Microinjection of the Sand Fly Lutzomyia longipalpis, a Vector for Leishmaniasis

Two recent papers, Jefferies et al (2018) and Martin-Martin et. al (2018), describe experiments to develop and optimise a sand fly microinjection protocol.

There are 2 authors to this post: Alla Madich, Simon Collier, technical constraints on this website allow only one name in the byline.

alla Madich
Dr. Alla Madich is a Microinjection Technician in the Fly Facility at the University of Cambridge. https://www.flyfacility.gen.cam.ac.uk/
me
Dr. Simon Collier is Manager of the Fly Facility at the University of Cambridge.
https://www.flyfacility.gen.cam.ac.uk/

The phlebotomine sand fly Lutzomyia longipalpis transmits parasitic protozoa which  cause Leishmaniasis in humans and animals. Leishmaniasis has been estimated to affect over a million people each year resulting in tens of thousands of deaths. The ability to edit the sand fly genome using the CRISPR/Cas9 system should allow improved understanding of vector-parasite interactions at a molecular level and lead to potential disease control strategies. However, the adoption of CRISPR/Cas9 is complicated by the unique challenges of microinjecting sand fly embryos.

Lutzomyia longipalpis-sandfly.jpg

Blood-fed Lutzomyia longipalpis sandfly. Photo Credit:
Ray Wilson, Liverpool School of Tropical Medicine,
doi:10.1371/image.ppat.v05.i08

In Drosophila and mosquito embryos, there is a short window for successful microinjection of less than 90 minutes before the formation of pole cells, the germ line progenitors, at the posterior of the egg. In contrast, pole cells only form after 36 hours in the sand fly embryo. Instead, the time limiting factor for sand fly microinjection is the melanisation process which occurs progressively after egg laying. Martin-Martin et al. conclude that the window for microinjection is between 90 minutes, by which time the chorion is sufficiently hard to allow successful microinjection, and 3 hours after which it is difficult to penetrate the chorion with the injection needle. Jefferies et al. note that full melanisation is observed at 4 hours, but that melanised embryos can seen in 45 minute egg collections, and also in gravid female abdomens, suggesting egg laying is not necessarily synchronised.

To encourage synchronised laying, Martin-Martin et al held blood-fed females in cages for 5-7 days before initiating 1 hour egg collections in the dark. In contrast, Jefferies et al. counted the number of injectable eggs collected at 3, 4 and 5 days after blood-feed and found that the maximum number was observed in the first collection. Jefferies et al also compared three different substrates for oviposition plates, plaster of paris, apple juice agar and agar alone. They found the greatest hatch rates on agar alone which were over twice as high as on plaster of paris. 

Eggs of Lutzomyia longipalpis (Lutz and Neiva), a sand fly.

Eggs of Lutzomyia longipalpis (Lutz and Neiva), a sand fly. Photograph by Cristina Ferro, Instituto Nacional de Salud, Colombia.

To test their microinjection protocol, Jefferies et al. injected the endosymbiotic bacterium Wolbachia sourced from Drosophila eggs as a ‘marker’ for successful injection. They injected a total of 1,815 embryos which produce just 6 fertile adult females. However, three of these females tested positive for Wolbachia by PCR, and a low level of maternal transmission was observed from two of these flies for several generations.

hypothesise that achieving high density Wolbachia infection in sand flies might inhibit parasite development and vector competence as has been observed in mosquitos.

Genetic Technology Delivery by Embryo Microinjection
A generalized image of insect microinjection.

Martin-Martin et al. injected a CRISPR mix containing Cas9 mRNA and two sgRNAs to target the sand fly Yellow (LuloYLW) gene, the Drosophila yellow gene orthologue. They injected 775 embryos and saw hatch rates over 14% and larval survival of around 10%. However, they do not report follow the injected flies further and so could not verify CRISPR genome editing.

These two papers document the challenges of embryo microinjection in sand flies, but also present evidence that these may be overcome paving the way for the development  of control strategies for Leishmaniasis.

Jeffries CL, Rogers ME, Walker T. Establishment of a method for Lutzomyia longipalpis sand fly embryo microinjection: The first step towards potential novel control strategies for leishmaniasis. Wellcome Open Res. 2018 May 9;3:55

Martin-Martin I, Aryan A, Meneses C, Adelman ZN, Calvo E. Optimization of sand fly embryo microinjection for gene editing by CRISPR/Cas9. PLoS Negl Trop Dis. 2018 Sep 4;12(9):e0006769.

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