Harrell, R. A. | bioRxiv Science

bioRxiv2024

Rapid generation and screening of transgenic black soldier fly (Hermetia illucens)

BackgroundThe black soldier fly (BSF), Hermetia illucens is a widely used, and mass-produced insect that fulfils an important role in both the management of organic waste and as a component of animal feed formulations. They also have significant potential as a platform for converting organic waste into high-value proteins, and lipids for the production of biofuels. Applying synthetic biology to BSF provides even more potential for improvement through the generation of transgenic BSF to enhance animal feed, produce and fine tune high-value industrial biomolecules, and to expand their waste conversion capabilities. ResultsTo enable the rapid generation and screening of transgenic BSF, we utilised microinjections of piggyBac mRNA with donor plasmids. We have found preliminary screening of G0 BSF to identify mosaics for outcrossing can be completed less than 2 weeks after microinjection. Stable transgenic lines were reliably generated with effective transformation rates of 30-33%, and transmission of the transgene could be confirmed 3 days after outcrossing the G0 adults. We also present a protocol for identifying the location of integrated transgenes. ConclusionsThe methods presented here expedite the screening process for BSF transgenesis and further expand the toolkit for BSF synthetic biology.

synthetic biology↗

bioRxiv2021

Knockout of Anopheles stephensi immune gene LRIM1 by CRISPR-Cas9 reveals its unexpected role in reproduction and vector competence

PfSPZ Vaccine against malaria is composed of Plasmodium falciparum (Pf) sporozoites (SPZ) manufactured using aseptically reared Anopheles stephensi mosquitoes. Immune response genes of Anopheles mosquitoes such as Leucin-Rich protein (LRIM1), inhibit Plasmodium SPZ development (sporogony) in mosquitoes by supporting melanization and phagocytosis of ookinetes. With the aim of increasing PfSPZ infection intensities, we generated an A. stephensi LRIM1 knockout line , {Delta}aslrim1 , by embryonic genome editing using CRISPR-Cas9. {Delta}aslrim1 mosquitoes had a significantly increased midgut bacterial load and an altered microbiome composition, including elimination of commensal acetic acid bacteria. The alterations in the microbiome caused increased mosquito mortality and unexpectedly, significantly reduced sporogony. The survival rate of {Delta}aslrim1 and their ability to support PfSPZ development, were partially restored by antibiotic treatment of the mosquitoes, and fully restored to baseline when {Delta}aslrim1 mosquitoes were produced aseptically. Deletion of LRIM1 also affected reproductive capacity: oviposition, fecundity and male fertility were significantly compromised. Attenuation in fecundity was not associated with the altered microbiome. This work demonstrates that LRIM1s regulation of the microbiome has a major impact on vector competence and longevity of A. stephensi. Additionally, LRIM1 deletion identified an unexpected role for this gene in fecundity and reduction of sperm transfer by males.

microbiology↗