InteractiveFly: GeneBrief

Odorant receptor 22a and Odorant receptor 22b: Biological Overview | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

Animals are able to sense and discriminate among a remarkable number of odors. Olfactory information is received and encoded by olfactory receptor neurons (ORNs). These neurons encode the quality and intensity of odors, as well as aspects of their spatiotemporal distribution. The code is in the form of action potentials and is based on the differential responses of ORNs to different olfactory stimuli. The signals generated by ORNs are transmitted to the brain, where processing takes place (Dobritsa, 2003).

ORNs vary in their odor specificity, sensitivity, and response dynamics. The cellular basis of the odor code has been explored in detail in Drosophila, whose relatively simple olfactory system allows precise physiological measurements of individual ORNs in vivo. Flies contain two olfactory organs, the antenna and the maxillary palp, which contain ~1200 and ~120 ORNs, respectively (Stocker, 1994; Shanbhag, 1999, 2000). These ORNs are compartmentalized in olfactory sensilla, which divide into morphologically distinct classes, including large basiconic sensilla, small basiconic sensilla, trichoid sensilla, and coeloconic sensilla. Each sensillum contains up to four neurons, whose activities can be defined by extracellular electrophysiological recordings (Dobritsa, 2003 and references therein).

In Drosophila, extensive recordings have revealed that ORNs fall into distinct functional classes based on their odor response spectra. Sixteen functional classes of ORNs, each with a unique response spectrum to a panel of 47 odors, were identified from recordings of antennal basiconic sensilla (De Bruyne, 2001). These ORNs exhibit diverse response dynamics, including excitatory and inhibitory responses, and various modes of termination kinetics. The 16 ORN classes are found in stereotyped combinations in seven functional types of basiconic sensilla, each mapping to a defined subregion of the antennal surface (Dobritsa, 2003).

Functional differences among ORN classes are believed to arise from the expression of different odor receptors. A family of at least 60 seven-transmembrane-domain receptor genes, the Or genes, was discovered in Drosophila and proposed to encode odor receptors (Clyne, 1999a; Gao, 1999; Vosshall, 1999, 2000). Individual Or genes are expressed in different subsets of ORNs. A mutation that alters the expression of a subset of Or genes alters the odor specificity of a subset of ORNs (Clyne, 1999b), and direct evidence (Störtkuhl and Kettler, 2001; Wetzel, was recently found for the involvement of one Or gene in olfactory signaling (Dobritsa, 2003).

The isolation of Or genes and the functional identification of discrete ORN classes by physiological analysis now allows a critical problem to be explored in Drosophila: the integration of the molecular and cellular maps of the olfactory system. Three means of mapping the receptor repertoire to the neuronal repertoire are demonstrated in this study. Moreover, since the odor specificities of the neurons are defined, the results by extension map receptor space to odor space. This approach allows an integrated molecular and cellular definition of the basis of odor coding (Dobritsa, 2003).

Individual receptors are demonstrated to map to individual neuronal classes through a genetic and molecular analysis of two Or genes, Or22a and Or22b. There are three functional types of large basiconic sensilla, ab1, ab2, and ab3, defined on the basis of electrophysiological recordings from the ORNs they contain. The sensilla expressing Or22a and Or22b contain an A neuron whose strongest responses are to ethyl butyrate, pentyl acetate, and ethyl acetate and a B neuron whose strongest responses are to heptanone, hexanol, and octenol. The Or22a receptor is shown to map to the ab3A neuron, by using the Or22a promoter and the GAL4-UAS system to drive expression of GFP or the cell death gene reaper, followed by physiological recordings from individual sensilla. The Or22a receptor is thereby linked to the odor ethyl butyrate, to which ab3A is highly sensitive (Dobritsa, 2003).

Analysis of a mutant lacking Or22a, together with rescue experiments using an Or22a transgene, confirm the mapping of Or22a to the ab3A neuron. This genetic analysis provides direct evidence that an Or gene is required in vivo for normal odor detection (Dobritsa, 2003).

Evidence is provided that Or22b is coexpressed with Or22a in the same cell but that Or22b is neither necessary nor sufficient for ab3A function; rather, the broad response spectrum of the ab3A neuron is accounted for by a single receptor. Ectopic expression of another receptor, Or47a, in the mutant ab3A neuron is used to identify the ORN from which Or47a derives and to determine its odor specificity. These results show that the odor response spectrum of an ORN in Drosophila depends on the Or gene that it expresses. Expression of Or47a in a wild-type ORN shows that two receptors are able to function in a single cell (Dobritsa, 2003).

Finally, the possibility of a developmental role for Or genes is addressed, a possibility that has not been systematically investigated by expression analysis and that can be rigorously determined only by functional analysis. The ab3A ORN is able to navigate toward its target in the CNS if Or22a and Or22b are deleted or substituted by other receptor genes (Dobritsa, 2003).

The demonstration that deletion mutants lacking Or22a and Or22b are defective in odor response and that the response is restored upon introduction of an Or22a transgene provide direct evidence that Drosophila Or genes are in fact critical components of olfactory signal transduction. The effect of the deletion mutation is specific: the mutation has a profound effect on the ab3A neuron but no other ORN among the large basiconic sensilla. The response of the ab3A cell is eliminated for all odors tested. Most ab3A neurons in mutant flies exhibited spontaneous electrical activity, albeit at a low rate, and thus the absence of Or22a/b does not lead to the immediate death of these neurons. The loss of Or22a/b does not appear to lead to the de novo expression of another functional receptor, suggesting that the process of receptor gene choice does not include a receptor-mediated negative feedback mechanism (Dobritsa, 2003).

A central problem in odor coding concerns the distribution of odor receptors among ORNs. A priori, there are several ways of distributing n receptor types among m functional classes of ORNs. Each receptor type could be expressed in a single ORN class, or, by contrast, in multiple, distinct ORN classes. If expressed in a single ORN class, then a receptor, R_i, could in principle be the only receptor expressed in its class, or it could be one of multiple receptors, e.g., (R_i, R_j), that are invariably coexpressed in that class. If a receptor is expressed in multiple, distinct ORN classes, then the ORN classes may be functionally distinct either because they express different combinations of receptors, e.g., (R_i, R_j) versus (R_i, R_k), or, conceivably, because each expresses the same receptor in different molecular contexts, (R_i; X) versus (R_i; Y), containing ORN class-specific differences in local OBPs, RAMPs, or heterodimerization partners, for example (Dobritsa, 2003).

The expression of the Or22a receptor was found to be limited to a single morphological subtype of olfactory sensillum (LB-I), a single functional type of sensillum (ab3), and a single class of ORN (ab3A). The mapping of Or22a to a single functional type of neuron argues against a model in which different neuronal classes acquire their diverse identities through the combinatorial expression of different receptors, or through the expression of a single receptor in different molecular contexts; according to these models, an individual receptor would be expressed in multiple, distinct neuronal classes (Dobritsa, 2003).

ORNs vary in the breadth of their odor response spectrum. Physiological recordings from individual ORNs have shown that some are narrowly tuned, whereas others are broadly tuned with respect to a panel of test odors (De Bruyne, 1999, 2001). For example, the ab5A cell responds to only one of 47 odors tested at relatively high doses, whereas the ab3A neuron responds to a variety of esters, alcohols, ketones, and other odors of varying chain lengths. The broad tuning specificity of ORNs such as ab3A could in principle be due either to the expression of multiple receptors or to the expression of a single receptor that is broadly tuned. The finding that deletion of Or22a and Or22b eliminates response to all tested odors, and that the full response spectrum can be rescued by Or22a alone, suggests that the broad response spectrum documented for ab3A can be attributed to one receptor, Or22a (Dobritsa, 2003).

The results with Or47a are also consistent with a model in which a single receptor accounts for the odor response profile of a particular ORN class. When expression of a single Or gene, Or47a, is driven in an ab3A cell lacking expression of Or22a and Or22b, Or47a confers an odor response profile like that of ab5B. These results support an interpretation in which the odor response profile of ab5B derives from the expression of a single Or gene, Or47a. Functional analysis of other Or genes will be required to determine the generality of these results, but they are consistent with observations made with mammalian ORNs, which are able to respond to diverse odors, apparently by virtue of expression of a single odor receptor in many (Malnic, 1999; Araneda, 2000; Bozza, 2002), if not all (Rawson, 2000; Spehr, 2002), cases (Dobritsa, 2003).

While these results are consistent with a model in which Or22a is the only receptor that functions in the ab3A neuron, the data force consideration of the possibility that Or22a is not the only receptor that is expressed in the ab3A neuron. Both Or22a-GAL4 and Or22b-GAL4 drive expression of GFP in ab3 sensilla and of rpr in ab3A neurons. The driver constructs were designed to mimic the expression of the endogenous Or22a or Or22b genes, respectively. In each construct, GAL4 coding sequences replace the coding sequences of the respective Or genes, and they include a substantial amount of DNA upstream of either Or22a or Or22b (8.2 and 10.3 kb, respectively). While it is formally possible that the expression of Or22b-GAL4 does not mimic the expression of Or22b in vivo, there is no evidence for expression of Or22b in ORNs of the fly other than ab3A: (1) in situ labeling with Or22b probes revealed no labeling outside the region of the antenna that contains ab3 sensilla; (2) immunolabeling showed no staining of sensilla other than LB-I in the wild-type antenna, nor the maxillary palp nor the larval olfactory organ. At the same time, it is clear that the antibody can recognize an Or22b product, since immunolabeling is observed in antennae lacking Or22a but expressing Or22b. Moreover, Or22b transcripts have been amplified from the antenna by RT-PCR or found in antennal/maxillary palp cDNA libraries in multiple laboratories (Clyne, 1999a; Vosshall, 1999). The simplest interpretation of all these data, taken together, is that Or22b is coexpressed with Or22a in ab3A neurons, but that Or22b is neither necessary nor sufficient for response to the odors used in this study (Dobritsa, 2003).

A functional role for Or22b is nonetheless suggested by the observation that an Or22b ortholog is present in D. simulans, which diverged from D. melanogaster ~2.5 million years ago. Most important, in neither species has the gene accumulated stop codons, frameshift mutations, or deletions. One possibility is that Or22b recognizes odors not tested in this study, such as pheromones. Another possibility is that it functions only under a specific set of epigenetic, e.g., environmental, conditions (Dobritsa, 2003).

Does the odor response spectrum of a cell depend exclusively on its receptor expression or on a more complex molecular context? In the case of Or47a, the substitution of Or47a for Or22a and Or22b engenders a transformation of the response spectrum from that of ab3A to that of ab5B. These results are consistent with evidence from other organisms that the odor response spectrum of an ORN depends on the odor receptor gene that it expresses (Dobritsa, 2003).

ORNs in insects are intimately associated with each other in sensillar compartments. In this study the activity of an ORN was measured following genetic manipulation of its neighbor. When the function of ab3A was severely compromised, either by mutation of Or22a and Or22b or by expression of the cell death gene rpr, the neighboring ab3B cell showed strong responses to odors. Thus, the ability of ab3B to respond to odors does not depend absolutely on the presence of a functional neighboring cell (Dobritsa, 2003).

At the same time, however, in both cases a large increase was noted in the response of ab3B to pentyl acetate. It is formally possible that this effect may arise to some extent from difficulties in counting the small ab3B spikes during intense activity of the ab3A cell. However, another interpretation is that in wild-type the activity of ab3A inhibits the response of ab3B; when this inhibition is relieved, ab3B exhibits an increased response. In some insects, neighboring ORNs in certain sensilla have been shown to respond to odors whose behavioral significance is related (Wojtasek, 1998; Grant, 1998). It seems plausible that information transmission between adjacent ORNs represents an early step in the processing of information carried by neighboring ORNs (Dobritsa, 2003 and references therein).

An emerging body of evidence in mammals indicates that odor receptor expression is essential to normal axonal pathfinding in the vertebrate olfactory system. The results of this study indicate, however, that the ab3A neuron finds its normal glomerular target in a mutant that lacks Or22a and Or22b expression, demonstrating that these receptors are not required for targeting. Moreover, ectopic expression of either of two other receptors did not cause alterations in the targeting. The possibility that Or22a and Or22b play a subtle role cannot be excluded, or that ab3A expresses an additional Or gene that plays a role in pathfinding, such as Or83b, which is expressed widely among ORNs (Vosshall, 1999, 2000) and whose function is unknown. However, the simplest interpretation of these results is that ab3A finds its glomerular targets through mechanisms independent of Or expression (Dobritsa, 2003).

One striking difference between the insect and mammalian olfactory systems is that in mammals, but not insects, ORNs are regenerated throughout adult life. Thus, pathfinding of mammalian ORNs toward their target glomeruli occurs during adult life, whereas in insects axonal pathfinding occurs only during development. Insect ORN pathfinding likely depends on a system of navigational cues that are expressed in an orchestrated temporal and spatial program. Perhaps the mammalian dependence on Ors, which are expressed both during development and during adult life, reflects the evolution of a mechanism designed to operate independently of signals that occur only transiently in development (Dobritsa, 2003).

A second difference between insect and mammalian olfactory systems is the greater numerical complexity of mammals. The number of ORNs and glomeruli in mammals exceeds that of Drosophila by more than an order of hmagnitude, and it is likely that the mammalian olfactory system accordingly requires more information to specify the larger number of connections. Perhaps the use of extant Ors to provide developmental cues may have been the most economical means of expanding the informational content of the navigational system during evolution (Dobritsa, 2003).

Wild african Drosophila melanogaster are seasonal specialists on marula fruit

Although the vinegar fly Drosophila melanogaster is arguably the most studied organism on the planet, fundamental aspects of this species' natural ecology have remained enigmatic. This study has investigated a wild population of D. melanogaster from a mopane forest in Zimbabwe. These flies are closely associated with marula fruit (Sclerocarya birrea), and it is proposed that this seasonally abundant and predominantly Southern African fruit is a key ancestral host of D. melanogaster. Moreover, when fruiting, marula is nearly exclusively used by D. melanogaster, suggesting that these forest-dwelling D. melanogaster are seasonal specialists, in a similar manner to, e.g., Drosophila erecta on screw pine cones. It was further demonstrated that the main chemicals released by marula activate odorant receptors that mediate species-specific host choice (Or22a) and oviposition site selection (Or19a). The Or22a-expressing neurons-ab3A-respond strongly to the marula ester ethyl isovalerate, a volatile rarely encountered in high amounts in other fruit. Or22a differs among African populations sampled from a wide range of habitats, in line with a function associated with host fruit usage. Flies from Southern Africa, most of which carry a distinct allele at the Or22a/Or22b locus, have ab3A neurons that are more sensitive to ethyl isovalerate than, e.g., European flies. Finally, the possibility is discussed that marula, which is also a culturally and nutritionally important resource to humans, may have helped the transition to commensalism in D. melanogaster (Mansourian, 2018).

The vinegar fly Drosophila melanogaster displays preference toward certain fruit and strongly favors citrus for egg laying. The presence of a distinct host partiality is intriguing and implies that D. melanogaster during its evolutionary history likely has had a close association with a specific fruit, or group of fruit, with characteristics akin to citrus. This ancestral host is, however, likely not found among members of the Asian genus Citrus, but rather among fruit found within the Miombo and Mopane forests of the fly's predicted Urheimat in Southern Africa, more precisely in present day Zimbabwe and Zambia, and displays physical and chemical properties that fit with the known preference of D. melanogaster. In brief, marula has a thick rind similar to that of citrus, which encloses a sugary (and highly fermentable) juicy pulp, with a pH similar to that of orange, features all favored by D. melanogaster. Marula emits terpenes and esters, which in terms of total emission contribution, as well as in numbers, are the primary chemical components, as determined via gas chromatography-mass spectroscopy analysis of headspace collections. The two main chemicals, ethyl isovalerate (an ester) and β-caryophyllene (a sesquiterpene), together make up ~55% of the headspace. Both terpenes and esters are known to be important and ecologically relevant olfactory cues for D. melanogaster. In short, marula fulfills the criteria on essentially all counts and is accordingly a good candidate ancestral host (Mansourian, 2018).

Do flies from native habitats then use marula? To answer this question, an expedition was mounted to Southern Africa in search of forest-dwelling D. melanogaster and marula. Specifically, mopane woodlands of the Matopos national park in Southwestern Zimbabwe, a site situated within the predicted ancestral range, was searched. The Matopos covers 424 km², hosts no permanent human habitation, and is covered in Mopane and kopje woodlands (Mansourian, 2018).

Once in the Matopos, marula trees, as well as fruiting trees with fermenting fruit below, were localized. among which fly traps baited with marula were placed. Over the next days, these traps caught numerous D. melanogaster. Traps placed under an additional 5 marula trees yielded another 67 D. melanogaster specimens. At all examined sites, though, D. simulans outnumbered D. melanogaster. These flies will be referred to as 'wild,' in line with their presence in undisturbed wilderness, with the caveat that their ultimate origin remains unknown (Mansourian, 2018).

The forest flies were provided with a choice of marula versus orange, the favorite breeding substrate of domestic D. melanogaster. Paired traps, containing either marula or orange, were placed under a fruiting marula tree. Similar to the laboratory strain, the wild D. melanogaster showed a strong preference for marula. Interestingly, though, D. simulans displayed no such preference, indicating that the marula preference is exclusive to D. melanogaster and, moreover, that marula is not simply overall a more suitable fruit resource to Drosophila spp. Marula was dissected in search of fly eggs and larvae, and in all fruit examined, drosophilid larvae were located, from which D. melanogaster adults later emerged. In short, wild African D. melanogaster are drawn to the odor of marula, prefer marula to orange, and use marula as breeding substrate (Mansourian, 2018).

To investigate the general distribution of D. melanogaster in the Matopos, traps (baited with fermenting marula) were placed at five locations with no fruiting marula trees nearby, but with otherwise similar vegetation (including other fruiting trees). Strikingly, D. melanogaster was absent, or very sparse, in traps at these locations. On the other hand, D. simulans was as abundant at sites with marula as it was in sites without. The distribution pattern of D. melanogaster in the Matopos hence indicates niche confinement and, in turn, a specialized lifestyle. D. melanogaster as a seasonal fruit specialist would actually not be surprising given. (2) the observed presence of a distinct egg-laying preference, and (3) the fact that host specialization is a prevalent feature in the melanogaster subgroup. Drosophila sechellia exclusively breeds in noni fruit, whereas Drosophila erecta and Drosophila orena are seasonal specialists on Pandanus cones and Syzygium waterberries respectively. Drosophila teissieri is closely associated with Parinari fruit, which limits its geographic range. A nonrandom subset of olfactory genes is associated with host preference in the fruit fly Drosophila orena, whereas Drosophila santomea is found with figs from Ficus clamydocarpa trees. Thus, seasonal host specialization in D. melanogaster would fall into the pattern displayed by most (if not all) of its close relatives. Outside of marula season, these forest flies may go into diapause, much like they do in temperate regions, or switch to opportunism, utilizing alternate breeding substrates. One such alternative could be figs, which are present year-round in the Matoposand in terms of biomass are even more abundant than marula. D. melanogaster has moreover been reared from figs in Africa, which are also an alternate host for the seasonal specialist D. erecta outside of Pandanus season (Mansourian, 2018).

Wild African D. melanogaster hence not only utilize marula for parts of the year, marula appears to be exclusively utilized. It was asked how domestic flies react to this fruit. To this end, a two-choice assay to examine egg-laying preference in Canton-Special (Canton-S) wild-type flies. The Canton-S strain was established sometime before 1916 from a population in Canton, Ohio, well outside the sub-Saharan range of marula. The citrus preference of these flies was verified in the oviposition assay. Given a choice between orange and banana, the flies clearly preferred citrus as oviposition substrate. Having confirmed the assay, orange versus marula was tested, and indeed, flies provided this choice strongly preferred marula, similar to Wild African D. melanogaster. The ancestral marula preference is accordingly conserved in non-African flies (Mansourian, 2018).

Which chemicals then mediate the marula preference? The same two-choice assay was used and the major chemical components of the headspace were tested individually. Previous work has shown that fly food spiked with terpenes confers positive egg-laying site selection, and thus the main terpene (β-caryophyllene), which as expected generated preferential oviposition, was tested. The main ester component, ethyl isovalerate, also conferred oviposition preference, as well as attraction in a T-maze assay. The preference of marula over orange may hence be mediated by the high presence of esters in the former. In line with this reasoning, flies provided with a choice of orange spiked with ethyl isovalerate against marula failed to make a choice (Mansourian, 2018).

In D. sechellia and D. erecta, host specialization is linked to the Or22a circuit, which in both species is activated by distinct esters from the respective hosts. It was thus asked whether the primary marula ester ethyl isovalerate also activates Or22a-expressing olfactory sensory neurons (OSNs) in D. melanogaster. To investigate this issue, functional imaging of the antennal lobe was performed in flies expressing the calcium reporter GCaMP6m. Stimulation with ethyl isovalerate yielded strong calcium signals in the DM2 glomerulus (the target of the Or22a-expressing OSNs) already at 10^-7 dilution. In line with its chemistry, marula odor also triggered strong Ca²⁺ signals from DM2, whereas orange odor triggered weak to no activity from the same glomerulus. Thus, similar to its specialized siblings, the main ester from the preferred host activates Or22a. Silencing of the Or22a pathway via Or22a-Gal4>UAS-TNT did not, however, abolish the marula oviposition preference, suggesting that additional pathways are involved in this behavior. Rather than mediating egg-laying preference, the primary function of Or22a may instead be locating the host over distance. Hence, up-wind flight navigation toward marula of flies with Or22a silenced (via Or22a-Gal4>UAS-TNT) was examined in a wind tunnel assay. Flies with non-functional Or22a input showed a reduced ability to localize marula compared to control flies, suggesting that these neurons' predominant function is to assist the fly in locating its host over distance. The importance of these neurons in this context is also evident from D. sechellia, which has a numerical increase of Or22a-expressing OSNs, which likely affords an improved ability to find noni fruit over distance (Mansourian, 2018).

Since marula is restricted to sub-Saharan Africa, most D. melanogaster have to make do with alternative hosts. If Or22a indeed is linked to the specific chemistry of the host, local adaptation of the Or22a locus would be expected between D. melanogaster populations from diverse environments that may utilize disparate hosts. Thus, local genetic differentiation (as indexed by F_ST was estimated within the OR family between genomes from 10 African populations, plus one European. For each window centered on an olfactory receptor gene, the F_ST quantile was evaluated for each pairwise population comparison (the proportion of all windows on the same chromosome arm that showed stronger allele frequency differences [higher F_ST]) between these same two populations. The Or22a locus, and the adjacent tandem paralog Or22b, shows striking genetic differentiation between almost all population pairs, in stark contrast to most of the other ORs, for which little or no sign of local adaptation can be discerned (Mansourian, 2018).

In cases where other ORs did show strong F_ST outliers (quantiles < 0.0001), differentiation in one or a few populations was often most apparent. These genes included Or33a, Or65b, and Or67a. Interestingly, these receptors also appear to have important functions. Or33a has unknown function, but like Or22a, it shows variable expression across species and has undergone serial duplication in Drosophila suzukii and Drosophila biarmipes. Or65b is expressed in pheromone-sensing neurons, but its function has not been established. In short, unlike most members of the OR family in D. melanogaster, Or22a (and its closely linked paralog, Or22b) shows strong signs of local adaptation, in line with a function associated with host-specific chemistry (Mansourian, 2018).

At the molecular level, Or22a (and Or22b) thus differs between populations, but does this local differentiation also translate into functional changes in the ab3A neurons where these genes are expressed? The most conspicuous alteration among the investigated populations in the Or22a/Or22b locus is a deletion allele, whereby a segment stretching from the second exon of Or22a to the start of the second exon of Or22b has been deleted, generating a chimeric receptor, Or22ab. In light of the chimeric appearance of Or22ab, this variant appears to be a derived deletion (following a more ancient duplication to create these paralogs), rather than a representation of the ancestral state of the Or22 locus (Mansourian, 2018).

The data support a prior suggestion that the Or22ab fusion variant is quite ancient. This variant is at a very high frequency within the ancestral range (e.g., 88% in Zambia). Nucleotide diversity of flanking sequences, which should accrue on the order of 4 N_e ~ 10 million generations in this species, is at or above typical levels among Zambia haplotypes carrying this deletion. Hence, it is likely that the fusion variant existed well before the species expanded beyond its ancestral range on the order of 150,000 generations ago, or ~10,000 years ago. In contrast, putatively ancestral full-length Or22a/Or22b haplotypes from Zambia show strongly reduced diversity across the deletion region. This pattern could reflect a low long-term population size of the full-length allele, in accordance with its current rarity in the ancestral range. In some populations, such as in Europe or the Ethiopian highlands, the full-length allele has become predominant. Many of these haplotypes show identical or nearly identical sequences, in line with prior evidence for positive selection linked to the Or22a/Or22b haplotype in Europe. It is noted that some populations with similarly high frequencies of the fusion variant are strongly differentiated from each other at the Or22a/b locus, which could imply either parallel increases of the fusion variant on distinct haplotypes or additional variants under spatially varying selection at this locus (Mansourian, 2018).

Consequently, most D. melanogaster in Southern Africa will likely carry the Or22ab allele, which prompts the question: do their ab3A neurons respond to the marula ester? A strain in which Or22ab is fixed (RG18N) was selected, and single-sensillum recordings (SSRs) were performed. Measurements from ab3A neurons revealed strong responses to stimulation with ethyl isovalerate. The ab3A neurons in RG18N actually responded more strongly to ethyl isovalerate than to ethyl hexanoate-the primary ligand of Or22a, where ethyl hexanoate yielded a stronger response than ethyl isovalerate. In short, African D. melanogaster not only detect ethyl isovalerate, but also are even more sensitive to this marula compound than flies from outside Africa. It is noted that the distribution of populations with a high frequency of Or22ab overlaps with the distribution of marula. However, whether the Or22ab allele is an adaptation toward marula remains to be shown. Heterologous expression and detailed functional characterization of this interesting receptor variant will be a topic for future studies (Mansourian, 2018).

The Matopos is best known for its elaborately painted caves-made by now-vanished San tribes during Late Pleistocene to Early Holocene. For these tribes, marula played a pivotal role, and archeological excavations of their cave homes have uncovered enormous quantities of marula stones. From the Pomongwe cave alone, remains of at least 24 million marula stones were recovered, which only represents the carbonized remains, and hence but a fraction of the marula that must have once been brought into this cave. The San evidently spent considerable time collecting and processing marula, which would have been the staple food item during many months of the year. Thus, just like D. melanogaster, these San tribes appear to have been seasonal specialists on marula as well (Mansourian, 2018).

The marula-San link offers a plausible scenario by which D. melanogaster became a human commensal. The smell of the stored marula emanating from the caves would have attracted flies from far and wide. Flies would have found a steady supply of marula and fermenting leftovers inside the caves, long after the fruit's presence in the surrounding woodlands had diminished. In other words, the time frame for using the optimal breeding substrate would have been increased considerably. Inside the caves, the flies would also have benefitted from a reduced risk of predation, as well as protection from adverse weather conditions. Over time, the cave flies would have accumulated adaptations helpful for human commensalism. Relevant traits may have included a willingness to enter darker enclosures and an increased tolerance of ethanol, both of which differentiate D. melanogaster from its closest relatives. Thus, it was asked whether D. melanogaster actually enter these caves. To this end, four traps baited with fermenting marula wkere placed along the far wall of the Nswatugi cave. Over three days, these traps caught a number of D. melanogaster specimens, but no D. simulans, in contrast to the closest traps (n = 3) placed under fruiting marula trees outside the cave, where D. simulans greatly outnumbered D. melanogaster (Mansourian, 2018).

The archeological record indicates that systematic and intensive marula use began ~12,000 years ago. At ~9,500 years ago, marula harvesting reached massive proportions, finally ebbing out ~8,000 years ago . These dates coincide with demographic data from D. melanogaster, which point to a within-Africa expansion starting ~10,000 years ago, an expansion presumably representing the dispersal of the commensal population throughout its new niche. In short, archeological and demographic data would support the notion that marula use by the San may have been a factor in turning the woodland species D. melanogaster into the cosmopolitan species of today (Mansourian, 2018).

This study has demonstrated that D. melanogaster from a mopane forest within the predicted ancestral range are seasonal specialists on marula fruit. The odor of this seasonally abundant and widely distributed fruit activates select key odorant receptors previously implicated as having particular importance to D. melanogaster, and it is argued that marula is the ancestral primary host of the fly. Flies from sub-Saharan Africa were shown to carry a specific allele of one of these odorant receptors and are also more responsive to a key marula chemical. Finally, it is speculate that the marula specialization might have been important in driving commensalism (Mansourian, 2018).

The finding of a woodland population of D. melanogaster within the ancestral habitat opens up a range of interesting questions to be addressed. For example, how do these flies differ from their commensal relatives, i.e., which genetic factors underlie this shift in lifestyle? The finding that D. melanogaster appears to have a close association with a single host fruit will furthermore facilitate studies relating to host specific chemosensory adaptations, which so far have had to be conducted in other insects in which the wealth of tools available in D. melanogaster are unavailable (Mansourian, 2018).

DEVELOPMENTAL BIOLOGY

Many of the Drosophila OR genes are expressed in the antenna and not in the maxillary palp. The 47E.1 probe hybridizes to 40 cells in a broad area across the antenna, including both anterior and posterior faces, similar to the distribution pattern of small sensilla basiconica. A probe from the 25A.1 gene hybridizes to fewer cells (16) but in a region of the antenna similar to that of 47E.1 staining, as judged by reconstruction of serial sections. The 22A.2 probe hybridizes to 22 cells in a different distribution, clustered in the dorsomedial region of the antenna. This pattern matches the distribution of the large sensilla basiconica (Clyne, 1999a).

Demonstrating the expression of a single receptor gene in individual sensory neurons within the antenna is difficult since this olfactory organ expresses 32 members of the DOR gene family in ~1000 sensory cells. The lateral distal domain of the antenna contains about 300 neurons, 50 of which express Or47b. This same domain expresses 15 other receptors, including Or47a, Or23a, Or67a, Or43a, Or88a, Or49b, Or98a, and Or83c. In pairwise experiments, antennal sections were annealed with an Or47b antisense RNA probe and either individual or mixed probes for these eight additional receptors. RNA probes were labeled with either fluorescein or digoxigenin and visualized with antibodies that distinguish the two types of probes. Or47b is expressed in a subpopulation distinct from that expressing any of these eight receptors. A similar experiment was performed to examine DOR gene expression in the medial proximal region of the antenna. Or22a probes were labeled with fluorescein and a mix of probes complementary to five additional genes expressed in this domain (Or7a, Or56a, Or85b, Or42b, and Or59b) was labeled with digoxigenin. Despite the interspersion of Or22a cells with cells expressing other DOR genes in the domain, there is no overlap in the expression of Or22a with any of these five DOR genes. Taken together, these data provide strong support for a model reminiscent of the mammalian olfactory system, in which individual sensory neurons express only a single receptor. This conclusion must be tempered by the previous description of an odorant receptor gene, Or83b, that is expressed in all olfactory sensory neurons in both the antenna and maxillary palp. If Or83b does indeed recognize odorous ligands, then this data would indicate that all sensory neurons express two receptors rather than one: Or83b and one additional gene from the family of DOR genes (Vosshall, 2000).

These experiments suggest that there are 39 distinct neuronal cell types within the Drosophila olfactory organs. In the mammalian olfactory system, neurons expressing the same receptor project their axons to two spatially invariant glomeruli. It was therefore determined whether cells expressing a given receptor in Drosophila converge upon spatially defined loci in the antennal lobe, the first relay station for olfactory information in the fly brain. Axons from the antenna and maxillary palp have been found to synapse with the dendrites of projection neurons in the antennal lobe. There are 43 morphologically distinct synaptic structures or glomeruli in the antennal lobe that are invariant in position and size in individual flies. The number of antennal lobe glomeruli dedicated to olfactory input (41) approximates the number of different sensory neurons identified in the two olfactory organs (39), suggesting that olfactory neurons expressing a given receptor may converge on a single glomerulus (Vosshall, 2000).

Genetic experiments were performed that permit the visualization of the axonal projections from neurons expressing a given receptor. Transgenic flies were generated in which DOR gene promoters direct the expression of the yeast transcriptional activator, Gal4. These flies were then crossed with stocks bearing a transgene in which the Gal4-responsive promoter, UAS, drives the expression of either beta-galactosidase (LacZ) or a C-terminal fusion of green fluorescent protein (GFP) to neuronal synaptobrevin (nsyb-GFP). The expression of LacZ in specific subpopulations of sensory neurons allows the visualization of cell bodies, whereas the expression of nsyb-GFP, which selectively labels synaptic vesicles in nerve terminals, allows the visualization of terminal axonal projections (Vosshall, 2000).

Two to eight kilobases of DNA immediately upstream of the putative translation start from five DOR genes were fused to the coding sequence of Gal4. To demonstrate that these transgenes recapitulate the expression of the endogenous gene, the DOR-Gal4 strains were crossed with UAS-lacZ responders. The progeny of these crosses express LacZ in spatially defined subsets of cells in the antenna or maxillary palp that mirror the pattern of expression of the endogenous receptor. To confirm that these cells are sensory neurons, it was demonstrated that cells expressing LacZ are also labeled with an antibody that recognizes the neuron-specific RNA binding protein Elav. As a further test for the fidelity of expression of the promoter fusions, it was demonstrated that all cells expressing LacZ from the transgenes also express the corresponding endogenous odorant receptor RNA. In these experiments, RNA in situ hybridization was used to detect endogenous receptor RNA; immunofluorescence localized the expression of LacZ protein. Each of five DOR promoter fusions recapitulates the pattern of expression of the endogenous receptor gene (Vosshall, 2000).

The DOR-Gal4 transgenes were used to drive the expression of UAS-nsyb-GFP to visualize the projections of different populations of sensory neurons. Flies carrying five different DOR-Gal4 transgenes were crossed with animals bearing a UAS-nsyb-GFP transgene, and the brains of the adult progeny were then examined for localization of GFP. Immunofluorescence was performed on whole-mount brain preparations with antibody directed against GFP and with the monoclonal antibody, nc82, which labels neuropil in the fly brain and identifies the individual glomeruli in the antennal lobe. Four DOR-Gal4 promoter fusions (Or47a, Or47b, Or22a, and Or23a) are expressed in subpopulations of antennal neurons. When these flies are crossed with flies carrying the UAS-nsyb-GFP transgene, GFP labeling is seen in spatially invariant subsets of glomeruli within the antennal lobe. Or47a, for example, is expressed in 20 lateral distal neurons and their axonal projections converge on a single bilaterally symmetric glomerulus located at the dorsal medial limit of the antennal lobe. Or47b is expressed in 50 lateral distal neurons and these cells project axons that converge on a single large glomerulus that lies at the ventral lateral edge of the antennal lobe. Neurons expressing Or22a converge upon one dorsal glomerulus. The projections of one of the seven different subpopulations of sensory neurons from the maxillary palp have also been visualized. Sensory neurons expressing the palp receptor, Or46a, project to a single glomerulus that resides ventrally in the antennal lobe (Vosshall, 2000).

These four subpopulations of olfactory sensory neurons therefore project axons to single spatially invariant, bilaterally symmetric glomerulus within the antennal lobe. Cells expressing Or23a, however, send processes to two glomeruli. Or23a fibers enter the brain and initially converge upon a small dorsal glomerulus. Axons are seen extending more ventrally into the anterior of the antennal lobe where they converge upon a larger glomerulus. These two Or23a glomeruli are bilaterally symmetric and positionally invariant in multiple independent transgenic lines. It is not possible at present to determine whether individual fibers branch to form synapses within the two glomeruli or if Or23a-expressing neurons sort such that individual subpopulations project to either one of the two glomeruli (Vosshall, 2000).

The topographic map of all five populations of olfactory sensory projections in the antennal lobe is unrelated to spatial domains of receptor expression in the antenna. For instance, while the 20 neurons that express Or22a in a proximal medial domain of the antenna converge upon a dorsal medial glomerulus, the Or47a glomerulus is situated directly adjacent to the Or22a glomerulus but receives input from cells in the lateral distal domain of the antenna. These precise patterns of glomerular convergence were observed in at least 20 individuals derived from multiple independent transgenic lines obtained for each of the four constructs. At a low frequency (1/100 flies), Or47a-expressing olfactory neurons project to two bilaterally symmetric medial glomeruli in addition to the OR47a glomerulus. The basis for this variation in axon targeting is unknown (Vosshall, 2000).

The relationship between Or expression and sensillum type

In order to test the hypothesis that Or22a and Or22b encode odor receptors, and to examine the relationship between Or expression and sensillum type, the cellular and subcellular distribution of the Or22a and Or22b proteins were examined. For this purpose a polyclonal antibody was raised against a 16 amino acid sequence common to the N terminus of both proteins; this region was selected because it was predicted by bioinformatic analysis to generate effective antibodies. If Or22a and Or22b are in fact odor receptors, they would be expected to be localized to the dendritic membranes of ORNs. It was of interest to determine whether the labeled sensilla all belonged to the same morphological type of olfactory sensilla. In situ hybridization with Or22a and Or22b probes show labeling of cells in the dorso-medial region of the antenna but is of insufficient resolution to determine with precision the morphological types of sensilla associated with the labeled cells (Dobritsa, 2003).

The antibody to Or22a and Or22b common sequence stains a subset of the large basiconic sensilla in the dorso-medial region of the antenna. Labeling is clearly visible in the sensillum shaft, where the dendrites of ORNs are located. No labeling was observed in cell bodies or axons. No labeling was detected in the maxillary palp, the other adult olfactory organ, or in the larval antenno-maxillary complex, which mediates both larval olfaction and taste. Sexual dimorphism was observed: although both males and females show similar spatial patterns of labeling, the number of labeled sensilla in females, 29 ± 2, is greater than that in males, 18 ± 2 (Dobritsa, 2003).

Immunoelectron microscopy confirms that the labeled sensilla are large basiconic sensilla and allows a precise definition of their morphological subtype, LB-I. Sensilla of this subtype are characterized by a relatively thin cuticle, and they contain two ORNs per sensillum. In agreement with these findings, this subtype exhibits sexual dimorphism, with 33 LB-I sensilla present in females and 19 in males (Shanbhag, 1999). Immunoelectron microscopy further reveals that the label is distributed on the surfaces of dendrites, consistent with the expression of membrane receptors (Dobritsa, 2003).

Having determined that Or22a and Or22a localize to a particular morphological type of sensillum, the large basiconic sensilla, attempts were made to localize them to a functional type of sensillum. The seven well-characterized functional types of antennal basiconic sensilla show overlap in their distributions, but each type is restricted to a particular spatial domain on the antennal surface (De Bruyne, 2001). There are three functional types of large basiconic sensilla, ab1, ab2, and ab3, defined on the basis of electrophysiological recordings from the ORNs they contain. ab1 sensilla are easily recognized in such recordings because they contain four ORNs with distinct spike amplitudes or shapes, whereas ab2 and ab3 each house two ORNs (De Bruyne, 2001). All three sensillum types can be distinguished by measuring the response of their neurons to a panel of odors. Throughout this study ORN activity was monitored by recording action potentials, which provide a direct measure of ORN signaling (Dobritsa, 2003).

To determine in which functional type or types of sensilla Or22a/b are expressed, physiological recordings were made from live flies in which the Or22a/b-expressing sensilla were labeled with GFP. In brief, two strains of transgenic flies were generated in which the presumed promoters of Or22a or Or22b drive expression of the yeast transcription factor GAL4, which in turn drives expression of GFP. Recordings were then made from GFP-labeled sensilla, which allows the correlation of a particular receptor gene with a particular sensillum type (Dobritsa, 2003).

Specifically, to drive GAL4 under the control of the Or22a promoter, an 8.2 kb region upstream of the Or22a translational start codon was isolated and fused to the coding sequence of GAL4 to generate a construct referred to as 22a-GAL4. To drive GAL4 under the control of the Or22b promoter, a 10.3 kb region upstream of Or22b was isolated to generate 22b-GAL4. These 10.3 kb include the 8.2 kb upstream of Or22a, the Or22a coding sequence, and the intergenic region between Or22a and Or22b. Flies carrying the 22a-GAL4 or 22b-GAL4 transgenes were crossed to flies carrying UAS-GFP to yield progeny in which GAL4 binds to a UAS and activates transcription of GFP. The particular GFP derivative used was mCD8-GFP, which contains sequences of the mouse lymphocyte surface marker CD8 and which accordingly localizes to membranes. This derivative is hereafter referred to as 'GFP' for simplicity (Dobritsa, 2003).

It was found that 22a-GAL4 drives expression of GFP in a subset of large basiconic sensilla in the dorso-medial region of the antenna, and 22b-GAL4 shows an indistinguishable pattern. To confirm that GFP expression recapitulates the endogenous Or22a/b expression pattern, double-labeling experiments were performed with anti-Or22a/b and antibodies directed against GFP. All sensilla expressing GFP were found also to express Or22a/b in the case both of the 22a-GAL4 driver and the 22b-GAL4 driver (Dobritsa, 2003).

The GFP-labeled sensilla are visible in live animals, thereby allowing the sensilla to be distinguish and allowing them to be electrophysiologically recorded. Recordings from labeled sensilla of 22a-GAL4; UAS-GFP flies revealed that these sensilla house two neurons, as expected of ab2 or ab3, but not ab1. The labeled sensilla were tested with a diagnostic set of 11 odors that distinguish among the different types of large basiconic sensilla. The sensilla labeled with 22a-GAL4 are homogeneous in their response spectrum. They contain an A neuron whose strongest responses are to ethyl butyrate, pentyl acetate, and ethyl acetate (like the A neuron of the ab3 sensillum) and a B neuron whose strongest responses are to heptanone, hexanol, and octenol (like the B neuron of the ab3 sensillum). In the 22b-GAL4; UAS-GFP flies, the labeled sensilla are also homogeneous: they contain two ORNs each, and the ORNs yield a response pattern similar to that of ab3 (Dobritsa, 2003).

These results indicate that both 22a-GAL4 and 22b-GAL4 drive expression in the ab3 sensillum. It is noted further that the total number of ab3 sensilla estimated for the male antenna in the physiological studies of De Bruyne (2001) is 18, a number that agrees well with the number of sensilla labeled in males with the anti-Or22a/b antibody, 18 ± 2, and the number (19) of LB-I sensilla in males as determined in ultrastructural studies (Dobritsa, 2003).

Although all sensilla expressing GFP reacted with the anti-Or22a/b antibody, some anti-Or22a/b-reactive sensilla did not show expression of GFP (24.6% for 22a-GAL4; 9.6% for 22b-GAL4). A simple interpretation of this observation is that these two GAL4 lines are not completely expressive, an interpretation also drawn from experience with several other Or-GAL4 lines. In support of this interpretation, no physiological differences were found between GFP⁺ and GFP^- ab3 sensilla in either 22a-GAL4; UAS-GFP or 22b-GAL4; UAS-GFP lines (Dobritsa, 2003).

To increase the resolution of mapping from sensillum type to neuron type, a modified strategy was adopted. Or promoter-GAL4 constructs and single-unit electrophysiology were used, but rather than using GAL4 to drive GFP, GAL4 was used to drive the cell death gene reaper (rpr). Specifically, it was asked whether Or22a-GAL4; UAS-rpr or Or22b-GAL4; UAS-rpr antennae lack a particular ORN (Dobritsa, 2003).

Recordings from ab3 sensilla of Or22a-GAL4; UAS-rpr flies do not show the large spikes characteristic of the ab3A neuron. By contrast, the small spikes characteristic of ab3B are present. To characterize the effect of rpr expression more fully, both Or22a-GAL4; UAS-rpr and Or22b-GAL4; UAS-rpr flies were tested with the entire panel of odors. In both genotypes, there is no response of the ab3A neuron to any tested odor. The ab3B neuron, however, responds strongly to all of the odors that elicit a response from a control line, Or22a-GAL4; UAS-GFP. The ORNs of the other types of large basiconic sensilla appeared normal in limited testing. These results indicate that both Or22a and Or22b drivers direct expression in the ab3A neuron (Dobritsa, 2003).

It is noted with interest that for some odors, most notably pentyl acetate, mean response of the ab3B neuron in the rpr lines is greater than that in the control. Further investigation will be needed to establish whether this effect reflects an inhibitory role of the ab3A cell on the activity of the neighboring ab3B cell in wild-type sensilla (Dobritsa, 2003).

The relationship between Or expression and ORN response spectrum

The Deltahalo mutant, which is deleted for Or22a and Or22b, was used to investigate the relationship between Or expression and ORN response spectrum. One simple model is that the odor response spectra of ORNs are dictated solely by the Or genes they express. Alternatively, the response spectrum might be determined by an ensemble of molecules that includes not only an Or protein but also other proteins that are differentially distributed among the various sensilla and ORNs of the system. Such molecules could include odor binding proteins (OBPs) that might bind and deliver odors to the Or, other GPCRs that might heterodimerize with the Or, or other molecules such as RAMPs (receptor activity-modifying proteins) that might modulate the ligand specificity of the Or. To address this issue, the ab3A neuron of the Deltahalo mutant was used as a recipient for the expression of other Or genes (Dobritsa, 2003).

An antennal gene, Or47a, was expressed under the control of 22a-GAL4 in the Deltahalo mutant so as to drive its expression in ab3A cells. Large basiconic sensilla that contain ORNs with a response spectrum characteristic of the adjacent ab3B cell were then identified. The response of the cell neighboring the ab3B cell was analyzed and it was found that it responded to a subset of 11 odors tested. This neighboring cell responds most strongly to pentyl acetate, followed by 2-heptanone, a pattern similar to that of the ab5B neuron, which has been defined previously (De Bruyne, 2001). To test further the cell's similarity to ab5B, its response was measured to a panel of odors structurally related to pentyl acetate and to 3-methylthio-1-propanol, which elicits a much stronger response from ab5B than from any other characterized ORN on the antenna. The neuron adjacent to ab3B in flies ectopically expressing Or47a was found to exhibit a response spectrum similar to that of ab5B and distinct from that of ab3A. The simplest interpretation of all these results is that Or47a is expressed in ab5B and that it confers a response pattern similar to that of ab5B in the ab3A neuron when substituted for Or22a/b (Dobritsa, 2003).

Having established that Or47a is capable of functioning when driven by 22a-GAL4 in a Deltahalo background, i.e., in a cell that apparently contains no other functional receptors, it was asked whether Or47a can function when driven by 22a-GAL4 in a wild-type background, i.e., in a cell containing at least one other functional receptor, Or22a. Accordingly, a UAS-47a construct was expressed under the control of 22a-GAL4 and then response was tested across a broad concentration range to ethyl butyrate, which elicits a strong response from Or22a but a much weaker response from Or47a, and pentyl acetate, which elicits a strong response from Or47a but a weaker response from Or22a (Dobritsa, 2003).

Recordings from cells designed to express both Or22a and Or47a yielded a dose-response curve for ethyl butyrate that is identical to that of cells expressing Or22a but not Or47a. These results suggest that the expression of Or47a does not interfere with the expression and function of Or22a. Responses to pentyl acetate are greater in the cells expressing Or47a and Or22a than in the cells expressing Or22a alone, indicating that Or47a is able to function in a cell that is also expressing Or22a. The response to pentyl acetate of cells expressing both Or47a and Or22a is lower than that in cells expressing Or47a alone; one interpretation of this finding is that Or22a, which has a moderate response to pentyl acetate, competes with Or47a, which has a strong response to pentyl acetate, and the integrated signal sent by the cell in the form of action potentials is lower than in cells expressing only Or47a. In any case, the differing results observed in the cell when stimulated with ethyl butyrate versus pentyl acetate are informative: in the former case, the expression of Or47a appears to have no effect on Or22a, indicating that the introduction of Or47a does not block expression of Or22a or inhibit the cell nonspecifically, whereas in the latter case it appears to engender a response greater than that of Or22a alone, indicating that Or47a is expressed in a form that is capable of contributing to olfactory signaling, despite the presence of another receptor (Dobritsa, 2003).

REFERENCES

Search PubMed for articles about Drosophila Odorant receptor 22a or Drosophila Odorant receptor 22b

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date revised: 25 April 2019

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