CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major

Abstract

Sand flies are the natural vectors for the Leishmania species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/Leishmania interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the Phlebotomus papatasi sand fly, a natural vector for Leishmania major, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with L. major. Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for Leishmania parasites. IMPORTANCE Sand flies are the natural vectors of Leishmania parasites. Studies of sand fly/Leishmania interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible null mutant alleles of relish, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with L. major, showing that the sand fly immune response impacts its vector competence for this pathogen.

Modulation of IMD, Toll, and Jak/STAT Immune Pathways Genes in the Fat Body of Rhodnius prolixus During Trypanosoma rangeli Infection

“…and Leishmania sp.) showed that the expression of Relish and Cactus regulates infection prevalence and intensity, highlighting the importance of the IMD and Toll pathways in anti-parasitic immune responses in vectors ( Meister et al., 2005 ; Frolet et al., 2006 ; Hu and Aksoy, 2006 ; Antonova et al., 2009 ; Louradour et al., 2019 ). In this regard, a recent report demonstrated a reduction in T. cruzi loads in the midgut of R. prolixus treated with the drug IMD-0354, which impedes the activation of NF-κB TFs ( Vieira C. S. et al., 2018 ).…”

Aedes aegypti Infection With Trypanosomatid Strigomonas culicis Alters Midgut Redox Metabolism and Reduces Mosquito Reproductive Fitness

“…numbers in the gut of sand flies. Similarly, the mutagenesis of Rel2 by CRISPR/Cas9 generates a more permissive phenotype in insect vector, presenting higher loads of promastigotes (Telleria et al, 2012;Louradour et al, 2019). Hamilton et al (2015) described that Jaenimonas drosophilae, a trypanosomatid found naturally infecting Drosophila fallen, is able to establish infections in larvae of different species of Drosophila.…”

Chemical control and insecticide resistance status of sand fly vectors worldwide

“…papatasi and L . longipalpis , aiming to target leishmanial vector competence, has been recently attempted [ 110 ]. Furthermore, Phlebotomus and Lutzomyia species have been found naturally infected with Wolbachia strains [ 111 ], and a potential for the use of Wolbachia , alone or in combination with sterile insect technique, has been indicated [ 112 ].…”