The “Nose” Knows: Olfactory Receptor Losses Explain the Transition to Herbivory in Flies

By Madison Dipman (Pomona College) [Edited by Lars Schmitz, as part of BIOL 167 “Sensory Evolution”, an upper division class at the W.M. Keck Science Department. Written for educational purposes only].

A puzzling discrepancy exists in the world of insects. Although herbivorous species make up half of all known insect orders, the switch to a plant-only diet evolved in merely one-third of all living orders. Why is this the case, and what does it mean for insects desiring a change in diet?

Scientists have long hypothesized that this incongruity may be associated with the inherent challenges of colonizing plants. To avoid predation and parasitism by insects, plants have evolved a series of toxic chemical and physical defenses that impair the ability of insects to take advantage of nutrient-rich structures and potential homes for their offspring. Switching from feeding on yeast and other microbes on decaying plant tissues to targeting living plants requires an intricate combination of physiological, morphological, and behavioral adaptations—certainly a bit trickier than giving up meat on a whim or for a New Year’s resolution. Identifying the functional genomic changes that underlie the transition to herbivory in insects may reveal how this novel innovation arose from preexisting chemosensory circuits and why it has caused such massive adaptive radiation events in the paleontological record.

No nose?! But how do insects smell?

Like other high order animals, flies sense odors with olfactory organs located on their heads. Although fly “noses” on the antennae and maxillary palp look quite different from mammalian noses, the underlying odorant receptor neurons (ORNs) used for smell are morphologically similar to vertebrate ORNs (Vosshall and Stocker, 2007; Figure 1).

The position of olfactory (pink) and gustatory (blue) neurons on the fly (left). Scanning electron image of a fly head with major chemosensory organs (antenna and maxillary palp) labeled (right). [Figure 1a,b of Vosshall et al., 2007]

The position of olfactory (pink) and gustatory (blue) neurons on the fly (left). Scanning electron image of a fly head with major chemosensory organs (antenna and maxillary palp) labeled (right). [Figure 1a,b of Vosshall et al., 2007]

In Drosophila, odorants are detected by distinct subsets of olfactory receptor neurons (ORNs), which are localized in sensilla that cover the surface of the two olfactory organs: the antennae and maxillary palp. A sensillum is a broad term that describes an arthropod sensory organ associated with an insect’s tough outer covering. After odors are recognized, the ORNs send action potentials via axons to the antennal lobe of the brain, which processes olfactory information and initiates a behavioral response (de Bruyne et al., 2009; Hansson et al., 2010).

The responses of most insect ORNs rely on members of a large family of odorant receptor (OR) genes, which constitutes one of the most diverse gene families in insects. Despite significant variation in OR sequences and divergence within the genus Drosophila for 40 million years, functional ORN responses have been conserved across millions of years. For example, the OR gene Or42b is highly conserved across species, and is necessary for attraction and orientation to chemical structures in yeast volatiles. Because similar compounds activate Or42b across many Drosophila species, researchers postulate that volatile cues and the associated receptors for yeast detection are conserved across the Drosophilidae (Goldman-Huertas et al., 2014).

What does this have to do with herbivory?

Insect olfactory systems have evolved to monitor volatile chemicals in the environment and respond to olfactory-triggered cues that vary depending on the needs and habitat of the insect species. Previous studies have found that the diversification of the chemosensory repertoire from a relatively stable number of genes may reinforce or encourage adaptations, especially if these genes evolve to confer novel functions or are redeployed in new developmental contexts (Cande, Prud’homme, and Gompeil, 2013).

Goldman-Huertas and other researchers at the University of Arizona applied the idea that chemonsensory genes may diversify and encourage adaptations to the aforementioned herbivory issue and set out to test the hypothesis that the functional loss of chemosensory genes played a critical role in the transition to herbivory in insects. Changing the genetic repertoire, they argued, would in turn reorganize neurological processes involved in detection of plant volatiles, thereby drastically affecting behavior.

Drosophilidae makes an excellent study system for examining herbivory evolution in insects, since it boasts the ultimate genomic model for olfactory studies, Drosophila melanogaster—the fruit fly—as well as several well-documented transitions to herbivory. This 2014 study focuses on Scaptomyza flava, an herbivorous close relative of D. melanogaster. The ancestral niche for the genus Scaptomyza is microbe-feeding, but Scaptomyza species use decaying leaves and stems rather than fermenting fruit for olfactory-directed behaviors, such as feeding and oviposition (i.e. laying eggs) (Lapoint, O’Grady, and Whiteman, 2013; Figure 2).

Adult female S. flava fly with green abdomen after feeding on mustard leaf tissue. This behavior is one example of how S. flava uses plants for survival. [Figure 1 of Goldman-Huertas et al., 2014]

Adult female S. flava fly with green abdomen after feeding on mustard leaf tissue. This behavior is one example of how S. flava uses plants for survival. [Figure 1 of Goldman-Huertas et al., 2014]

Based on a time-calibrated Bayesian phylogeny, it appears that herbivory evolved only a single time within the genus about 13.5 million years ago and may have followed close association of flies with decaying plant tissues (Lapoint, O’Grady, and Whiteman, 2013; Figure 3). A Bayesian phylogeny incorporates the prior probability of an event occurring and the likelihood of an event occurring, allowing it to quickly produce both a complex tree estimate and measures of uncertainty for the groups on the tree (Holder and Lewis, 2003).

Time-calibrated Bayesian phylogeny of Drosophilidae species, including Drosophila and Scaptomyza species. Pie graphs at each node indicate the probability of a change to herbivory (green) or retention of ancestral microbe-feeding (white) traits. Herbivorous taxa are indicated by the leaf and bracket. [Figure 1 of Goldman-Huertas et al., 2014]

Time-calibrated Bayesian phylogeny of Drosophilidae species, including Drosophila and Scaptomyza species. Pie graphs at each node indicate the probability of a change to herbivory (green) or retention of ancestral microbe-feeding (white) traits. Herbivorous taxa are indicated by the leaf and bracket. [Figure 1 of Goldman-Huertas et al., 2014]

Since each ORN has characteristic spike amplitude, it is possible to determine the activity of a single neuron with electrophysiological assays (Goldman et al., 2005). In a single-unit electrophysiology, a sensillum (and the ORNs in the surface) is stimulated by application of an odor. A fine-tipped electrode is then inserted into the sensillum to measure the electrical activity of the OR neurons by recording the action potentials from the cell (Figure 4). Using this technique and genomic mapping, researchers have been able to functionally describe the OR gene family in Drosophila species.

Head of a fly showing fluorescence of the maxillary palp (left). Micrograph of the fly maxillary palp, demonstrating the use of an Or promoter to drive expression of a gene that confers fluorescence (middle). Single-unit electrophysiology (right), in which a recording electrode is placed through a fluorescently-labeled sensillum. [Figure 1B of Goldman et al., 2005]

Head of a fly showing fluorescence of the maxillary palp (left). Micrograph of the fly maxillary palp, demonstrating the use of an Or promoter to drive expression of a gene that confers fluorescence (middle). Single-unit electrophysiology (right), in which a recording electrode is placed through a fluorescently-labeled sensillum. [Figure 1B of Goldman et al., 2005]

Goldman-Huertas et al. collaborated with neuroscientists to perform these assays on S. flava and D. melanogaster. They measured the electrical responses in the insects’ antennae generated by olfactory receptors following presentation of two different scents: yeast and live plant (Arabidopsis) volatile compounds, which are both emitted in nature and would indicate the presence of a viable food source contingent on feeding preferences. The antennae of S. flava were more strongly stimulated by Arabidopsis volatiles than yeast, and the antennae of D. melanogaster were significantly more responsive to yeast volatiles than Arabidopsis (Figure 5). They also tested other simpler compounds associated with yeast and leaf tissue, and determined that overall, S. flava was less sensitive to short chemical compounds associated with yeast strains (e.g. aliphatic esters), which may explain S. flava’s lack of attraction to yeast volatiles.

Boxplot results from the electroantennogram assay. S. flava had diminished antennal responses to yeast volatiles and enhanced responses to plant-related Arabidopsis volatiles, whereas D. melanogaster was more sensitive to yeast volatiles than Arabidopsis. [Figure 2b (top) and 2c (bottom) of Goldman-Huertas et al., 2014).

Boxplot results from the electroantennogram assay. S. flava had diminished antennal responses to yeast volatiles and enhanced responses to plant-related Arabidopsis volatiles, whereas D. melanogaster was more sensitive to yeast volatiles than Arabidopsis. [Figure 2b (top) and 2c (bottom) of Goldman-Huertas et al., 2014).

This experiment was supplemented by a behavioral test carried out with a four-field olfactometer apparatus that created four independent airfields, two of which were exposed to yeast volatiles. Gravid adult females were placed in the arena, and their presence in either yeast or control fields was recorded. D. melanogaster flies spent significantly more time in yeast-volatile fields than S. flava, while S. flava did not spend more time in yeast-volatile fields, dividing time evenly between the yeast and control fields (Figure 6).

Apparatus for the four-field olfactometer assay, in which filtered air is blown through four corners of an arena and establishes four independent airfields that flies can choose between, [Figure 1 of Lehrman et al., 2013] (left). Two of these airfields were exposed to yeast volatiles. Results from the behavioral observation, indicating herbivorous S. flava flies did not spend a significantly higher proportion of time in yeast fields, consistent with a loss of attraction to yeast volatiles, [Figure 2a of Goldman-Huertas et al., 2014] (right).

Apparatus for the four-field olfactometer assay, in which filtered air is blown through four corners of an arena and establishes four independent airfields that flies can choose between, [Figure 1 of Lehrman et al., 2013] (left). Two of these airfields were exposed to yeast volatiles. Results from the behavioral observation, indicating herbivorous S. flava flies did not spend a significantly higher proportion of time in yeast fields, consistent with a loss of attraction to yeast volatiles, [Figure 2a of Goldman-Huertas et al., 2014] (right).

Neurological assays and behavioral observations revealed that the smell of yeast, which is abundant on rotting fruit, is minimally detected by antennae of S. flava and does not attract the flies. This is in stark contrast to the response of D. melanogaster. As any person who has left a piece of fruit out for too long has observed, fruit flies are drawn to this compound with extreme fervor. Although S. flava flies were not attracted to these yeast volatiles, their antennal chemoreceptors were sensitive to the Arabidopsis compound responsible for the smell of freshly cut grass, which is common in leafy plants.

Based on the lack of attraction and minimal electrical response to yeast volatiles in S. flava, Goldman-Huertas and collaborators predicted that olfactory genes important for sniffing out yeast must have been lost or altered in herbivorous Scaptomyza species. To characterize the changes in the OR gene family responsible for this behavioral modification, they sequenced the genome and annotated OR genes in herbivorous S. flava and then compared the findings to the thoroughly documented functional ORs in D. melanogaster. In the Scaptomyza lineage, only four widely conserved ORs were uniquely lost (Or22a, Or85d) or pseudogenized, indicating they are no longer functional (Or9a, Or42b). As expected, these ORs that function in microbe-feeding flies and are lost in herbivorous species play a role in detecting yeast volatiles, leading them to conclude that loss-of-function mutations were critical for the transition to herbivory in insects.

The researchers then set out to confirm their hypothesis that functional losses of portions of the OR gene family implicated in detecting yeast volatiles played a role in the ecological transition to herbivory in insects. This would be supported if OR gene losses coincided with the transition to herbivory in Scaptomyza according to their maximum likelihood ancestral state reconstruction model, which assumes the phenotypes that developed were statistically most likely but does not assume that all events are equally likely to happen. According to their model, one OR gene loss coincided with the evolution of herbivory, but losses of two other OR genes preceded the switch to plant-feeding (Figure 7). In addition, the researchers found no evidence of accelerated chemosensory gene loss in S. flava compared with other microbe-feeding Drosophila species, but this may be due to insufficient loss events to parameterize the complex model or the involvement of other gene families.

OR gene losses mapped onto a Scaptomyza phylogeny based on the results from PCR screens and genomic data. Three of the four OR genes lost coincided with or preceded the evolution of herbivory in the lineage ca.13.5 mya. [Figure 3a of Goldman-Huertas et al., 2014]

OR gene losses mapped onto a Scaptomyza phylogeny based on the results from PCR screens and genomic data. Three of the four OR genes lost coincided with or preceded the evolution of herbivory in the lineage ca.13.5 mya. [Figure 3a of Goldman-Huertas et al., 2014]

Taken together, these findings suggest that ancestral Scaptomyza had already lost conserved yeast-volatile sensors and possibly gained new olfactory pathways, which were later co-opted by herbivorous lineages to aid in the colonization of plant species. Examples of these ancestral but non-herbivorous species may include flies that live within decaying leaves or in tunnels in leaves produced by other insects (Goldman-Huertas et al., 2014). Sister groups of many herbivorous insect lineages also feed on dead organic material and fungi, which may be a precursor to full-fledged herbivory. None of these species are herbivorous, yet they are closely associated with plants, signifying the first steps of a major trophic shift.

The transition to herbivory in Scaptomyza likely involved many changes in olfactory cues, and loss-of-function mutations are not sufficient to explain this novel behavioral shift, since losing the ability to detect yeast does not inherently lead to colonizing plants. But what is missing in the story? The researchers tested for evidence of episodic positive selection in S. flava OR genes and found two ORs with a signature of positive selection. Other experimental and functional tests are needed to verify whether positive selection fixed changes to chemosensory genes in the Scaptomyza lineage, and if so, how these amino acid changes contributed to the development of herbivorous behaviors. Because it is still unclear where host-finding behaviors arose, further research on the annotated library of ORs is crucial, but potential candidates include the ORs with signatures of episodic positive selection.

But why does this matter?

The finding that the functional loss of chemosensory genes contributed to trophic shifts may be a major theme in the tree of life and one that facilitates rampant species radiation events. This study provides a valuable framework for tackling challenging questions of how these novel choices came to be as a result of subtle, targeted changes to a portion of chemosensory gene families. Armed with this knowledge, researchers may be able to answer similar questions in other insect lineages as well as animal species whose feeding preferences and behaviors diverge from those of their ancestors.

Understanding how trophic transitions are mediated by changes to the chemoreceptor repertoire also has numerous practical implications. There are many insect species (including S. flava itself) that are considered pests due to their negative impact on plant or animal lives. The “easy” solution for controlling insect pests since the Green Revolution of the 1960s and 70s has been the use of insecticides. However, abundant scientific research has demonstrated pesticides are not only harmful to other organisms and the environment, but also often fail in the long-term due to the evolution of pesticide resistance. By applying pesticides and other insect repellants to our crops and our bodies, we as humans may be changing the course of insect evolution.

The progression of experiments used in the Goldman-Huertas study may be applied to these organisms to better understand how insects made the shift from feeding on microbes to a particular plant species that humans rely on as an economically valuable crop. From these studies, researchers may be able to inform integrated pest management strategies, which seek to control pests but also minimize disruption to agroecosystems. Similar studies would also be valuable if they targeted vectors of human diseases, such as malaria-carrying mosquitoes, with the aim of deducing the genetic basis behind the trophic transition to consumption of human blood and ultimately designing repellants that could protect human health. Changes in insect behavior are intricately linked to the health and economy of humans, and there is still much to be learned about human-insect-plant relationships that may be gleaned from studying evolution in insects.

References

Cande, J., Prud’homme, B., Gompeil, N. 2013 Smells like evolution: the role of chemoreceptor evolution in behavioral change. Current Opinion in Neurobiology 23, 152-158. (DOI 10.1016/j.conb.2012.07.008)

de Bruyne, M., Smart, R., Zammit, E., Warr, C.G. 2009 Functional and molecular evolution of olfactory neurons and receptors for aliphatic esters across the Drosophila genus. J Comp Physiol A 196(2), 97-109. (DOI 10.1007/s00359-009-0496-6)

Goldman, A.L., van der Goes van Naters, W., Lessing, D., Warr, C.G., Carlson, J.R. 2005 Coexpression of two functional odor receptors in one neuron. Neuron 45(5), 661–78. (DOI 10.1016/j.neuron.2005.01.025)

Goldman-Huertas, B., Mitchell, R.F., Lapoint, R.T., Faucher, C., Hildebrand, J.G., Whiteman, N.K. 2014 Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors, and ancestral diet. PNAS 112(10), 3026-3031. (DOI 10.1073/pnas.1424656112)

Hansson, B.S., Knaden, M., Sachse, S., Stensmyr, M.C., Wicher, D. 2010 Towards plant-odor related olfactory neuroethology in Drosophila. Chemoecology 20(2), 51-61. (DOI 10.1007/s00049-009-0033-7)

Holder, M., Lewis, P.O. 2003 Phylogeny estimation: traditional and Bayesian approaches. Nature Reviews Genetics 4, 275-284. (DOI 10.1038/nrg1044)

Lapoint, R.T., O’Grady, P.M., Whiteman, N.K. 2013 Diversification and dispersal of the Hawaiian Drosophilidae: The evolution of Scaptomyza. Molecular Phylogenetics and Evolution 69, 95-108. (DOI 10.1016/j.ympev.2013.04.032)

Lehrman, A., Boddum, T., Stenberg, J.A., Orians, C.M., Bjorkman, C. 2013. Constitutive and herbivore-induced systemic volatiles differentially attract an omnivorous biocontrol agent to contrasting Salix clones. AoB Plants 5, plt005. (DOI 10.1093/aobpla/plt005)

Vosshall, L.B., Stocker, R.F. 2007 Molecular Architecture of Smell and Taste in Drosophila. Annual Review of Neuroscience 30, 505-533. (DOI 10.1146/annurev.neuro.30.051606.094306)

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