Germline Modification of Ants

Hongmei Li-Byarlay, Ph.D. is an NRC Research Fellow in the Department of Entomology at North Carolina State University. She is working on working on insect genomics, stress, and social behavior.
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Trible et al 2017 reported in BioRχiv the first study of genetically modified ants using genome editing. Clonal raider ants (Ooceraea biroi) are potential genetic models to study social complexity and social evolution.

It is known that social insects such as ants use odor cues for communication and their social interactions. The gene orco encodes for a co-receptor protein found in odorant receptors cells, and is essential for detecting pheromones that ants use to communicate.

Trible et al developed a CRISPR/Cas9 protocol involving the injection of O. biroi embryos with a mixture of 100ng/uL Cas9 (from PNAbio) and 10ng/L gRNA (reported in Kistler et al., 2015).  About 3300 injected embryos resulted in 42 G0 adults from which they recovered orco mutants.  Because O. biroi reproduce parthenogenetically recovering  orco-/-  homozygotes required a bi-allelic editing event creating two frameshift alleles that supposedly resulted in complete loss-of-function mutants. PCR amplicons of orco mutants were examined by Sanger and Illumina sequencing. It was interesting to see that  somatic CRISPR mutagenesis in G0s displayed similar behavioral phenotype as the G1 ants, which indicated that G0 ants may be useful for functional studies in these ants.

http://lab.rockefeller.edu/kronauer/assets/image/IMG_1591_ES.jpg

Ooceraea biroi, The queenless parthenogenetic ant shows army ant-like behavior and is one of the main study systems in our group. The species is a native of Asia and has been introduced globally on tropical and subtropical islands. Image and text from the lab website of Daniel Kronauer.

In the ant brain, each type of odorant receptor is clustered in glomeruli. Mutants of orco in the antennal lobes.  Mutant ants had impaired glomeruli and reduced volume of the antennal lobes. Ants with orco mutations started wandering at an earlier age than normal young adult ants. They also were not able to follow pheromone trails left by other ants. Finally, longer term studies showed orco mutants had reduced lifespans and reduced fecundity.

Other social insects have been used for functional genetic studies via CRISPR/Cas9 as well. In honeybees, Kohno et al 2016 has reported mutants in major royal jelly protein 1 (mrjp1) displayed normal male development, which indicated mrjp1 is not critical for drone pupal development. However, it is known that establishing a stable mutant line is difficult in honeybees due to the haplodiploid system of their genetics as Schulte et al (2014) reported.

Buck Trible attended the IGTRCN Technical Workshop in 2015 and honed his embryo microinjection skills. This is a video of O. biroi injections recorded during the Workshop.

The studies of Trible et al. provided important evidence that support the use of genetic technologies in social insects for mechanistic studies of behavioral biology, brain function, and sociality.

Trible et al, 2017. orco mutagenesis causes loss of antennal lobe glomeruli and impaired social behavior in ants. BioRxiv preprint first posted online Feb. 28, 2017; doi: http://dx.doi.org/10.1101/112532.

Kistler et al., 2015. Genome engineering with CRISPR-Cas9 in the mosquito Aedes aegypti. Cell Rep., 11: 51–60.

Kohno et al., 2016. Production of knockout mutants by CRISPR/Cas9 in the European honeybee, Apis mellifera L. Zoological Science, 33: 505-512.

Schulte, C. et al. (2014). Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera). Proc. Natl. Acad. Sci. U.S.A., 111: 9003– 9008.

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