Taste Sensory Responses in Mosquitoes

Abstract

Analysis of taste sensory responses has been a powerful approach for understanding principles of taste detection and coding. The shared architecture of external taste sensing units, called sensilla, in insects opened up the study of tastant-evoked responses in any model of choice using a single-sensillum tip recording method that was developed in the mid-1900s. Early studies in blow flies were instrumental for identifying distinct taste neurons based on their responses to specific categories of chemicals. Broader system-wide analyses of whole organs have since been carried out in the genetic model insect Drosophila melanogaster, revealing principles of stereotypical organization and function that appear to be evolutionarily conserved. Although limited in scope, investigations of taste sensory responses in mosquitoes showcase conservation in sensillar organization, as well as in groupings of functionally distinct taste neurons in each sensillum. The field is now poised for more thorough dissections of mosquito taste function, which should be of immense value in understanding close-range chemosensory interactions of mosquitoes with their hosts and environment. Here we provide an introduction to the basic structure of a taste sensillum and functional analysis of the chemosensory neurons within it.

THE MOSQUITO TASTE SYSTEM

Taste sensitivity in insects has been a topic of investigation for about 100 years, beginning with observations of feeding responses upon stimulation of distal leg segments (tarsi) in the nymphalid butterflies Pyrameis atlanta Linn. and Vanessa antiopa Linn. (Minnich 1921). Insects rely on taste, or contact chemoreception, for behaviors such as feeding, mate recognition and courtship, and oviposition, which are essential for their survival and reproductive success. Some of the very same behaviors are drivers of an enormous economic and health burden caused by pests and disease vectors, including anthropophilic, or human-seeking, mosquitoes. Studies of taste cues and how they are sensed provide important insight into the biology and evolution of insect taste-guided behaviors.

Insects possess taste sensory units, called sensilla, in many different locations across the body surface: on mouthparts, tarsi, wing margins, ovipositors (female-specific structures used to lay eggs), and, in some cases, the antennae and maxillary palps. The external mouthparts of blood-feeding mosquitoes include a labellum on the distal part of the labium that acts as a sheath to a set of six needle-like structures or stylets that the mosquito uses to pierce the skin of its host (Fig. 1A). Of the six stylets, the labrum is the only one that is used to pierce the host skin and locate blood via chemosensory sensilla. Two types of taste sensilla can be found in mosquitoes: apical/subapical sensilla that are small, straight, hair-like structures that lie flat against the tip of the labrum of hematophagous (blood-feeding) mosquito species, and longer trichoid sensilla that are located throughout external taste organs. Olfactory sensilla are also located on the mosquito labellum and are easily distinguished from the larger and more numerous trichoid taste sensilla. The mosquito cibarium, an internal taste organ that is likely to serve as a checkpoint for evaluating meal quality before ingestion, is coated with small taste papillae in addition to a few trichoid taste sensilla and mechanosensory campaniform sensilla (Lee 1978). Comparative studies suggest that numbers of cibarial papillae may be lower in non-blood-feeding species (Lee 1978). For this reason, they are of great interest in terms of investigating blood-sensing function, however, they are difficult to access for electrophysiological analysis.

Figure 1.

(A) Schematic showing the mosquito’s taste organs (colored in) while probing for a blood meal. Upon landing on a host, the mosquito will use its tarsi and labellum to survey the surface of the host skin. Once a suitable bite site is found, the female mosquito will apply pressure to the skin, causing the labial sheath to retract and expose the stylets. Although the stylets have the appearance of a single feeding tube structure, there are in fact six stylets (L, labrum; H, hypopharynx; M, mandibles; Mx, maxillae) that stack together neatly to form it. The mosquito uses its collection of stylets to cut through the host skin, but the labrum is the only one inserted through the skin to locate host blood. Once imbibed, the blood passes through the feeding tube formed by the stylets and through the cibarium, an internal structure, which is positioned to be a last taste checkpoint before the blood meal enters the midgut. Image credits: (Inlet) Teodros Hailye/KQED based on research by Young-Moo Choo and colleagues. (B) Schematic showing the basic structure of a uniporous trichoid sensillum. In mosquitoes, the trichoid sensilla can be innervated by up to five gustatory neurons (colored) and a single mechanosensory neuron (black).

Despite the morphological differences between the trichoid and apical sensilla, these along with the papillae in the cibarium are categorized as taste sensilla due to the presence of a single pore that allows the dendrites of sensory neurons to contact tastants in the environment. The basic architecture of a trichoid taste sensillum, also known as a taste bristle or taste hair, is conserved in arthropods and consists of an external cuticular shaft that is socketed at the base and contains two compartments, one of which is innervated by the dendrites of a number of taste sensory neurons housed in the sensillum (Fig. 1B). Each sensillum also includes a single mechanosensory neuron with a dendrite that terminates below the shaft. The cell bodies of all sensory neurons lie below the socket and are enveloped by accessory cells that serve to electrically insulate the sensory unit and to supply components of the sensillar lymph that bathes the dendrites. The single pore at the tip of the cuticular hair serves as a conduit between the taste sensors and soluble chemicals in the environment, making them easily accessible for electrophysiological analysis.

The arrangement and morphology of external taste sensilla has been mapped for only a handful of the thousands of mosquito species. As described in Drosophila and other flies, sensilla are found in a stereotypical organization that is characteristic for a species (Hill 1999). In Aedes aegypti and Anopheles gambiae, topographic maps of the labellum have been generated with unique names for each of the trichoid sensilla identified by morphology and location (Hill 1999, Kessler 2015). Trichoid sensilla are also found throughout the tarsi. However, higher numbers are seen particularly on the forelegs and midlegs, in A. aegypti and possibly other species as well (Baik and Carlson 2020). While tarsal sensilla have not been mapped to the same degree of completeness as those in the labellum, it is clear that differently sexed individuals of the same species show consistent patterns of sensilla (Seenivasagan et al. 2009). In addition, sexual dimorphism has been observed in the number of trichoid sensilla in some organs of some species. Females of A. aegypti, for example, have many more trichoid sensilla on their legs than males, but those on the labellum are the same in the two sexes (McIver 1978).

ANALYSIS OF TASTE SENSORY RESPONSES

The responsiveness of taste neurons within a sensillum can be assessed using the extracellular tip recording method, developed in blow flies, Phormia regina, by Edward S. Hodgson in 1955 (Hodgson 1955). This method simultaneously captures the activity of all neurons housed in the sensillum, visualized as train(s) of action potentials. In this method the mosquito, or indeed any insect, is immobilized and then its body is pierced with a reference electrode. With this live restrained preparation, any water-soluble stimulus can be delivered to the open pore at the tip of a selected external taste sensillum via a glass micropipette electrode that also records neural responses. The activity of individual neurons can often, but not always, be parsed by amplitude of the action potential and sensitivity to “diagnostic” tastants. The strength of a neuron’s response is then quantified by measuring action potential frequency, typically in the first second or part thereof after contact of the sensillum tip with the tastant solution. In our associated protocol, we introduce such a protocol (see Protocol: Single-Sensillum Taste Recordings in Mosquitoes<prot108195> [Lomelí & Dahanukar 2023]).

Selected labellar and tarsal sensilla in a few different mosquito species, including both hematophagous and non-hematophagous species, have been tested for responsiveness to sugars, bitter compounds, salts, amino acids, and water. Compounds in each of these categories are found in plant nectar, which is a food source for both sexes of hematophagous and non-hematophagous mosquito species (Lomelí and Dahanukar, 2022). Additionally, anthropophilic mosquitoes encounter sugars, salts, and free amino acids as human sweat and skin-associated tastants, as well as various components in blood. In Culex pipiens, hematophagous female-specific apical and sub-apical labral sensilla have been tested with tip recordings for responses to ATP, which is a blood component, and related nucleotides such as ADP, AMP, adenylyl-imidodiphosphate (AMP-PNP) and GTP (Liscia et al. 1993). In A. aegypti, calcium imaging has been used to examine neurons innervating these same sensilla for responses to blood, as well as individual components such as ATP, glucose, salt (NaCl) and sodium bicarbonate (NaHCO₃) (Jové et al. 2020). In the labellum, taste neuron groupings similar to those described in Drosophila were found in Culiseta inornata, with neurons responding to water, sugars, and salts at low and high concentrations (Pappas and Larsen 1976). Bitter sensitivity has also been explored in A. aegypti and Anopheles quadrimaculatus, and a taste neuron activated by bitter compounds is indeed found in labellar sensilla (Sanford et al. 2013, Sparks 2016). Moreover, as found in Drosophila (Meunier et al. 2009, French et al. 2015, Zhang et al. 2013), bitter compounds and salts inhibit sugar and water taste responses in mosquitoes (Kessler et al. 2013). The observed inhibition of neuronal activity is not a permanent effect, and the dampened activity of the neuron recovers when the inhibitory stimulus is removed and the activating stimulus is re-introduced to the tip of the sensillum. These neuronal properties can be correlated with behavior, exemplifying the importance of understanding how not only pure compounds but also mixtures are encoded by the insect taste system.

CONCLUSION

The external distribution of taste sensilla in insects makes them easily accessible for functional analyses, and, if body size is not a limiting factor, the physiological and behavioral activity of even a single sensillum can be measured (Minnich 1926, Dethier 1976). This feature presents a tremendous advantage for performing high-resolution system-wide analyses of peripheral taste sensitivity. Responses to diverse sets of chemicals have been mapped to taste neurons in individual sensilla in entire taste organs in Drosophila, which allows comparison of how chemicals are distributed in the “tastant space” of different organs. Clearly, similar analyses in anthropophilic mosquitoes would offer a glimpse into the similarities and disparities in taste coding in a blood-feeding dipteran as compared to the extensively studied phytophagous, or plant-feeding, Drosophila. Such analyses would also afford a comprehensive view of taste sensory input that can be evaluated for correlation with behavior.