Implications of orco on olfaction, neural development, and social behavior in : A review of the literature

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Authors Agosttini, Joseph Anthony

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Link to Item http://hdl.handle.net/10150/626733 IMPLICATIONS OF ORCO ON OLFACTION, NEURAL DEVELOPMENT, AND

SOCIAL BEHAVIOR IN ANTS: A REVIEW OF THE LITERATURE

By

JOSEPH ANTHONY AGOSTTINI

______

A Thesis Submitted to The Honors College

In Partial Fulfillment of the Bachelors degree With Honors in

Neuroscience & Cognitive Science

THE UNIVERSITY OF ARIZONA

D E C E M B E R 2 0 1 7

Approved by:

______

Dr. Wulfila Gronenberg Department of Neuroscience Abstract Olfaction is one of the most essential sensory modalities amongst . Not only is this behaviorally observed, but it is also anatomically evidenced. A range of social behaviors, including communication, foraging, and reproduction to name a few, immensely rely upon chemosensation. However, studies examining the genetics and neuroethology underlying this sociality in eusocial species, such as bees and ants, have been scarce, thus limiting our understanding of the molecular mechanisms needed for these species to thrive. This review examined the current literature on the orco-encoding gene and the effects that its mutagenesis had on olfaction, neural development, and behavior, specifically on Harpegnathos saltator and biroi ants. Results from these few but insightful studies included an observed decrease in the number of olfactory sensory neurons and glomeruli; reduced antennal lobe size, an inability to detect foraging and reproductive pheromones; non-cooperative behavior; and diminished egg laying and longevity, indicating that orco is a crucial protein needed for normal ant development and sociality. These findings show that ants make a prime model to study the sociobiology of insects and to highlight differences in neural development between eusocial and solitary species.

Key words: ant, eusocial, neuroethology, sociality, olfaction, chemosensation, pheromone, olfactory sensory neurons (ORNs), antennal lobes, glomeruli, orco, mutagenesis, CRISPR-Cas9, Harpegnathos saltator, Ooceraea biroi

Introduction Regarded as one of the most cooperative and socially sophisticated insects, ants – a family (Formicidae) of – are a biological marvel, working together in unison, each with an allocated task, in an effort to make their colonies flourish. These eusocial colonies, which are hierarchical in nature, are sustained by sterile female workers whose roles include preserving the integrity of the colony, protecting the queen (typically the sole reproducing adult female in the colony), and foraging for food. Such complex behaviors are made possible through the use of tactile, auditory, and olfactory communication methods, with the latter being the most notable (Jackson and Ratnieks, 2006). For this reason, olfaction (specifically in ants) will serve as the central focus of this review along with the role that the orco gene plays in this sensory modality and consequentially in neuroethology. The olfactory, neural developmental, and behavioral implications observed in ants when this gene is knocked out using gene-editing techniques, will also be explored. Before delving into the genetics underlying sociality in ants, an overview of their olfactory pathway is necessary.

Neurophysiology of the Olfactory Pathway Attached at the forefront of an ant’s head are two long, slender, and mobile appendages known as antennae, which detect vibrations, touch, air currents, and function as the organism’s olfactory sensory organs (indisputably the most vital of its sensory modalities). Antennae not only enable ants to perceive odors within their environment, but also to distinguish fellow nest-mates from foreign intruders by recognizing cuticular hydrocarbons (CHCs), pheromones found along the body and on the antennae themselves (d’Ettorre and Moore, 2008). Other pheromones – secreted hormones that elicit specific social behaviors – alert nest-mates of potential danger and inform other colony members of the location of food via trails (Jackson and Ratnieks, 2006).

These faculties are made possible by the hair-like structures on antennae called sensilla, which are comprised of one or more olfactory sensory neurons (OSNs) that express a single type of olfactory receptor protein (OR). These proteins are activated when bound to particular chemical compounds (e.g., specific odorants and pheromones). Since ants, specifically female ants, predominantly navigate the world around them using their sense of smell, some species have Fig. 1: An odorant binding to an olfactory over 400 olfactory receptor proteins (Zube and receptor protein on a sensillum housing an olfactory receptor neuron (Suh et al., 2014). Rossler, 2008), a considerably larger number than the fruit fly, (Drosophila melanogaster) who has a mere 42 receptors (Laissue and Vosshall, 2008). Although olfactory receptors come in a variety of different flavors with specific ones responding to carbon dioxide levels (Kleineidam et al., 2000) and others responding to sex pheromones, they behave in a similar manner. That is, when receptor proteins are activated by a ligand, a signal is transmitted down the axons of olfactory sensory neurons, whose terminal buttons synapse onto globular-shaped structures known as glomeruli in the antennal lobes of the brain. The net combinatory pattern of activated glomeruli allows for the signal to be processed and interpreted as a certain odorant by high-order brain regions like the mushroom bodies and lateral protocerebrum.

The Function of Orco and its Proposed Signaling Pathways Fig. 2: Frontal view of glomeruli in Or83b (otherwise known as orco), is an essential Componotus rufipes ants, stained with cobalt chloride (Gronenberg, 2008). heterodimeric seven-transmembrane domain co-receptor protein found in insects that is encoded by the orco gene (Nakagawa et al., 2012). Orco co-operates with most olfactory receptors and, upon ligand binding, opens its ion channel allowing Ca2+ and Na+ ions to enter the cell and K+ ions to exit the cell, in turn causing olfactory sensory neurons to depolarize (Benton et al., 2006). Two probable signaling pathways by which this occurs have been proposed. The first is a short and immediate pathway – the direct pathway – whereby Orco’s ion channel instantly opens upon odorant binding (Sato et al., 2008). The second, a more prolonged and slower signal transduction (the indirect pathway), proposes that ligand binding initiates a signaling cascade that causes the activation of

Glutamine Synthetase (GS), a G-protein, via the hydrolysis of GTP to GDP (Kaupp, 2010; Wicher

et al., 2008). GS then binds to the enzyme Adenylyl Cyclase (AC), which prompts ATP to convert to cyclic AMP (cAMP), a second messenger. cAMP Fig. 3: a) Direct orco-mediated pathway whereby odorant binding immediately results in will then bind to the appropriate binding site on depolarization of ORN; b) Indirect orco-mediated Orco, opening its ion channel and enabling the pathway whereby odorant binding produces a signal transduction necessary for ORN depolarization of OSNs (Wicher et al., 2008). depolarization facilitation. (Kaupp, 2010).

Previous transgenic studies conducted on Drosophila and mosquitos have shed light on Orco’s function. Larsson et al. (2004) discovered that when orco is mutated, Drosophila experience a loss of chemosensation that results in behavioral and electrophysiological changes. It did not, however, lead to a decreased number of glomeruli in the antennal lobe, suggesting that glomeruli number in fruit flies is not influenced by sensory input or a lack thereof (Larsson et al., 2004). Mutagenic orco proteins also affected female mosquitos’ ability to detect lactic acid – a chemical present in sweat that attracts mosquitos (Davis and Sokolove, 1976) – in the absence of CO2 thereby preventing them from scouting out the blood needed to fertilize their eggs (DeGennaro et al., 2013).

Methods Prior to 2017, the molecular mechanisms underlying sociality in ants had not been examined in large part because doing so was impractical (Leonhardt et al., 2016). As social insects, the success of the colony heavily relies upon the cooperation of other nest-mates, specifically that of female worker ants, whose responsibilities include raising the fertilized eggs laid by the queen (Hölldobler and Wilson, 1990, p. 351, 372). Despite the queen regularly laying eggs, barring the hibernation period, 40 to 60 days are generally required for an egg to hatch and the larva to be raised into an adult worker ant (Hölldobler and Wilson, 1990, p. 732). For this reason, genetically modifying ants is a challenging task, as obtaining a sufficient quantity of mutant ants would be tedious and a lengthy process. In August of 2017, laboratories at New York University and Rockefeller University discovered two ways to bypass this limitation using the gene-editing technique CRISPR-Cas9. Before reviewing their work, an explanation of this technique will prove helpful.

CRISPR-Cas9 Technique in Orco Mutagenesis Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) is a gene-editing technique that knocks out a target gene by inactivating it. Modeled after the immune system in bacteria (Lillestol et al., 2006) and modified to work in and human embryos (Zhu et al., 2014), CRISPR-Cas9 involves the use of four macromolecules: (1) CRISPR-associated protein 9 (Cas9), an endonuclease; (2) guide RNA (gRNA); (3) a DNA sequence containing the target gene of interest; and (4) a DNA endonuclease, a DNA repair enzyme. In these experiments, Cas9 and the gRNA (a segment of RNA, synthesized in vitro, containing a complementary base pair sequence to that of the orco gene) were first injected into ant eggs (Trible et al., 2017; Yan et al., 2017). In vivo, the gRNA will attach itself to the Cas9 protein and search for the sequence of DNA that complements it (Jinek et al., 2012). Once identified, the gene encoding orco is fed through Cas9 and is cleaved 3 nucleotides upstream of a highly conserved TGG sequence – known as the protospacer-adjacent motif (PAM) – by Cas9’s “pincers,” thus rendering the gene inactive (Jinek et al., 2012). The cell machinery will recognize that damage to the DNA has occurred and a DNA endonuclease repair enzyme will attempt to reverse the damage (Lees-Miller and Meek, 2003). This enzyme, however, is unaware if any genetic information was lost or added upon cleavage and must guess which occurred (Jeggo, 1998). Using a process called non-homologous end joining (NHEJ), the DNA Fig. 4: A visual representation of the CRISPR- Cas9 knockout technique (“CRISPR/Cas9 is repaired by means of an insertion of deletion (indel; Guide,” https://www.addgene.org/crispr/guide/). Jeggo, 1998). Because this leads to a frameshift mutation, often times one that engenders a nonsense mutation, the gene product – orco in this context – is truncated and therefore nonfunctional, resulting in a mutant phenotype (Perez et al., 2008).

Results Native to India, female Harpegnathos saltator ants (more commonly known as Indian Jumping ants) are facultatively sterile (Peeters and Hölldobler, 1995). That is, in the presence of a queen, they do not reproduce; however, in the absence of one, all female worker ants are capable of becoming Fig. 5: Harpegnathos saltator (Wild, gamergates (female workers capable of mating and sexually “Harpegnathos saltator”). reproducing). When such is the case, the female worker ant becomes a “pseudoqueen” and establishes a new colony, typically one that is small with few members (Peeters and Hölldobler, 1995). This ability for female worker ants to become fertile and bare brood is what attracted Yan and colleagues (2017) to choose the species for their investigation of the role that orco has on ant olfaction, neural development, and sociality. As previously mentioned, the life cycle from egg to worker ant is a prolonged process (Hölldobler and Wilson, 1990, p. 732). However, by isolating several female workers at once, facilitating their transition to pseudoqueens, Yan et al. (2017) were able to propagate large quantities of eggs whose genotypes could then be mutated using the CRISPR-Cas9 technique with the orco gene as the target.

Eggs were initially injected with CRISPR reagents (Cas9 and synthesized gRNA), and placed on an agar plate until hatching into larvae. Larvae were then introduced into a colony of wild type ants to be nursed into mature adults at which point female workers were isolated in order to

become pseudoqueens (F0). After laying her eggs and too allowing them to mature into adults, the queen was crossed with her haploid mutant male

-/-- offspring (orco ) [F1], hence producing a hemizygous strain of future pseudoqueens (orco+/-

) [F2]. This process was repeated with each Fig. 6: Propagation method of Orco mutagenesis performed by Yan et al. (2017). subsequent generation of females being crossed

with mutant males. By the F4 generation, a 1:1 ratio of hemizygous pseudoqueens to homozygous ones emerged (orco-/-), with the latter serving as the desired strain for this study. Mass spectroscopy was used to confirm the absence of the orco protein in mutant queens (Yan et al., 2017).

The results of Yan et al.’s 2017 experiments reveal that a lack of orco expression in ants not only leads to decreased chemosensation, but it also causes various neuroanatomical and behavioral changes. Both mutant male and female ants showed a decreased number in olfactory sensory neurons, of glomeruli in the antennal lobes as well as a reduced size in the antennal lobes themselves. Specifically, males expressed 31 of their 79 glomeruli, while females only expressed 62 of their 275

Fig. 7: Comparison of glomeruli number in glomeruli. This is a sharp contrast to the phenotype of orco-/- H. saltator worker ants relative to wild type (Yan et al., 2017). mutant Drosophila, which do not experience a reduction in glomeruli in the absence of orco (Larsson et al., 2004). Like Drosophila however, ants also exhibited abnormalities in social behaviors. For instance, infant ants would regularly wander outside of the nest and failed to cluster with its fellow nest-mates, suggesting an inability to communicate with them. Additionally, these ants were inept at foraging and would eat food foraged by other colony members such as crickets. Moreover, when more than one H. saltator female is present in a colony and a queen is absent, females will duel with one another to determine the next pseudoqueen (Penick et al., 2014). Mutant ants failed to engage in this behavior. Lastly, female mutants did not groom male ants upon becoming gamergates (a common mating behavior) and scarcely mated (Böröczky et al., 2013; Yan et al., 2017). When they would Fig. 8: Comparison of glomeruli become fertile, however, they laid fewer eggs relative to wild type number in orco-/- O. biroi worker -/- females and would not tend to them (Yan et al., 2017). ants relative to orco Drosophila (Trible et al., 2017).

Trible and colleagues (2017) conducted a similar study on Clonal Raider ants (Ooceraea biroi) with the hope of replicating Yan et al.’s results in a different species of ant. Clonal Raider ants are characteristic in that they lack a queen and reproduce parthenogenetically – that is, every female worker lays unfertilized eggs that develop into adult ants Fig. 9: Ooceraea biroi (Wild, “ biroi”). (Oxley et al., 2014). By extension of this asexual reproduction, embryos, and in turn themselves, are genetically identical (Oxley et al., 2014). This species native to Asia that feeds on the brood of other ant species (Hölldobler and Wilson, 1990, p. 732; Morisita et al., 1989, p. 42) made the ideal model for Trible and colleagues to use in this effort, as a great deal of mutants can be propagated by altering the genome of a single female ant also using CRISPR-Cas9 in a shorter period of time, thus eliminating the need to perform multiple crosses between progeny. Ergo, the methodology in this study was less complex than that of the former.

Eggs were firstly collected and injected with CRISPR-Cas9 reagents. They were subsequently incubated for 14-15 days at 25-30 oC and larvae were introduced into a wild type colony for nursing upon hatching. This process was repeated until a sufficient number of homozygous mutant ants were spawned. Antibody staining was used to substantiate the lack of orco proteins (Trible et al., 2017).

The results of Trible et al.’s (2017) were consistent with those of Yan et al.’s. orco-deficient ants demonstrated a loss of olfactory sensation, particularly in detecting pheromones, and too exhibited a reduced number of olfactory sensory neurons and glomeruli with only 18% of glomeruli being expressed. Decreased antennal lobe sizes were also seen, with lobes weighing 1/3 that of wild type ants. Similarly, infant mutant ants almost Fig. 10: An orco-deficient ant wandering immediately began to wander around the agar dish and from its nest-mates (Trible et al., 2017). segregated themselves from nest-mates. They also failed to follow pheromone trails and were not repelled when exposed to the scent emitted by a Sharpie marker, a behavior that is normally seen. Likewise, mutagenic ants laid fewer eggs in comparison to wild type females and showed a decrease in fitness and longevity with increased mortality rates. Discussion Yan et al. (2017) and Trible et al.’s (2017) findings establish a positive link between orco expression and neural development in ants. Relative to wild type, orco-deficient ants displayed a reduction in antennal lobe size, and diminished olfactory sensory neuron and glomeruli numbers, indicating that orco directly influences the development of ants’ nervous system (Trible et al., 2017; Yan et al., 2017). These results are comparable to work done with orco-/- Drosophila such that, like ants, they demonstrated a decreased chemosensory phenotype that elicited behavioral and electrophysiological changes (Larsson et al., 2004). Contrary to ants, however, mutant Drosophila did not show a reduction in glomeruli numbers suggesting that evolutionary genetic and neuroanatomical differences exist between the two species (and perhaps others; Larsson et al., 2004). This notion is corroborated by the observation that despite both species experiencing decreased olfactory sensory input as a result of their mutations, ants were the only ones whose glomeruli numbers dropped. This suggests that olfactory stimulation is needed for normal neural development in the antennal lobes or possibly that orco is needed to maintain glomeruli. If the former is the case, then lack of input to the higher-order structures that process olfactory information and are predominantly involved in olfactory memory (e.g., the mushroom bodies) may also atrophy due to lack of stimulation. Further research would be needed to confirm this hypothesis, however.

Although evolutionary differences between ants and fruit flies are evident, there also appears to be some evolutionary commonalities between the two. That is, despite orco – a co-receptor needed for general chemosensation – being knocked out in mutant ants and Drosophila, both still expressed and maintained a small number of glomeruli (Trible et al., 2017; Yan et al., 2017), suggesting that the two additional kinds Fig. 11: The three classes of chemoreceptors (IRs, ORs, and GRs) in insects, and the specific odorants that bind to them; of orco-independent chemoreceptors general odorants and pheromones pertinent to Drosophila, mosquitos, and moths are depicted here as an illustration (Suh found on antennae: ionotropic and et al., 2014). gustatory receptors. Ionotropic receptors (IRs) respond to acids and amines, compounds that are often times poisonous and toxic (Abuin et al., 2011), and undergo a conformational change in shape upon ligand binding that opens the receptor’s ion channel and subsequently depolarizes the neuron. Gustatory receptors (GRs) are

analogous to taste buds in humans in that they differentiate between tastes, but also respond to CO2 some pheromones (Brays and Amren, 2003; Dahanukar et al., 2001; Montell, 2013). The fact that sensory input from these sparser, less dominant receptors in the olfactory system was enough to conserve the presence of some glomeruli in the antennal lobes is most fascinating.

The studies reviewed in this paper also reiterated the significance of olfaction in eusocial insects by demonstrating the abnormal behavioral consequences that occur in null-orco ants. These behaviors are the result of an inability for pheromones – the chemical molecules ants use to communicate – to bind to olfactory receptors given the absence of its obligate co-receptor (orco). Without the ability to sense and perceive these behavior-governing signals, ants lose their collectivistic nature and adopt a more individualistic disposition: they fail to forage, yet eat prey captured by other nest-mates, and tend to leave the nest and wander around (Trible et al., 2017; Yan et al., 2017). Mutant H. saltator ants, in particular, will not participate in dueling in the absence of a queen, and pseudoqueens seldom engage in pre-mating ritualistic grooming behaviors (Yan et al., 2017). Additionally, these orco-deficient ants lay fewer eggs relative to wild type and display increased mortality rates (Yan et al., 2017). These anomalies reaffirm the importance of orco-dependent olfactory receptors in social behavior.

These discoveries, though fascinating and enlightening, are merely a microcosm. That is, the quest to understand the molecular genetics underlying development and behavior is far from over. While the role that orco plays on ant olfaction has largely been established by the studies considered in this paper, much remains to be known about its wider special implications, if any, namely: Are these outcomes conserved across all species of ants? Do the same phenotypic outcomes occur in other eusocial insects (e.g., wasps, bees, termites) when orco is knocked out? And, how do these effects compare and contrast to those when orco is unexpressed in solitary insects like caterpillars?

Based on the consistent behavioral and structural findings observed when orco is knocked out in Harpegnathos saltator and Ooceraea biroi, it is likely that all orco –deficient ants will be affected in a similar manner across species, despite inter-special variations in social structure, since the same mutant phenotype was seen in ant species that did and did not have a queen. Given orco’s ubiquitous presence in olfaction and the commonalities in social organization that eusocial species share, a lack of orco expression in other eusocial insects like wasps, bees, and termites is expected to yield the same abnormal results as in ants, though, these outcomes may be expressed differently depending on these insects’ respective social structures. For instance, bees may fail to leave odor trails in space when foraging for nectar rather than on the ground like ants do. The end result is the same, but the manner by which it was carried out differs based on the niche of the species. Solitary species are also expected to exhibit some of these behaviors (such as being unable to detect pheromones emitted by prey); however, they are more apt to die in shorter periods of time since they do not have a colony of nest-mates to sustain them.

Further research on orco across species will aid in our understanding of cross-specific evolutionary genetic differences and the ramifications these differences have had on sociality. In doing so, other insects besides Drosophila can more effectively be used as models in the laboratory, and, more broadly, our understanding of the role that genetics has on social behavior, in general, will advance, likely revealing the genetic mechanisms underlying social impairment disorders in humans.

Acknowledgements I would like to thank Dr. Gronenberg for his exceptional mentorship, patience, contagious passion for neuroethology, and inspiring me to continue researching ants beyond graduation.

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