Mutagenesis of the orco odorant receptor co-receptor impairs olfactory function in the malaria vector Anopheles coluzzii

Abstract

Mosquitoes rely heavily on their olfactory systems for host seeking, selection of oviposition sites, and avoiding predators and other environmental dangers. Of these behaviors, the preferential selection of a human blood-meal host drives the vectorial capacity of anthropophilic female Anopheles coluzzii mosquitoes. Olfactory receptor neurons (ORNs) are dispersed across several appendages on the head and express an obligate odorant receptor co-receptor (Orco) coupled with a "tuning" odorant receptor (OR) to form heteromeric, odor-gated ion channels in the membrane of these neurons. To examine the mechanistic and functional contributions of Orco/OR complexes to the chemosensory processes of An. coluzzii, we utilized CRISPR/Cas9 gene editing to create a line of homozygous, Orco-knockout, mutant mosquitoes. As expected, orco-/-ORNs across both adult and larval stages of An. coluzzii display significantly lower background activity and lack nearly all odor-evoked responses. In addition, blood-meal-seeking, adult female, orco-/-mutant mosquitoes exhibit severely reduced attraction to human-and non-human-derived odors while gravid females are significantly less responsive to established oviposition attractants. These results reinforce observations in other insects that Orco is crucial in maintaining the activity of ORNs. In that light, it significantly influences a range of olfactory-driven behaviors central to the anthropophilic host preference that is critical to the vectorial capacity of An. coluzzii as a primary vector for human malaria.

A dual-target molecular mechanism of pyrethrum repellency against mosquitoes

“…In insects, in addition to individual Ors that are responsible for recognizing specific odorants, an obligate co-receptor (Orco) is required for detection of diverse odorants 28 – 30 and can be used to infer whether insect attraction or avoidance response is Or-mediated. No obvious degenerate DAPI-stained ORNs were detected from orco −/− Anopheles mosquitoes 31 . We found that pyrethrum repellency was significantly reduced in orco −/− Ae.…”

Neuronal odor coding in the larval sensory cone of Anopheles coluzzii: Complex responses from a simple system

“…Indeed, it has been shown to be a potent attractant for anopheline larvae and specifically recognized by Or2 and Or10, both of which are expressed in the larval antennae ( Xia et al, 2008 ). Lastly, the inhibitory responses to several volatiles uncovered by orco −/− mutant sensory cones ( Sun et al, 2020 ) and the inhibition of background spiking, as well as the relative sparsity of excitatory responses characteristic of D, E, and F group neurons reported here, may, in part, reflect the impact of ephaptic interactions between neurons ( Figure 4 ) within the closely packed chemosensory neurons of the larval antennae, as has been observed in Drosophila ORNs ( Su et al, 2012 ).…”

“…Previous work has established the critical role of the OR co-receptor (Orco) in the chemical ecology and OR-dependent olfactory responses of An. coluzzii larvae ( Sun et al, 2020 ; Liu et al, 2010 ). To begin with, we examined the background neuronal activity across the larval sensory cone in orco −/− mutant larvae in which background activity, as well as the responses to 2-methylphenol and acetophenone and 72 other volatiles, were dramatically reduced relative to wild-type controls ( Figures 7A and 7B ) ( Sun et al, 2020 ).…”

“…coluzzii larvae ( Sun et al, 2020 ; Liu et al, 2010 ). To begin with, we examined the background neuronal activity across the larval sensory cone in orco −/− mutant larvae in which background activity, as well as the responses to 2-methylphenol and acetophenone and 72 other volatiles, were dramatically reduced relative to wild-type controls ( Figures 7A and 7B ) ( Sun et al, 2020 ). To further determine the role of Orco in the neuronal and behavioral responses to these volatiles, we also utilized VUAA1, a highly specific Orco agonist ( Jones et al, 2011 ) that can be volatized by heat ( Ferguson et al, 2020 ; Yang et al, 2019 ; Pask et al, 2017 ), to challenge the larval sensory cone.…”

“…To accomplish this, electrophysiological recordings of the An. coluzzii larval antennal sensory cone were conducted in the air using an adaptation of the well-established SSR technique ( Sun et al, 2020 ; Liu et al, 2013 ). Here, 3rd instar larvae selected based on large body size and aggressive activity ( Xia et al, 2008 ; Liu et al, 2010 ) were hand transferred via their abdomen with fine forceps (#5 Dumont, Switzerland) from rearing pans to KimWipes (Kimberly Clark, Fisher Scientific) for 30 s to remove excess liquid while avoiding rolling or otherwise damaging the antennae.…”