Gene editing reveals obligate and modulatory components of the CO2 receptor complex in the malaria vector mosquito, Anopheles coluzzii

Abstract

The sensitivity to volatile carbon dioxide (CO2) produced by humans and other animals is a critical component in the host preference behaviors of the malaria vector mosquito Anopheles coluzzii. The molecular receptors responsible for the ability to sense CO2 are encoded by three putative gustatory receptor (Gr) genes (Gr22,23,24) which are expressed in a distinctive array of sensory neurons housed in maxillary palp capitate peg sensilla of An. coluzzii. Despite the identification of these components and subsequent studies, there is a paucity of understanding regarding the respective roles of these three GRs in the mosquito's CO2 transduction process. To address this, we have used CRISPR/Cas9-based gene editing techniques combined with in vivo electrophysiological recordings to directly examine the role of Gr22,23,24 in detecting CO2 in An. coluzzii. These studies reveal that both Gr23 and Gr24 are absolutely required to maintain in vivo CO2 sensitivity while, in contrast, Gr22 knock out mutants are still able to respond to CO2 stimuli albeit with significantly weaker sensitivity. Our data supports a model in which Gr22 plays a modulatory role to enhance the functionality of Gr23/24 complexes that are responsible for CO2 sensitivity of mosquitoes.

Identification of a gustatory receptor tuned to sinigrin in the cabbage butterfly Pieris rapae

“…Using Sf9 cells and RNAi methods, PxutGr1 has been shown to respond to synephrine, an oviposition stimulus for P. xuthus [39]. In addition, a complex formed by two CO 2 receptors has been shown to be necessary for CO 2 detection in cotton bollworms and mosquitoes in oocytes and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) systems [62][63][64].…”

Mutagenesis of the Ammonium TransporterAcAmtReveals a Reproductive Role and a Novel Ammonia-Sensing Mechanism in the Malaria Vector MosquitoAnopheles coluzzii

“…A complete AcAmt null mutant strain was generated using CRISPR/Cas9 gene editing via embryonic microinjection of two targeting plasmids expressing Cas9 and dual sgRNAs along with a homology template to knock-in a 3xP3-DsRed eye-specific red fluorescence marker between two AcAmt DSB sites (Liu et al 2020). The two sgRNAs targeted sequences at the start of both Exon 1 and Exon 4 to remove the majority of the three exons in between the DSBs of the AcAmt coding region ( Supplementary Table 1 ).…”

“…CRISPR/Cas9 gene editing in An. coluzzii was carried out as previously described (Liu et al 2020), with minor modifications. The CRISPR gene-targeting vector was a kind gift from Dr. Andrea Crisanti of Imperial College London, UK (Hammond et al 2016).…”

Transcriptome Profiles of Anopheles gambiae Harboring Natural Low-Level Plasmodium Infection Reveal Adaptive Advantages for the Mosquito

“…gambiae gustatory receptors (AgGRs) (Matthews et al, 2016; Pitts et al, 2011; Rinker et al, 2013a). In contrast, AgGR22 (AGAP00999), AgGR23 (AGAP003098), and AgGR24 (AGAP001915), which make up a complex that is required for volatile carbon dioxide (CO2) detection critical for blood-feeding behaviors, have been well characterized (Liu et al, 2020). The differential transcriptome profiles for AgGR22 (0.28-fold), AgGR23 (0.31-fold), and AgGR24 (0.34-fold) are only slightly more abundant in sporozoite-infected head transcriptomes than in controls (Figure 7A, red bar) and, not surprisingly, these changes fail to meet the rigorous significance thresholds employed here.…”