**3.1 Gustatory receptors of** *Drosophila*

Like mammals, GRs in *Drosophila* also detect taste compounds. *Drosophila* utilize ion channels (Ionotropic receptors) to detect salts and sour (acid) compounds. Putative gustatory receptors in *Drosophila* and mammals were discovered almost simultaneously and detect sugars and bitter substances. Mammalian taste receptors belong to the large super family of GTP-binding (G) protein-coupled receptors (GPCRs), but fly GRs share no significant sequence similarity with them [4, 30–38]. A total of 68 *Gr* genes were found in *Drosophila* by analyzing the *Drosophila* genome database using algorithms that identify multi-transmembrane proteins or by performing reiterated Basic Local Alignment Search Tool searches with *Drosophila* olfactory receptor proteins as query sequences [4, 32, 38, 39]. The *Gr* genes are remarkably diverse having similarity between most receptor pairs only 20% or less

#### **Figure 2.**

*(A) Taste neurons send projections from the periphery (neurons from different taste sensilla L,I and S) including pharyngeal areas (LSO-labral sense organ, VCSO-ventral cibarial sensory organ, and DCSOdorsal cibarial sensory organ) to sub esophageal zone (SEZ) in the brain which is the main taste processing center. The taste information flow from SEZ to the antennal and mechanosensory motor center (AMMC). Mushroom body (MB), calyx and lateral horn (LH) are learning and memory centers. Antennal lobes (AL) get information from the olfactory neurons present on antennal surface in Drosophila. (B) GRNs from the legs of an adult fly (Red) send projections to SEZ and some GRNs project to thoracic ganglia only (shown in light blue). GRNs from the wing margins send axonal projections to thoracic ganglia (taste cells present in the wing shown in Green).*

**133**

TRPM5 [43].

receptors [48].

*3.2.1 Sweet receptors*

*Understanding Taste Using* Drosophila melanogaster *DOI: http://dx.doi.org/10.5772/intechopen.89643*

devoted to the detection of bitter-tasting and toxic compounds.

into their complex cellular expression [6, 7, 32, 38].

generating second messengers IP3, DAG and H+

channels leading to membrane depolarization [50].

**3.2 Types of taste receptors in mammals and** *Drosophila*

(at the amino acid sequence level). There are several gene clusters containing up to six genes, exhibit significantly higher similarity to each other (up to 70%). *Gr* genes with greater than 30% sequence similarity have been grouped into several subfamilies [32]. The domain that is most conserved among all *Gr* genes is located in the region encoding the putative seventh transmembrane domain at the carboxy terminus, a domain that is also shared with the olfactory receptors (ORs) genes this domain was used as a signature motif in one study that lead to the discovery of the fly taste receptors [32]. GRs within a subfamily detect structurally similar taste compounds. For example, the sugar receptor *Gr5a* subfamily consisting of *Gr5a* encoding a trehalose receptor [3, 40, 41] *Gr61a*, and *Gr64a*–*f* share sequence similarity in the range of 60% and detect diverse sugars [18]. Bitter compounds cover a vast chemically much more diverse structural space than sugars and majority of remaining GRs are

Well established GAL4/UAS system, transgenic expression methods helped visualizing the expression of various *Gr* genes [42]. *Gr* gene promoters drive the expression of the yeast transcription factor GAL4 (regulate genes induced by galactose) and GAL4 in turn activates transcription with high specificity via the cisregulatory element upstream activating sequence (UAS), cloned upstream of green fluorescence protein (GFP), or β-galactosidase reporter genes [42]. Expression analyses of *Gr* genes suggest that they are expressed in distinct subpopulations of GRNs, supporting their role as chemosensory receptors and providing first insights

The three tastes, bitter, sweet and umami taste are mediated by taste-specific GPCRs, which are expressed in distinct subsets of taste receptor cells in mammals [13]. These three taste employ a canonical G protein phosphoinositide-based pathway, where receptors activate a taste cell-specific G protein that activates PLCβ2,

to release Ca2+ from intracellular stores, and Ca2+ gates the membrane channel

*Drosophila* taste receptors bear no sequence relationship to mammalian taste receptors. The majority of bitter and sweet taste receptors in insects are members of a large protein superfamily of gustatory receptors [4, 32, 38, 39]. The 68 members of *Drosophila* taste receptors have seven transmembrane domains, but they share no sequence relationship to GPCRs. Rather, they are distantly related to *Drosophila* olfactory receptors (ORs), which have an opposite membrane topology from GPCRs and form ligand-gated ion channels [44–46]. Insect GRs including *Drosophila* have an inverted topology like ORs relative to GPCRs [47] and may form ionotropic

Highly concentrated sugars (100–500 mM), artificial sweeteners, and small number of sweet-tasting proteins elicit the sweet taste in mammals. The heterodimer of T1R2 and T1R3 constitutes the sweet receptor [49]. Animals also sense energy-rich foods and various sugars through a mechanism similar to that used by pancreatic β-cells to detect blood glucose [50]. According to this hypothesis, the metabolism of sugars by sweet cells produces ATP, which closes ATP-sensitive K+

Flies are attracted to many of the same sugars as humans [9, 51] although they respond most robustly to disaccharides (such as sucrose and maltose) and

. IP3 acts on the IP3 receptor (IP3R3)

*Understanding Taste Using* Drosophila melanogaster *DOI: http://dx.doi.org/10.5772/intechopen.89643*

*Animal Models in Medicine and Biology*

**3.1 Gustatory receptors of** *Drosophila*

(**Figure 2**) [24, 25].

of foods.

the esophagus and the digestive system. Peripheral labial palp GRNs, the internal sensillum, and some leg GRNs project their axons to the sub esophageal zone (SEZ), whereas the wing and a minority of leg GRNs project to the thoracic ganglion

A single MSN and several support cells are also present in the taste sensilla together with the GRNs (**Figure 1**) [26]. These MSNs translate mechanical forces into electrical signals and mediate hearing, positional awareness, and the coordination of movements [27, 28]. The MSNs sense the hardness and viscosity of food [29] similar to the ability of the human tongue to determine the consistency and texture

Like mammals, GRs in *Drosophila* also detect taste compounds. *Drosophila* utilize

ion channels (Ionotropic receptors) to detect salts and sour (acid) compounds. Putative gustatory receptors in *Drosophila* and mammals were discovered almost simultaneously and detect sugars and bitter substances. Mammalian taste receptors belong to the large super family of GTP-binding (G) protein-coupled receptors (GPCRs), but fly GRs share no significant sequence similarity with them [4, 30–38]. A total of 68 *Gr* genes were found in *Drosophila* by analyzing the *Drosophila* genome database using algorithms that identify multi-transmembrane proteins or by performing reiterated Basic Local Alignment Search Tool searches with *Drosophila* olfactory receptor proteins as query sequences [4, 32, 38, 39]. The *Gr* genes are remarkably diverse having similarity between most receptor pairs only 20% or less

*(A) Taste neurons send projections from the periphery (neurons from different taste sensilla L,I and S) including pharyngeal areas (LSO-labral sense organ, VCSO-ventral cibarial sensory organ, and DCSOdorsal cibarial sensory organ) to sub esophageal zone (SEZ) in the brain which is the main taste processing center. The taste information flow from SEZ to the antennal and mechanosensory motor center (AMMC). Mushroom body (MB), calyx and lateral horn (LH) are learning and memory centers. Antennal lobes (AL) get information from the olfactory neurons present on antennal surface in Drosophila. (B) GRNs from the legs of an adult fly (Red) send projections to SEZ and some GRNs project to thoracic ganglia only (shown in light blue). GRNs from the wing margins send axonal projections to thoracic ganglia (taste cells present in the wing* 

**132**

*shown in Green).*

**Figure 2.**

(at the amino acid sequence level). There are several gene clusters containing up to six genes, exhibit significantly higher similarity to each other (up to 70%). *Gr* genes with greater than 30% sequence similarity have been grouped into several subfamilies [32]. The domain that is most conserved among all *Gr* genes is located in the region encoding the putative seventh transmembrane domain at the carboxy terminus, a domain that is also shared with the olfactory receptors (ORs) genes this domain was used as a signature motif in one study that lead to the discovery of the fly taste receptors [32].

GRs within a subfamily detect structurally similar taste compounds. For example, the sugar receptor *Gr5a* subfamily consisting of *Gr5a* encoding a trehalose receptor [3, 40, 41] *Gr61a*, and *Gr64a*–*f* share sequence similarity in the range of 60% and detect diverse sugars [18]. Bitter compounds cover a vast chemically much more diverse structural space than sugars and majority of remaining GRs are devoted to the detection of bitter-tasting and toxic compounds.

Well established GAL4/UAS system, transgenic expression methods helped visualizing the expression of various *Gr* genes [42]. *Gr* gene promoters drive the expression of the yeast transcription factor GAL4 (regulate genes induced by galactose) and GAL4 in turn activates transcription with high specificity via the cisregulatory element upstream activating sequence (UAS), cloned upstream of green fluorescence protein (GFP), or β-galactosidase reporter genes [42]. Expression analyses of *Gr* genes suggest that they are expressed in distinct subpopulations of GRNs, supporting their role as chemosensory receptors and providing first insights into their complex cellular expression [6, 7, 32, 38].

#### **3.2 Types of taste receptors in mammals and** *Drosophila*

The three tastes, bitter, sweet and umami taste are mediated by taste-specific GPCRs, which are expressed in distinct subsets of taste receptor cells in mammals [13]. These three taste employ a canonical G protein phosphoinositide-based pathway, where receptors activate a taste cell-specific G protein that activates PLCβ2, generating second messengers IP3, DAG and H+ . IP3 acts on the IP3 receptor (IP3R3) to release Ca2+ from intracellular stores, and Ca2+ gates the membrane channel TRPM5 [43].

*Drosophila* taste receptors bear no sequence relationship to mammalian taste receptors. The majority of bitter and sweet taste receptors in insects are members of a large protein superfamily of gustatory receptors [4, 32, 38, 39]. The 68 members of *Drosophila* taste receptors have seven transmembrane domains, but they share no sequence relationship to GPCRs. Rather, they are distantly related to *Drosophila* olfactory receptors (ORs), which have an opposite membrane topology from GPCRs and form ligand-gated ion channels [44–46]. Insect GRs including *Drosophila* have an inverted topology like ORs relative to GPCRs [47] and may form ionotropic receptors [48].

#### *3.2.1 Sweet receptors*

Highly concentrated sugars (100–500 mM), artificial sweeteners, and small number of sweet-tasting proteins elicit the sweet taste in mammals. The heterodimer of T1R2 and T1R3 constitutes the sweet receptor [49]. Animals also sense energy-rich foods and various sugars through a mechanism similar to that used by pancreatic β-cells to detect blood glucose [50]. According to this hypothesis, the metabolism of sugars by sweet cells produces ATP, which closes ATP-sensitive K+ channels leading to membrane depolarization [50].

Flies are attracted to many of the same sugars as humans [9, 51] although they respond most robustly to disaccharides (such as sucrose and maltose) and oligosaccharides [8]. The fly sweet receptors belong to the same superfamily of receptors that includes most of the bitter receptors. In adult flies the three key receptors required for sensing sugars, except for fructose, are Gr5a, Gr64a and Gr64f [8, 40, 52–54]. These three receptors are co-expressed in the sugar-responsive GRNs in the labellum, along with five other related GRs that comprise the *Gr*-Sugar (*Gr*-S) clade [8, 52].

Gr5a and Gr64a sense structurally different sugars. Gr64a participates in the response to sucrose and maltose [8, 52], while Gr5a detect trehalose and melezitose [8, 40, 41]. Gr64f might act as a co-receptor for the responses for all sugars tested except fructose, and functions in concert with Gr5a and Gr64a [53]. Gr43a is the only receptor known to detect fructose [55].

#### *3.2.2 Bitter receptors*

Bitter taste allows animals to detect toxins in the environment and avoid consuming them. Compounds such as caffeine, cycloheximide (a protein synthesis inhibitor), denatonium (added to rubbing alcohol to discourage consumption), and quinine (a component of tonic water) taste bitter to humans, mice and flies. In vertebrates, bitter chemicals are detected by a small family of receptors (T2Rs), which are structurally related to rhodopsin, and range in number from 3 to 49, depending on the species [31, 34, 56]. In general, each bitter responsive taste receptor cell expresses multiple types of bitter receptors [57], but not all bitter receptors are expressed by every bitter cell [58], leading formally to the possibility that there are subclasses of bitter cells, as is the case in flies [59]. The chemical receptive field of the bitter receptors fall into two classes—"specialists" that detect one or a few bitter chemicals and "generalists" that detect many [60].

In contrast to vertebrate bitter detection, flies employ a much more complex strategy to sample bitter chemicals. In flies, bitter sensitive GRNs have distinct sensitivities. Based on the response profile to a panel of 16 bitter compounds, the L-, I- and S-type sensilla on the labella are classified into five groups, four of which are sensitive to bitter chemicals (**Figure 1**) [59]. Out of the four, two groups are narrowly tuned to distinct sets of bitter compounds (I-a and I-b). The other two groups respond broadly to bitter tastants but have variable patterns of activity (S-a, S-b). Analysis with a larger panel of bitter compounds may reveal more additional subgroups.

In flies, 33 out of 38 *Gr* genes that express in the labellum are found to be localized to bitter GRNs [59]. The roles of only a few of the bitter GRs have been dissected genetically so far. A minimum of 28 *Gr*s can be expressed by some GRNs in the labellum in adult fly. One of the larval GRN classes expresses at least 17 *Gr*s [59, 61]. Many GRs act as co-receptors responding to large numbers of aversive chemicals. *Gr32a*, *Gr33a* and *Gr66a* are needed for detection of most bitter chemicals [62, 63]. These three *Gr*s with additional *Gr*s (*Gr89a* and *Gr39a.a*) are expressed in all bitter responsive GRNs making this group of five GRs to be the "core-bitter GRs" [59]. Other GRs are very narrowly tuned and confer ligand specificity. These receptors are critical in defining the chemical specificity of the GRs, in combination with other GRs. Different combinations of complex sets of GR receptors may explain how a limited number of bitter GRs confer the capacity to respond to a vast collection of structurally diverse bitter compounds. Three TRP channels expressed in the labellum GRNs also contribute to the sensation of aversive compounds through mechanisms that are independent of GRs. TRPA1 show behavioral avoidance to aristolochic acid [64], a related TRPA channel Painless, is required for the behavioral avoidance to isothiocyanates (AITC;

**135**

*3.2.4 Sour taste*

ence from Na<sup>+</sup>

*Understanding Taste Using* Drosophila melanogaster *DOI: http://dx.doi.org/10.5772/intechopen.89643*

only the front of the tongue. Epithelial Na+

encoding salt taste has been proposed [77].

to low concentrations of salt [71].

sensitivity to camphor [66].

*3.2.3 Salt taste receptors*

and Li+

elicited by Na+

for Na+

wasabi) [65] and TRP-Like (TRPL) is both necessary and sufficient to confer

Moderate levels of salt is necessary to maintain the important physiological functions such as muscle contraction, action potentials and many other functions while excessive salt intake is deleterious and can lead to hypertension. Salty taste is

amiloride-insensitive. The amiloride sensitive component of salt taste is selective

trations of salts (<100 mM), and is generally appetitive [67]. Amiloride-sensitive salt taste occurs only in the front of the tongue [68]. Based on taste nerve recordings, there is a population of broadly tuned high-salt fibers that are insensitive to amiloride and activated by KCl and NaCl [69]. These fibers innervate both the front and back of the tongue, in contrast to the amiloride-sensitive fibers that innervate

three subunits—α, β and γ and α subunit is absolutely essential and forms part of the pore [70]. ENaC α has been suggested to be a component of the low salt sensor since a taste-cell specific knockout eliminates sensitivity and behavioral attraction

The cells that mediate the behavioral responses to high salts are not specifically dedicated to sensing high salt, but instead comprise at least two populations of cells with previously identified functions in sensing bitter and sour [72]. Inactivation of TRPM5 or PLCβ2, expressed by bitter cells, eliminates a component of the high salt response, while silencing PKD2L1-expressing sour cells eliminates the remaining components [72]. Remarkably, mice in which PKD2L1-expressing cells are silenced and TRPM5 is inactivated find high salt concentrations attractive, presumably due

Salt taste preferences in *Drosophila* are similar to those in mammals. Both larvae and adult fruit flies prefer low-salt foods, while they reject high-salt concentrations. Two ENaC channels family members, *ppk11* and *ppk19* are reported to be expressed in the terminal organ and required for sensing low salt [73] in *Drosophila* larvae. However, these channels do not appear to function in the salt response in adults [74]. A member of the ionotropic glutamate receptor (IR) family member, Ir76b, is required for low salt sensing in adult flies [74]. IRs were identified originally as a new class of olfactory receptor [75]. However, several IRs are also expressed in GRNs [76]. Ir76b is expressed in GRNs distinct from those that respond to sugars and bitter compounds, and the Ir76b GRNs extend their projections into a unique region of the SEZ [74]. Most recently combined activity of most of all GRN classes

Acidic pH and organic acids such as acetic acid evokes sour taste in the tongue. A subset of taste receptor cells in the tongue and palate epithelium that respond to acidic pH and weak organic acids with electrical activity detects the sour taste [78, 79]. PKD2L1-expressing cells respond are required for sensory response to acids [72, 78, 80] which is mediated by an unusual proton-selective ion channel [80]. Proton selectivity allows the cells to respond to acids without interfer-

, which may vary independently in concentration. The taste of

carbonation (CO2) is also detected by PKD2L1-expressing cells. This response is

over other monovalent cations such as K+

to activation of the amiloride-sensitive ENaC channels by high salt [72].

concentrations ranging from 10 to 500 mM. In humans, salt taste is

, is sensitive to low concen-

channels (ENaCs) are composed of

wasabi) [65] and TRP-Like (TRPL) is both necessary and sufficient to confer sensitivity to camphor [66].
