Biomedical subjects
Charles D Derby
Publications and source records attributed to Charles D Derby.
Gene expression and specificity in the mature zone of the lobster olfactory organ.
The lobster olfactory organ is an important model for investigating many aspects of the olfactory system. To facilitate study of the molecular basis of olfaction in lobsters, we made a subtracted cDNA library from the mature zone of the olfactory organ of Homarus americanus, the American lobster. Sequencing of the 5'-end of 5,184 cDNA clones produced 2,389 distinct high-quality sequences consisting of 1,944 singlets and 445 contigs. Matches to known sequences corresponded with the types of cells present in the olfactory organ, including specific markers of olfactory sensory neurons, auxiliary cells, secretory cells of the aesthetasc tegumental gland, and epithelial cells. The wealth of neuronal mRNAs represented among the sequences reflected the preponderance of neurons in the tissue. The sequences identified candidate genes responsible for known functions and suggested new functions not previously recognized in the olfactory organ. A cDNA microarray was designed and tested by assessing mRNA abundance differences between two of the lobster's major chemosensory structures: the mature zone of the olfactory organ and the dactyl of the walking legs, a taste organ. The 115 differences detected again emphasized the abundance of neurons in the olfactory organ, especially a cluster of mRNAs encoding cytoskeletal-associated proteins and cell adhesion molecules such as 14-3-3zeta, actins, tubulins, trophinin, Fax, Yel077cp, suppressor of profilin 2, and gelsolin.
Rosette-type tegumental glands associated with aesthetasc sensilla in the olfactory organ of the Caribbean spiny lobster, Panulirus argus.
The lateral antennular flagellum of decapod crustaceans bears unique olfactory sensilla, namely the aesthetascs, and other sensilla types. In this study, we identify a new major tissue in the lateral flagellum of the Caribbean spiny lobster, Panulirus argus, namely "aesthetasc tegumental glands" (ATGs), based on immunostaining with antibodies against CUB serine protease (Csp), in situ hybridization with csp-specific probes, labeling with the F-actin marker phalloidin, labeling with the nuclear marker Hoechst 33258, and staining with methylene blue. Each ATG has 12-20 secretory cells arranged in a rosette. Each secretory cell has a Csp-immunoreactive basal portion and an apical portion containing granular material (metachromatic staining indicative of acid mucopolysaccharides). At the center of each secretory rosette is a phalloidin-positive common locus that gives rise to a main drainage duct projecting toward the cuticle. Scanning electron and light microscopy show that thin ducts traverse the cuticle and connect to "peg pores" proximal to the bases of the aesthetascs, with 3.4 peg pores per aesthetasc. Since the number of common loci is correlated with the number of peg pores, we conclude that each pore represents the outlet of one ATG, and that the secretions are released from them. We conclude further that ATGs and aesthetascs are functionally linked. We hypothesize that ATG secretions have antifouling and/or friction-reducing properties, and that they are spread over the surface of the aesthetascs by antennular grooming. A review of the literature suggests that ATGs are common in decapod crustacean antennules, and that rosette glands and grooming might be functionally coupled in other body areas.
Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica.
Sea hares protect themselves from predatory attacks with several modes of chemical defenses. One of these is inking, which is an active release of a protective fluid upon predatory attack. In many sea hares including Aplysia californica and A. dactylomela, this fluid is a mixture of two secretions from two separate glands, usually co-released: ink, a purple fluid from the ink gland; and opaline, a white viscous secretion from the opaline gland. These two secretions are mixed in the mantle cavity and directed toward the attacking predator. Some of the chemicals in these secretions and their mechanism of action have been identified. In our study, we used western blots, immunocytochemistry, amino acid analysis, and bioassays to examine the distribution of these components: (1) an L-amino acid oxidase called escapin for A. californica and dactylomelin-P for A. dactylomela, which has antimicrobial activity but we believe its main function is in defending sea hares against predators that evoke its release; and (2) escapin's major amino acid substrates--L-lysine and L-arginine. Escapin is exclusively produced in the ink gland and is not present in any other tissues or secretions. Furthermore, escapin is only sequestered in the amber vesicles of the ink glandand not in the red-purple vesicles, which contain algal-derived chromophores that give ink its distinctive purple color. The concentration of escapin and dactylomelin-P in ink, both in the gland and after its release, is as high as 2 mg ml(-1), or 30 micromol ml(-1), which is well above its antimicrobial threshold. Lysine and arginine (and other amino acids) are packaged into vesicles in the ink and opaline glands, but arginine is present in ink and opaline at <1 mmol l(-1) and lysine is present in ink at <1 mmol l(-1) but in opaline at 65 mmol l(-1). Our previous results showed that both lysine and arginine mediate escapin's bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapin's antimicrobial effects require concentrations of lysine and/or arginine >1 mmol l(-1), our data lead us to conclude that lysine in opaline is the primary natural substrate for escapin in ink. Furthermore, packaging of the enzyme escapin and its substrate lysine into two separate glands and their co-release and mixing at the time of predatory attack allows for the generation of bioactive defensive compounds from innocuous precursors at the precise time they are needed. Whether lysine and/or arginine are substrates for escapin's antipredatory functions remains to be determined.
Source and specificity of chemical cues mediating shelter preference of Caribbean spiny lobsters (Panulirus argus).
Caribbean spiny lobsters display a diversity of social behaviors, one of the most prevalent of which is gregarious diurnal sheltering. Previous research has demonstrated that shelter selection is chemically mediated, but the source of release and the identity of the aggregation signal are unknown. In this study, we investigated the source and specificity of the aggregation signal in Caribbean spiny lobsters, Panulirus argus. We developed a relatively rapid test of shelter choice in a 5000-l laboratory flume that simulated flow conditions in the spiny lobster's natural environment, and used it to examine the shelter preference of the animals in response to a variety of odorants. We found that both males and females associated preferentially with shelters emanating conspecific urine of either sex, but not with shelters emanating seawater, food odors, or the scent of a predatory octopus. These results demonstrate specificity in the cues mediating sheltering behavior and show that urine is at least one source of the aggregation signal.
Sea hares use novel antipredatory chemical defenses.
Numerous studies have demonstrated that chemical defenses protect prey from predation and have often assumed that these defenses function by repelling predators. Surprisingly, few have investigated the mechanisms whereby predators are affected by these defenses. Here, we examine mechanisms of chemical defense of sea hares (Aplysia californica), which, when attacked by spiny lobsters (Panulirus interruptus), release defensive secretions from ink and opaline glands. We show that ink-opaline facilitates the escape of sea hares by acting through a combination of novel and conventional mechanisms. Ink-opaline contains millimolar quantities of amino acids that stimulate chemoreceptor neurons in the spiny lobster's nervous system. Ink stimulates appetitive and ingestive behavior, opaline can elicit appetitive behavior but can also inhibit ingestion and evoke escape responses, and both stimulate grooming. These results suggest that these secretions function by "phagomimicry," in which ink-opaline stimulates the feeding pathway to deceive spiny lobsters into attending to a false food stimulus, and by sensory disruption, in which the sticky and potent secretions cause high-amplitude, long-lasting chemo-mechanosensory stimulation. In addition, opaline contains a chemical deterrent that opposes appetitive effects. Thus, chemical defenses may act in more complex manners than palatability assays of prey chemistry may suggest.
Non-olfactory chemoreceptors in asymmetric setae activate antennular grooming behavior in the Caribbean spiny lobster Panulirus argus.
In the spiny lobster Panulirus argus the antennules carrying olfactory sensilla called aesthetascs and several types of other non-olfactory sensilla accompanying them are frequently groomed by the third maxillipeds in a stereotyped behavioral pattern. This behavior can be elicited by chemical stimulation with l-glutamate. Using selective sensillar ablations, we tested whether this behavior is driven by the numerous aesthetascs, which have been implicated as mediating this chemically elicited antennular grooming behavior in a previous investigation, or other, less numerous sensilla called asymmetric setae, which are tightly associated with aesthetascs. The selective sensilla ablations showed that the asymmetric setae are necessary and sufficient for driving chemically elicited antennular grooming. Bilateral elimination of the ca. 160 asymmetric setae almost completely abolished the behavior, whereas bilateral elimination of the ca. 2600 aesthetascs or of another type of sensilla associated with them (guard setae) did not cause a reduction in chemically elicited antennular grooming. Microscopical analysis of the morphological properties of the asymmetric setae revealed the presence of a terminal pore at the tip of the seta and a phalloidin-positive scolopale below its base. Since these structures have been identified in decapod crustaceans as modality-specific structures of bimodal chemo- and mechanosensory sensilla, we conclude that the asymmetric setae belong to this type of sensilla and thus have the appropriate features to function as chemoreceptors in the elicitation of antennular grooming. The identification of asymmetric setae and not aesthetascs as the drivers of chemically elicited antennular grooming suggests that it is not the olfactory pathway in the brain but a parallel pathway, constituted mainly by the lateral antennular neuropils, that is the neuronal substrate of this behavior. The lateral antennular neuropils receive non-olfactory sensory input from the antennule and contain the major arborizations of antennular motoneurons, allowing that direct sensory-motor coupling is involved in mediating the chemical elicitation of antennular grooming behavior.
Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid oxidase from ink of the sea hare Aplysia californica.
A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica was cloned and sequenced, and is the first sea hare antimicrobial protein to be functionally expressed in E. coli. Sequence analysis suggested that this protein is a flavin-containing l-amino acid oxidase (LAAO), with one predicted potential glycosylation site, although the glycosylation could not be experimentally confirmed. This protein, which we call ;escapin', has high sequence similarity to several other gastropod proteins. Escapin was verified by NMR, mass spectroscopy and HPLC to have FAD as its flavin cofactor. Escapin's antimicrobial effects, bacteriostasis and bactericidal, were determined using a combination of two assays: (1) incubation of bacteria on solid media followed by assessment of inhibition by direct observation of zones of inhibition or by turbidity measurements; and (2) incubation of bacteria in liquid media followed by counting viable colonies after growing on agar plates. Native escapin inhibited the growth of Gram-positive and Gram-negative bacteria, including marine bacteria (Vibrio harveyii and Staphylococcus aureus) and pathogenic bacteria (Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa). Escapin also inhibited the growth of yeast and fungi, with different efficacies. Escapin's antimicrobial activity was concentration dependent and did not decrease when stored for more than 5 months at room temperature. Escapin was bacteriostatic and not bactericidal in minimal media (e.g. salt media) with glucose, yeast extract, and a mixture of 20 amino acids each at 50 micromol l(-1), but was bactericidal in media enriched with Tryptone Peptone. Escapin was also strongly bactericidal in media with l-lysine at concentrations as low as 3 mmol l(-1) and slightly bactericidal in 50 mmol l(-1) l-arginine, but not in most other amino acids even at 50 mmol l(-1). Escapin had high oxidase activity (producing hydrogen peroxide) with either l-arginine or l-lysine as a substrate and little to no oxidase activity with other l-amino acids. Hydrogen peroxide alone (without escapin or amino acids) was strongly bacteriostatic but poorly bactericidal, similar in this respect to l-arginine but different from l-lysine in the presence of escapin. Together these results suggest that there are multiple mechanisms to escapin's antimicrobial effects, with bacteriostasis resulting largely or entirely from the effects of hydrogen peroxide produced by escapin's LAAO activity, but bactericidal effects resulting from lysine-dependent mechanisms not directly involving hydrogen peroxide. Recombinant escapin expressed in bacteria was also active against Gram-positive and Gram-negative bacteria, suggesting that glycosylation is not essential for antimicrobial activity.
Localized ablation of olfactory receptor neurons induces both localized regeneration and widespread replacement of neurons in spiny lobsters.
The peripheral olfactory system of the spiny lobster Panulirus argus--located on paired antennules--undergoes continual postembryonic development. This process includes continuous addition of olfactory receptor neurons (ORNs) related to indeterminate growth, continuous replacement, and regeneration when necessitated by damage. We have shown previously that new olfactory tissue is continually added to the proximal margin of these populations, called the proximal proliferation zone (PPZ). Here, we show that focal damage to mature portions of the olfactory system causes localized degeneration of ORNs over 1-10 days after damage. Studies using the cell proliferation marker 5-bromo-2'-deoxyuridine show that localized degeneration was followed by rapid and localized regeneration of olfactory tissue. Rapidly dividing cells were recorded up to 40 days after damage, with regeneration of ORN clusters complete within 80 days. Focal damage appeared to stimulate widespread cell replacement (cell death and proliferation) within mature, undamaged ORN clusters. This response was observed in ORN clusters outside the damaged zone, including mature clusters in the contralateral antennule. The degree of widespread cell replacement was less than local repair after local damage, but it increased with more extensive damage. However, changes in on-going proliferation in the PPZ were not detected, at least not 20 days or longer after damage, suggesting damage-induced widespread proliferation may be specific to mature populations of ORNs. We speculate that localized regeneration involves activity of resident precursor cells not destroyed by the ablation and that unidentified regulatory signals released in response to localized damage induce widespread ORN replacement.
Inducible transcript expressed by reactive epithelial cells at sites of olfactory sensory neuron proliferation.
The continuous replacement of cells in the spiny lobster olfactory organ depends on proliferation of new cells at a specific site, the proximal proliferation zone (PPZ). Using representational difference analysis of cDNA, we identified transcripts enriched in the PPZ compared to the mature zone (MZ) of the organ. The 12 clones identified included four novel sequences, three exoskeletal proteins, a serine protease, two protease inhibitors, a putative growth factor, and a sequence named PET-15 that has similarity to antimicrobial proteins of the crustin type. PET-15 mRNA was only detected in epithelial cells. It was abundant in all epithelial cells of the PPZ, but was only detected in the MZ at sites of damage to the olfactory organ. PET-15 mRNA was increased by types of damage that are known to induce proliferation of new olfactory sensory neurons in the olfactory organ. It increased in the PPZ after partial ablation of the olfactory organ and in the MZ after shaving of aesthetasc sensilla. These ipsilateral effects were mirrored by smaller increases in the undamaged contralateral olfactory organ. These contralateral effects are most parsimoniously explained by the action of a diffusible signal. Because epithelial cells are the source of proliferating progenitors in the olfactory organ, the same diffusible signal may stimulate increases in both cellular proliferation and PET-15 mRNA. The uniformity of expression of PET-15 in the PPZ epithelium suggests that the epithelial cells that give rise to new olfactory sensory neurons are a subset of cells that express PET-15.
Serine proteases in the spiny lobster olfactory organ: their functional expression along a developmental axis, and the contribution of a CUB-serine protease.
Several serine proteases and protease inhibitors have been identified in the crustacean olfactory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or inactivation) or in the development or survival of olfactory receptor neurons. To examine directly the function of proteases in the olfactory organ of the Caribbean spiny lobster Panulirus argus, we used different tissue fractions from the lateral flagellum in an enzyme activity assay with a variety of protease substrates and inhibitors. Trypsin-like serine protease activity occurs throughout the lateral flagellum but is enriched in the cell membranes from aesthetascs. Cysteine- and metalloprotease activities also occur in olfactory tissue, but are more abundant in tissue fractions other than aesthetascs. To assess the contribution of one of the olfactory serine proteases--CUB-serine protease (Csp)--Csp was immunoprecipitated using an antibody; results with the remaining fraction suggest that Csp accounts for at least 40% of the total serine protease activity in the olfactory organ. The amount of total serine protease activity follows a developmental axis in the lateral flagellum. Total protease activity is lowest in the proximal zone, which lacks aesthetascs, and the proliferation zone, where olfactory receptor neurons and associated cells are born, and highest in aesthetascs of the distally-located senescence zone, which has the oldest olfactory tissue.
Dual antennular chemosensory pathways can mediate orientation by Caribbean spiny lobsters in naturalistic flow conditions.
Benthic crustaceans rely on chemical stimuli to mediate a diversity of behaviors ranging from food localization and predator avoidance to den selection, conspecific interactions and grooming. To accomplish these tasks, Caribbean spiny lobsters (Panulirus argus) rely on a complex chemosensory system that is organized into two parallel chemosensory pathways originating in diverse populations of antennular sensilla and projecting to distinct neuropils within the brain. Chemosensory neurons associated with aesthetasc sensilla project to the glomerular olfactory lobes (the aesthetasc pathway), whereas those associated with non-aesthetasc sensilla project to the stratified lateral antennular neuropils and the unstructured median antennular neuropil (the non-aesthetasc pathway). Although the pathways differ anatomically, unique roles for each in odor-mediated behaviors have not been established. This study investigates the importance of each pathway for orientation by determining whether aesthetasc or non-aesthetasc sensilla are necessary and sufficient for a lobster to locate the source of a 2 m-distant food odor stimulus in a 5000-liter seawater flume under controlled flow conditions. To assess the importance of each pathway for this task, we selectively ablated specific populations of sensilla on the antennular flagella and compared the searching behavior of ablated animals to that of intact controls. Our results show that either the aesthetasc or the non-aesthetasc pathway alone is sufficient to mediate the behavior and that neither pathway alone is necessary. Under the current experimental conditions, there appears to be a high degree of functional overlap between the pathways for food localization behavior.
Mechanosensory neurons with bend- and osmo-sensitivity in mouthpart setae from the spiny lobster Panulirus argus.
The mouthparts of the spiny lobster Panulirus argus hold primarily two types of setae--simple setae and cuspidate setae. Mechanosensory neurons from these setae were examined by electrophysiological recordings. The population of simple setae contained two types of mechanosensory neurons: displacement-sensitive neurons, which responded to deflection at the setal base; and bend-sensitive neurons, which responded to bending of the setal shaft. Displacement-sensitive neurons, in general, responded phasically and only during actual displacement. Typically, their response changed with alteration of the direction, amplitude, and velocity/acceleration of the mechanical stimulus. Bend-sensitive neurons, in general, responded phaso-tonically and carried information on the direction and region of bending. This is the first experimental demonstration of bend sensitivity for arthropod setae. Cuspidate setae contain highly sensitive mechanosensory neurons; however, due to the rigid nature of these setae, whether they were bend sensitive or displacement sensitive could not be determined, and they were thus called "tactile neurons." Bend-sensitive neurons, but not displacement-sensitive neurons or tactile neurons, showed graded responses to changes in osmolarity. The osmosensitivity of these neurons could mediate behavioral responses to changes in the osmolarity of seawater or food.
Amputation-induced activity of progenitor cells leads to rapid regeneration of olfactory tissue in lobsters.
Lobsters have a self-renewing olfactory system and, like many animals, continuously replace old or dying olfactory receptor neurons. In addition, lobsters are able to regenerate the peripheral olfactory system even after complete loss. The olfactory sensors in lobsters are located distally on a pair of antennules. These antennules are often damaged, but this has little impact on the lobster's sense of smell because damaged olfactory tissue is rapidly replaced. In this study, we investigated damage-induced regeneration of the olfactory system by measuring cell proliferation following controlled amputation. We show that amputation-induced regeneration occurs as a result of up-regulating the normal development of olfactory sensors. A unique feature of up-regulated development is the formation of patches of proliferating cells within the antennular epithelium. Epithelial patches were typically formed between 3 and 10 days postamputation on the amputated side. They were characterized by their: proximal position with respect to developing clusters of olfactory receptor neurons (ORNs); tendency to form two discrete patches within the borders of each existing annulus; cell size, which was approximately twice that of mature ORNs; and location within the ventral epithelium. The development of epithelial patches was immediately followed by proliferation of clusters of ORNs and associated glial cells, and the level of this proliferation increased significantly during the premolt stage of the lobster's molt cycle. These epithelial patches may represent populations of precursor cells, because they develop in response to amputation and immediately precede development of cell clusters composed of ORNs and glia. Possible regulatory signals controlling epithelial patch development are discussed.
Hooded sensilla homologues: structural variations of a widely distributed bimodal chemomechanosensillum.
A diversity of sensilla has been described in crustaceans, both across species and within a given species. However, few homologous setal types have been identified in crustaceans. In this study we examined setae with features of the hooded sensillum, which is a class of bimodal chemomechanosensilla first identified on antennules of the Caribbean spiny lobster Panulirus argus. We examined the antennules of 13 species representing seven families of malacostracan crustaceans, and most body surfaces of P. argus, and compared the sensillar morphology from different species and from different body regions to identify interspecific and intraspecific homologues of hooded sensilla. Our results show that sensilla with morphological characteristics of antennular hooded sensilla are present and have a similar pattern of distribution on the antennules of reptantian species representing three families (Palinuridae and Scyllaridae of the Achelata and Nephropidae of the Homarida). Furthermore, hooded sensillar homologues are present on most body surfaces of P. argus. However, there are intraspecific and interspecific variations in the morphology of these sensilla. We present evidence that supports the idea that postembryonic changes in individual sensilla may be responsible for some of these morphological variations. Despite these variations, we conclude that the sensilla are homologues, because they have several common characteristics, similar positions on the body surface, similar substructures, a continuum to their morphological variations, and morphological variation that is correlated with phylogenetic similarity. Taken together these results support the idea that the hooded sensillum is a singular and biologically important sensillar type that has a broad distribution.
Ultrastructure and physiology of the hooded sensillum, a bimodal chemo-mechanosensillum of lobsters.
The antennules of decapod crustaceans are covered with thousands of chemosensilla that mediate odor discrimination and orientation behaviors. Most studies on chemoreception in decapods have focused on the prominent aesthetasc sensilla. However, previous behavioral studies on lobsters following selective sensillar ablation have revealed that input from nonaesthetasc antennular chemosensilla is sufficient for many odor-mediated behaviors. Our earlier examination of the setal types on the antennules of the Caribbean spiny lobster Panulirus argus revealed three types of nonaesthetasc chemosensilla. The most abundant and widely distributed of these is the hooded sensillum. The present study describes the detailed ultrastructure of antennular hooded sensilla and the physiological response properties of their receptor neurons. Light and scanning and transmission electron microscopy were used to examine structural characteristics, and electrophysiology was used to examine single-unit responses elicited by focal chemical and mechanical stimulation of antennular hooded sensilla. Hooded sensilla have a porous cuticle and are innervated by 9-10 chemosensory and 3 mechanosensory neurons whose dendrites project to the distal end of the sensillum. Hooded sensillar chemosensory neurons responded to waterborne chemicals, were responsive to only one of the six tested single compounds, and had different specificities. Hooded sensillar mechanosensory neurons were not spontaneously active. They had low sensitivity in that they responded to tactile but not waterborne vibrations, and they responded to sensillar deflection with phasic bursts of activity. These results support the idea that hooded sensilla are bimodal chemo-mechanosensilla and are receptors in an antennular chemosensory pathway that parallels the well-described aesthetasc chemosensory pathway.
Dual antennular chemosensory pathways mediate odor-associative learning and odor discrimination in the Caribbean spiny lobster Panulirus argus.
Chemosensory neurons in the antennular flagella of lobsters mediate long-range responses to chemicals. These neurons are part of two parallel chemosensory pathways with different peripheral and central components. Aesthetasc sensilla on the lateral flagella are innervated by chemosensory neurons that project to the olfactory lobes. A diversity of other 'non-aesthetasc' sensilla on both lateral and medial flagella are innervated by mechano- and chemosensory neurons, and most of these non-aesthetasc neurons project to the lateral antennular neuropils. We investigated the roles of these two pathways in odor-associative learning and odor discrimination by selectively removing either aesthetasc or non-aesthetasc sensilla from the spiny lobster Panulirus argus. Lobsters lacking both aesthetasc and non-aesthetasc antennular sensilla show very reduced or no odor-mediated searching behavior. We associatively conditioned lobsters using two paradigms: aversive conditioning with generalization testing (which reveals the similarity in the lobsters' perception of odorants) and discrimination conditioning (which reveals the lobsters' ability to discriminate odorants). Sham-control intact lobsters performed these tasks well, as did lobsters lacking either aesthetascs or non-aesthetasc setae. There was a strong but statistically non-significant trend that lobsters lacking either aesthetascs or non-aesthetasc setae generalized more between complex odor mixtures than did intact lobsters. After aversive conditioning with generalization testing, aesthetasc-ablated lobsters had more difficulty discriminating among the most closely related complex mixtures than did intact or non-aesthetasc-ablated lobsters. However, after discrimination conditioning, aesthetasc-ablated lobsters were as proficient as intact animals in discriminating highly similar mixtures. These results indicate overlap and redundancy in the function of these two chemosensory pathways in odor-associative learning and odor discrimination, but these pathways also complement each other to enable better discrimination. This study presents the first evidence for a role of non-aesthetasc chemosensory neurons in complex odor-mediated behaviors such as learning and discrimination.