defenses: A Perspective Comparing Dipteran Piwi-piRNA Pathways

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