Sawfly and Ladybird Beetle Gene Editing

Hatakeyama et al (2015) have successfully used gene editing technology in Athalia rosae (sawfly) and Harmonia axyridis (ladybird beetle), enlarging the range of insects in which these technologies have been applied.  These results are reported in Insect Molecular Biology.

Athalia rosae

Athalia rosae Image credit: Michael Becker

Hatakeyama et al. used TALENS to mutagenize transgenic lines of A. rosae and H. axyridis that had an EGFP transgene under the regulatory control of the eye/neuronal specific promoter 3XP3.  These lines had been created some time ago.

Cartoon of the structure of GFP. In the right-hand model the beta barrel has been cut away to reveal the chromophore.

The pair of TALENs used in this set of experiments targeted a region of EGFP adjacent to the chromophore region.

A cocktail of two mRNAs (one for each TALEN, each at 500ng/ul) was injected into preblastoderm embryos using methods developed previously by this group and, depending on where (anterior or posterior) they injected the embryos, obtained varying levels of mutagenesis.

A. rosae males are haploid and this facilitated the detection and recovery of mutants.  For example, when injected at the posterior pole, 15% of male embryos developed into adults with clear mosaicism in the expression of EGFP.  75% of the males showing mosaicism produced EGFP-negative G1 progeny.  Interestingly, a small percentage of the G0 males that did not show any somatic mosaicism also produced mutant G1 progeny.

Similar results were obtained with H. axyridis although somatic mosaicism could not be observed since these insects are diploid.  However, following injection of the TALEN cocktail at the posterior pole of the young embryos 25% of the fertile G0 individuals produced EGFP-negative progeny.

Harmonia axyridis, various color patterns.

Harmonia axyridis, various color patterns.

The authors also report conducting experiments in which they injected embryos at the anterior pole and while these injections also successfully produced mutants the frequencies were somewhat reduced, perhaps as expected.

So, this paper demonstrates that TALENs can be effective mutagens in the two species tested and, with appropriate mutation-detection capabilities, mutants can be readily recovered.  While these results are not particularly surprising, they illustrate the level of efficiency that one might expect in using this system and these levels are not unlike what has been reported by other investigators in other systems.  It is becoming increasingly clear that these programmable endonuclease systems are effective enough that creating mutations does not require producing particularly large numbers of G0 animals.  That is good because while screening for visible mutations is relatively easy, when the mutations are cryptic and must be detected using molecular tools this becomes feasible only if the rates of mutagenesis are relatively high.

Hatakeyama, M., Yatomi, J., Sumitani, M., Takasu, Y., Sekiné, K. et al., 2015  Knockout of a transgene by transcription activator-like effector nucleases (TALENs) in the sawfly, Athalia rosae (Hymenoptera) and the ladybird beetle, Harmonia axyridis (Coleoptera). Insect Mol Biol:  n/a-n/adoi: 10.1111/imb.12195.

 

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