Genetically Encoded Calcium Indicators for Functional Imaging of Mosquito Olfactory Neurons

Abstract

Mosquitoes transmit a multitude of diseases to humans and animals through biting and blood feeding. To locate their hosts, mosquitoes primarily use their sense of smell. Therefore, an understanding of mosquito olfaction will help develop strategies to control the diseases they transmit. A mosquito’s sense of smell is determined by the response of olfactory neurons on its peripheral olfactory organs. Traditionally, mosquito olfactory neuron activity has been examined using electrophysiological techniques such as electroantennography and single sensillum recordings. Electroantennography examines if an odorant is detectable by the ensemble of all antennal neurons. In contrast, single sensillum electrophysiology allows detailed recordings of the activity of two to three neurons at a time. However, single sensillum recording of olfactory neurons is difficult, laborious, and typically allows examination of only a few neurons on the antenna. A promising new approach is to use optical imaging techniques to provide a way to visualize the global response of olfactory organs to an odor, as well as the specific responses of several olfactory neurons to that odor. In particular, calcium imaging has progressed significantly, from the use of chemical calcium indicators to the development of genetically encoded calcium sensors. These advances have opened the way to study the mode of action of known mosquito attractants and repellents as well as a way to screen potential new attractants and repellents. Here, we provide an introduction to the different types of calcium indicators and their uses for investigating the function of mosquito sensory neurons.

MOSQUITO OLFACTION: AN INTRODUCTION

Mosquitoes transmit several diseases to humans such as West Nile virus (Farajollahi et al. 2011), yellow fever (Christophers 1960), and malaria (Russell 1959). Every year, malaria alone infects more than 200 million people and kills more than 400,000 people worldwide (WHO 2020). Female mosquitoes transmit diseases through blood feeding, which is required for egg production. Some mosquitoes, such as the Anopheles malaria mosquitoes (Costantini et al. 1998), prefer to blood-feed on humans (anthropophilic), making them particularly dangerous as vectors for human diseases. Understanding how such mosquitoes find humans and distinguish them from other animals is an important step in the effort to develop methods for preventing mosquito bites and the spread of diseases. The important role of olfaction in mosquito host seeking has been appreciated for decades (Rudolfs 1922). Recent studies have further highlighted the primary role olfaction plays, along with other senses, to find hosts for blood feeding (Potter 2014). In fact, most mosquito behaviors are guided by their sense of smell (Konopka et al. 2021), which suggests that understanding how a mosquito smells its environment can be used to control its behaviors.

The mosquito olfactory system consists of three sensory organs: the antennae, the maxillary palps, and the labella (Potter 2014; Lombardo et al. 2017; Konopka et al. 2021). These peripheral organs are covered with sensory hairs called sensilla, with the antenna containing the most sensilla. For example, the antenna of an Anopheles gambiae female is covered by more than 700 sensilla, while the maxillary palp contains only 67 sensilla (Mclver 1982). Each of these sensilla contains sensory neurons that express different types of odorant receptors (ORs), gustatory receptors (GRs), and/or ionotropic receptors (IRs) (Potter 2014; Lombardo et al. 2017; Konopka et al. 2021). Insect ORs are odor-gated ion channels that function as a complex of two proteins: the first is the “tuning” odorant receptor (ORx) that binds the odorant to induce activation, and the second component is an obligate odorant receptor coreceptor (Orco) that traffics the ORx to the membrane and helps form the receptor complex (Carey and Carlson 2011; Potter 2014).

ORx/Orco activation by an odorant leads to an influx of ions into the neuron and results in action potentials and spiking of that neuron. The electrical activity of the neurons can be monitored by electrophysiological approaches such as electroantennography (EAG) and single sensillum recording (SSR). EAG is a global yet low-resolution approach that records the summed responses of the antenna to an odor stimulus. In contrast, SSR is a higher-resolution approach, in which an electrode is directly inserted into a sensillum and used to record the extracellular potentials of the neurons within that sensillum. SSR has been used in some mosquitoes to survey responses to panels of odorants (Lu et al. 2007; Syed and Leal 2008, 2009), but it is a difficult and challenging technique, and a full survey of the antenna requires the testing of a large number of sensilla, one at a time.

CALCIUM IMAGING TO STUDY MOSQUITO OLFACTION

Neural activity leads to a significant increase in intracellular calcium (Baker et al. 1971). Calcium imaging depends on the use of calcium-sensitive molecules to serve as “sensors” for detecting changes in intracellular calcium, and hence neural activity. These calcium indicators are weakly fluorescent at rest conditions (“baseline activity”), and the intensity of their emitted fluorescence increases as intracellular calcium levels increase (Hendel et al. 2008). Calcium imaging is less sensitive at detecting weak neural activity, but unlike EAG and SSR, the use of calcium imaging allows for visualizing the activity of large numbers of individual neurons at once. In addition, the mosquito preparation for calcium imaging is simpler than that required for EAG or SSR.

Two types of calcium indicators are typically used. The first of these are chemical indicators, which are fluorescent calcium chelators that bind strongly to calcium ions to increase their fluorescence properties and can be used to measure changes in intracellular calcium concentrations. These chemicals are generally membrane impermeable but can be combined with acetoxymethyl ester (AM) to make them membrane permeable, allowing them to be added to the solution bathing the neuronal sample and thereby label many neurons at once (e.g., the Calcium Green AM dye). Once inside the cell, such indicators are freed from the acetoxymethyl ester by the action of cytosolic esterases (Galizia and Vetter 2004). Chemical indicators can also be used in their membrane impermeable form, without acetoxymethyl ester. In this case, they are directly injected into neurons, allowing more confidence in the identity of the neurons being imaged (e.g., staining projection neurons with Fura dextran dye). These chemical indicators have been successfully used to study olfactory responses in the brain in different insects such as honeybees, Drosophila, and moths (Galizia and Vetter 2004). However, they require loading of dye into neurons before each imaging session.

The second type of calcium sensors are genetically encoded calcium indicators (GECIs). These sensors are calcium-sensitive proteins that can be genetically engineered to be expressed in targeted neurons. The most commonly used GECI is GCaMP, a GFP-based calcium indicator that results in recordings with high signal-to-noise ratio (Nakai et al. 2001). GCaMP contains a circularly permuted eGFP molecule in the center, connected to the M13 fragment of myosin light chain kinase at the amino terminus, and calmodulin (CaM) at the carboxyl terminus. When the calmodulin subunit binds calcium, it goes through a conformational change that enables binding to M13. This leads the carboxyl and amino termini of the enhanced green fluorescent protein (eGFP) to come together and helps optimize the fluorescent activity of eGFP (Nakai et al. 2001). Improved versions of GCaMP continue to be developed, such the three variants of GCaMP6, named GCaMP6f, GCaMP6m, and GCaMP6s, that have fast, medium, and slow kinetics, respectively (Chen et al. 2013).

GECIS FOR MOSQUITO OLFACTION

GECIs can be expressed in a population of neurons, such as Orco or IR-expressing neurons, using a tissue-specific promoter. This allows control over which tissues will be examined by calcium imaging. Binary expression systems such as the Q-system (Riabinina and Potter 2016) can also be used to provide versatility in expressing GECIs in different populations of neurons. In brief, this is accomplished by combining two genetic components in the same animal: a QF transcription factor driver line expressed in a tissue-specific population, and a QF-responsive QUAS-GCaMP line that will express the calcium reporter in the QF-expressing population of neurons. Binary expression systems also allow for transcriptional amplification of GCaMP, which can boost the fluorescent signal. GECIs have been used for years in Drosophila to monitor sensory or olfactory functions (Simpson and Looger 2018) but were only recently introduced into mosquitoes. In one such experiment, the Orco promoter was used to drive expression of GCaMP6f in olfactory neurons of Anopheles coluzzii mosquitoes (Orco-QF2+ QUAS-GCaMP6f) (Afify et al. 2019). The method by which this was done is described in detail in our associated protocol (see Protocol: Calcium Imaging of Anopheles coluzzii Mosquito Antennae Expressing GCaMP6f [Afify and Potter 2022]). This allowed direct monitoring of the odor-induced neuronal responses for the majority of olfactory neurons on the peripheral organs of Anopheles mosquitoes and was used to perform calcium imaging to test Anopheles antennal olfactory neuron responses toward mosquito repellents. Natural repellents (e.g., lemongrass oil and eugenol) were found to activate discrete odorant receptor neurons on the antenna while synthetic repellents (DEET, IR3535, and picaridin) did not robustly activate Anopheles olfactory neurons, but instead acted to decrease the amount of human odorants that reached the mosquito antenna (Afify et al. 2019). Calcium imaging with these transgenic mosquitoes was also used to test the response of Anopheles mosquitoes to repellent blends and showed that mixing repellents does not necessarily produce a better repellent (Afify and Potter 2020). These studies, which involved testing the activity of hundreds of neurons on the mosquito antennae, maxillary palps, and proboscis, would have been exceedingly difficult to perform using electrophysiology approaches.

In Aedes aegypti, the Bruchpilot (Brp) promoter has been used to drive the expression of GCaMP6s in all neurons (Jové et al. 2020; Zhao et al. 2021). This transgenic line was used to examine the response of the mosquito stylet to sugar and blood. “Stylet” is a common term used for the piercing tubular “needle” inserted into the skin during blood feeding. In this experiment, stylet neurons were imaged ex vivo while ligands were delivered using a microfluidics device, an experiment that would have been very difficult to perform using a technique like SSR that is more sensitive to movements.

GECIs can also be used to perform calcium imaging of neurons in the brain. For example, GCaMP6s was expressed in all cells in Ae. aegypti mosquitoes using the polyubiquitin (PUb) promoter (Bui et al. 2019; Lahondère et al. 2019). This enabled the use of calcium imaging to study responses to floral odors in the antennal lobe, the brain region targeted by olfactory neurons. Another example is the expression of GCaMP6f in Orco⁺ neurons in Ae. aegypti and examining odor-induced responses in the antennal lobes. Although human, animal, and nectar odors consistently activated a “universal glomerulus,” human odors alone activated a “human-sensitive” glomerulus, suggesting a potential neural mechanism by which Ae. aegypti mosquitoes might be able to distinguish human hosts (Zhao et al. 2020). Calcium imaging of brain regions can help identify how odors are represented by the activities of neurons within the brain, a task that would be far more challenging to accomplish using electrophysiological approaches.

Calcium imaging of peripheral organs can be a powerful approach to characterize the response properties of sensory neurons and to potentially isolate and identify the sensory receptors expressed by these neurons that enable these responses. This could be applied, for example, to identify the olfactory receptors that are required for the response to known mosquito attractant and repellent odors. In addition, calcium imaging of peripheral organs can be used as a quick and easy screening method for the development or optimization of new odor-based attractants and repellents.