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Olfactory discrimination conditioning in the moth Spodoptera littoralis
Abstract
We used a proboscis extension reflex (PER) to study the olfactory discrimination capability in the moth Spodoptera littoralis. Already after a single experience, moths were capable to discriminate a rewarded from an unrewarded odor. In the first experiment, when rewarded and unrewarded odors were substituted for each other, moths were able to undergo reversal conditioning already after two experiences. Both shorter and longer inter-trial intervals (ITIs) supported high degrees of learning. In a second experiment, moths could solve both feature-positive and -negative discrimination tasks. Two hypotheses for the way in which these associations exert their discrimination performance are considered. The moth's olfactory physiology has been extensively studied. This animal thus provides a powerful system in which to study the neurobiology of olfactory discrimination and odor recognition.
... In order to accomplish species-specificity, these pheromone components are blended with variation in molecular chain length, unsaturation level, functional group and total number of compounds. The olfaction system of the moth Spodoptera littoralis has been studied extensively in recent years, both from a behavioral and neurophysiological view [3]. Since (Z,E)-9,11-tetradecadienyl acetate (ZE-9,11-14:OAc) forms the main component in the sex pheromone of this moth [4], we have employed this pheromone compound for the establishment of insect-based communication protocol in our work. ...
... A prototype of the insect-based pheromone detection system has been constructed as shown in the figure 7. The chemoemitter module consists of a neMESYS high-precision dual channel syringe pump (cetoni GmbH, Germany) that drives a micro-machined artificial gland, releasing pheromone into the 14x14x40 cm 3 Perspex odour chamber. The polymer coated 4-sensor dual SAWR array, with the related RF oscillator circuitries and interfaces make up the chemoreceiver module. ...
... The stimuli and responses must be selected carefully if any learning is to be shown. For example, the moth Spodoptera littoralis can be classically conditioned to associate an odour with the proboscis extension reflex (PER) when rewarded with sucrose solution (Fan, 2000). S. littoralis can also learn discrimination and discrimination reversals as well as feature positive and negative discriminations (Fan & Hansson, 2000). ...
... For example, the moth Spodoptera littoralis can be classically conditioned to associate an odour with the proboscis extension reflex (PER) when rewarded with sucrose solution (Fan, 2000). S. littoralis can also learn discrimination and discrimination reversals as well as feature positive and negative discriminations (Fan & Hansson, 2000). However, classical conditioning was not possible with S. littoralis when colour stimuli were used instead of odour (Fan, Kelber & Balkenius, unpublished study). ...
We describe a behavioural experiment with the hawkmoth Deilephila elpenor and show how its behaviour in the experimental situation can be reproduced by a computational model. The aim of the model is to investigate what learning strategies are necessary to produce the behaviour observed in the experiment. Since very little is known about the nervous system of the animal, the model is mainly based on behavioural data and the sensitivities of its photoreceptors. The model consists of a number of interacting behaviour systems that are triggered by specific stimuli and control specific behaviours. The ability of the moth to learn the colours of different flowers and the adaptive processes involved in the choice between stimulus-approach and place-approach strategies is also modelled. The behavioural choices of the simulated model closely parallel those of the real animal. The model has implications both for the ecology of the animal and for robotic systems.
... Sucrose-elicited PER has been described and implicated in associative learning in a variety of restrained insects, including moths (39)(40)(41), butterflies (42), and bees (43,44). To comprehend an insect's feeding and oviposition behavior, first attempt to understand the chemosensory code that underpins these behaviors (45) [49][50]. ...
The gustatory system in insects is composed of detectors screening different panels of ligands, which enable or suppress life behaviors depending on the context. Single sensillum electrophysiology recordings were conducted from the antennal sensilla chaetica of an adult female moth of Spodoptera littoralis to ascertain whether these sensilla have a gustatory function. Five test stimuli (NaCl, sucrose, ethanol, green cotton leaves extract, and conspecific larval frass extract) were used to stimulate each sensillum. Two types of gustatory sensilla chaetica located on the same flagellomere of the distal third of the antenna were characterized, each sensillum enclosed three gustatory receptor neurons (N1, N2, and N3). Overall, responses (spikes/s) were higher in the case of low concentration of sucrose and higher concentration of ethanol than of salt and higher concentration of sucrose. Individual differences were observed in the response patterns of these sensilla to the tested stimuli but functional sensillum types could not be identified. Higher concentrations of sucrose, ethanol extracts of green cotton leaves, or conspecific larval frass significantly stimulated the same neuron of ventral and lateral sensilla chaetica. Response patterns revealed that antennal gustatory sensilla contain gustatory neurons, which are possible receptors for host-plant recognition. Moreover, stimulation of the female's antennae with the phagostimulative sucrose evoked activation of the proboscis extension reflex with dose-dependent responses. Differences in sensilla distribution and their response patterns suggest that gustatory sensilla on the antennae of the female S. littoralis have a key role in adaptation and host plant recognition.
... Appetitive olfactory learning has also been shown for moths (Daly et al., 2001;Daly and Smith, 2000;Cunningham et al., 2004;Riffell et al., 2013). As honey bees, moths can be conditioned to release a PER to an odour previously associated with a sucrose reward (Hartlieb et al., 1999;Hartlieb, 1996;Fan and Hansson, 2001;Fan et al. 1997;Skiri et al., 2005). ...
Pheromones are chemical communication signals known to elicit stereotyped behaviours and/or physiological processes in individuals of the same species, generally in relation to a specific function (e.g. mate finding in moths). However, recent research suggests that pheromones can modulate behaviours, which are not directly related to their usual function and thus potentially affect behavioural plasticity. To test this hypothesis, we studied the possible modulatory effects of pheromones on olfactory learning and memory in Agrotis ipsilon moths, which are well-established models to study sex-pheromones. To achieve this, sexually mature male moths were trained to associate an odour with either a reward (appetitive learning) or punishment (aversive learning) and olfactory memory was tested at medium- and long-term (1 h or 1.5 h, and 24 h). Our results show that male moths can learn to associate an odour with a sucrose reward, as well as a mild electric shock, and that olfactory memory persists over medium- and long-term range. Pheromones facilitated both appetitive and aversive olfactory learning: exposure to the conspecific sex-pheromone before conditioning enhanced appetitive but not aversive learning, while exposure to a sex-pheromone component of a heterospecific species (repellent) facilitated aversive but not appetitive learning. However, this effect was short-term, as medium- and long-term memory were not improved. Thus, in moths, pheromones can modulate olfactory learning and memory, indicating that they contribute to behavioural plasticity allowing optimization of the animal’s behaviour under natural conditions. This might occur through an alteration of sensitization.
... This proboscis extension response (PER) first reflects the integration of gustatory perception and motivation for sugar and then allows feeding. Sucrose-elicited PER has been described and involved in associative learning in restrained insects including moths (Hartlieb, 1996;Fan et al., 1997;Hartlieb et al., 1999a,b;Fan and Hansson, 2001;Skiri et al., 2005;Jorgensen et al., 2007), butterflies (Kroutov et al., 1999), bees (Menzel, 1999(Menzel, , 2012Page and Erber, 2002;Sandoz, 2011;Giurfa and Sandoz, 2012;Giurfa, 2015) and flies (Fresquet, 1999;Chabaud et al., 2006); similar feeding-related responses exist in ants (Guerrieri and d'Ettorre, 2010;Perez et al., 2013), crickets (Matsumoto et al., 2015), and bugs (Vinauger et al., 2013;Labrousse et al., 2017). PER has been used by Scheiner and her colleagues to assess responsiveness to sucrose in bees and flies (Scheiner et al., 2004a(Scheiner et al., ,b, 2013Mujagic et al., 2010). ...
Adult moths need energy and nutrients for reproducing and obtain them mainly by consuming flower nectar (a solution of sugars and other compounds). Gustatory perception gives them information on the plants they feed on. Feeding and food perception are integrated in the proboscis extension response, which occurs when their antennae touch a sugar solution. We took advantage of this reflex to explore moth sugar responsiveness depending on different parameters (i.e., sex, age, satiety, site of presentation, and composition of the solution). We observed that starvation but not age induced higher response rates to sucrose. Presentation of sucrose solutions in a randomized order confirmed that repeated sugar stimulations did not affect the response rate; however, animals were sometimes sensitized to water, indicating sucrose presentation might induce non-associative plasticity. Leg stimulation was much less efficient than antennal stimulation to elicit a response. Quinine prevented and terminated sucrose-elicited proboscis extension. Males but not females responded slightly more to sucrose than to fructose. Animals of either sex rarely reacted to glucose, but curiously, mixtures in which half sucrose or fructose were replaced by glucose elicited the same response rate than sucrose or fructose alone. Fructose synergized the response when mixed with sucrose in male but not female moths. This is consistent with the fact that nectars consumed by moths in nature are mixtures of these three sugars, which suggests an adaptation to nectar perception.
... Prey items were presented sequentially with 15 s between each presentation. The order of the prey items presented was pseudo-randomized (Smith et al. 1991;Gerber et al. 1996;Fan and Hansson 2001;Fernandez et al. 2009;Pegram and Rutowski 2014) in the following sequence repeated three times: S+/S−/S−/S+/S−/S+/S+/S−. This sequence has a pattern that is assumed to be difficult for the predators to learn the pattern (as opposed to alternating; Gellerman 1933) and prevents the birds from generalizing their responses from presentation to presentation, as could happen if the same order was used repeatedly. ...
Many bright colors function as visual signals and are produced by structural mechanisms that result in their being iridescent. Iridescent colors differ from other types of animal coloration in that they vary in both hue and intensity, as the angle of viewing and illumination change relative to the color surface. Iridescent colors can serve as warning signals by deterring predation on distasteful animals. Due to their directional and mirror-like reflection, iridescent signals have the potential to (1) vary in appearance with each approach; (2) create a flashing signal; (3) under the right conditions, be more intense than diffusely reflecting signals; (4) display different hues with different positioning of the sun, predator, and prey; and (5) display angle-dependent camouflage, which could all influence warning signal effectiveness. Here, we examine how signal intensity, short-wavelength hue, and variation in appearance affect the response of domestic chickens (Gallus gallus domesticus) to warning signals. Variation did not affect their response. Higher intensity signals were more effective in terms of predator avoidance than were lower intensity, and blue-violet signals were more effective than were blue and blue-green. This could influence the effectiveness of an iridescent warning signal in nature if the prey displays more intense, blue-violet coloration. With the properties we tested, we found no cost to having warning colors that are iridescent and that there may be benefits in the ability to change hue and display a more intense signal. We suggest future research to examine the effects of iridescent flashing and angle-dependent camouflage on warning signal effectiveness.
Significance statement
Iridescent colors have only been recently shown to function as warning signals, but we know very little about how they function to deter predation. Here, we show that birds are more likely to change their behavior in response to higher intensity and blue-violet signals compared to low intensity and blue and blue-green signals. The intensity results are consistent with previous experiments, but the role of short-wavelength hue on warning signal effectiveness has never been tested before. Variation due to the shifting appearance of an iridescent signal did not influence the response to the warning signal, consistent with studies in long-wavelength signals. We found no cost to displaying an iridescent signal and potentially an adaptive benefit as iridescent warning signals have the potential to be brighter and display a different hue based on the arrangement of light source, signaler, and receiver.
... In experimental conditions using another syrphid, E tenax, an innate PER was triggered by visual stimuli offering spectral reflectance properties similar to pollen found in food plants [LUNAU & WACHT 1994;. In this study the observed feeding behaviour of hoverflies equated to a proboscis extension reflex (PER); this behaviour is well known in various insects, usually in response to odour cues as detected by chemosensillae on the legs, antennae and mouthparts (see for example, OMURA, HONDA & HAYASHI [2000] (butterfly); AMAKAWA [2001] (fly); FAN & HANSSON [2001] (moth)). The role of scent attractants from flowers in general, and from pollen in particular, also requires determination in relation to foraging choice and patterns in hoverflies. ...
Flower visitors from several insect taxa (bees, butterflies and some flies) are known to forage assortatively at flowers where floral colour change has occurred; these alterations are often dramatic and the flowers are retained on the plant. Here, a further example of floral colour change is reported and, for the first time, demonstrated how the incidence of such alteration affects foraging in syrphid flies. Feeding behaviour in Rhingia campestris and at least two other hoverfly species is strongly influenced by a striking and extremely localised colour change in the flowers of the wood forget-me-not, Myosotis sylvatica. Nearly 99 % of flower visits to pre-change flowers elicited a feeding response whereas only 13 % of visits to post-change flowers led to the same behaviour. Feeding times at post-change flowers were 2-3 times shorter compared to pre-colour-change residence time. Additionally, we confirm centrifugation as a suitable method for extracting low volumes of nectar from small tubular flowers; the change in syrphid feeding behaviour corresponds with both colour change and floral reward status.
... While there is evidence in vertebrates of how this reduction in the error signal may be implemented biologically, such evidence is still lacking in invertebrates. Conditioning using the PER has been so successful that it has been established in other insects such as moths and flies (Medioni and Vaysse, 1975;DeJianne et al., 1985;Holliday and Hirsch, 1986;Brigui et al., 1990;Fan et al., 1997;Fresquet, 1999;Hartlieb et al., 1999;Daly and Smith, 2000;Fan and Hansson, 2001;Skiri et al., 2005) and for use with mechanosensory stimuli (Giurfa and Malun, 2004). ...
Cognition is an integrating process that utilizes phylogenetic and individual memory, creates an internal representation of the world and a basis for expecting the future of the animal's own actions within the experienced environment. It thus allows the animal to decide between different options in reference to the expected outcome of its potential actions. All these processes occur as intrinsic properties of the nervous system and provide an implicit form of knowledge for controlling behavior. This article shows that such processes must be assumed to also exist in invertebrates with proper nervous systems (albeit over a large range of levels of complexity) to account for their behavior. We first consider forms of behavior that are dominated by innate components, and raise the questions of how innate and learned behavior are related and whether innate behavior involves cognitive components. Then we focus on experience-dependent forms of behavior and follow in the order of ascending complexity: from elemental forms of associative learning to rule learning and observatory learning in navigation and communication. We discuss both aspects of memory, as a storing device and in the form of working memory, the implicit form of representation which may provide the substrate for neural operations underlying decision making in relation to the expected outcome of the animal's actions.
... In S. littoralis, these three sugars are detected also by tarsal taste sensilla (Blaney and Simmonds, 1990). Stimulating the legs or the antenna with sucrose triggers proboscis extension and associative Chapter 3 Sugar and salt detection by sensilla chaetica on the antennae of the egyptian cotton leafworm learning in S. littoralis ( Fan and Hansson, 2001) and in other Lepidoptera (Daly and Smith, 2000; Romeis and Wackers, 2000; Skiri et al., 2005). While slight differences exist in the respective sensitivities of antennal and tarsal taste sensilla, the antennal input is likely to be most important because it is the first appendage used to explore food located in the immediate vicinity of the animal. ...
Taste is one of the fundamental senses by which animals can detect food sources (sugars, salts, lipids, amino acids) but also noxious compounds dissolved in aqueous solution or adsorbed on surfaces (leaf, cuticle). Unlike olfaction, where only cephalic organs are involved in the detection of volatile compounds, in insects, gustatory sensilla are located on different parts of the insect body (mouthparts, legs, wings, ovipositor) which results in the precise spatial location of the stimuli which excite them. These sensilla are involved in different behaviours and might therefore be tuned to different types of contact chemosensory stimuli. These functional constraints imply a different organisation of the nervous centres processing the information received from gustatory receptor neurons. Whereas projections from olfactory receptor neurons are clearly chemotopic, comparatively little is known on how gustatory neurons project to the central nervous system and how signals are encoded and processed by central neurons. In different insect species, including Lepidoptera, responses of gustatory receptor neurons situated on the tarsae and the abdomen have been described. However, physiological characteristics of antennal gustatory sensilla and the behavioural context in which they are used are only starting to be investigated. The objectives of this thesis were to study the gustatory neurons of contact chemosensory sensilla present on the antennae of adult Spodoptera littoralis using two different approaches: an electrophysiological approach of testing soluble chemicals and recording the firing pattern of these neurons; and a neuroanatomical approach of staining their pathways and target regions in the brain. Our electrophysiological observations show that taste sensilla possess neurons that respond to sugars like sucrose, fructose and glucose and to NaCl. We could not identify a gustatory receptor neuron responding to bitter compounds or amino acids, but the range of tested substances was limited and nothing is known on the behavioural significance of such compounds. We were able to test the sensitivity along the antenna of the sensilla located on the lateral side of the antenna but no differences were noticed. However, sensilla in males and females differed in sensitivity. In females, the intensity of responses was found to be weaker for the sensilla on the dorsal side of the antennae than for those on the ventral side. Antennation is a behaviour frequently described before mating or egg laying. The precise role of contact chemoreceptors in this kind of behaviours is however, unknown. For a conclusive interpretation of our data on the neuronal coding and central representation of taste information from the antennae, the involvement of antennal gustatory receptors in mating behaviour, host-plant detection and oviposition and their possible interactions with olfactory receptor neurons remains to be investigated. A scanning electron microscopic study showed no sexual dimorphism in the distribution of taste sensilla on the antennae. Mass fills of antennal afferents and backfills of individual contact chemoreceptive sensilla using Neurobiotin revealed 4 distinct projection areas of antennal gustatory sensilla. Two areas are within the deutocerebrum: the antennal motor and mechanosensory centre (AMMC) and a region situated posteriorly to the antennal lobes. The two other areas are in the tritocerebrum/suboesophageal ganglion complex. As our electrophysiological investigations showed that different neurons in the same sensillum respond to different stimuli, including mechanical stimuli for one of the neurons, it can be hypothesized that the projection areas are functionally distinct. No evidence for somatotopy of sensillar afferents originating from different parts of the antenna was found, with the methods used. A more detailed analysis of branching patterns within each target zone might reveal some form of somatotopy, however.
... The similarity of the mechanisms of associative activity in members of these two phylogenetic superphyla was explained by Lobashev [4] as pointing to a common genetic basis for their ancestors. Experiments on members of the orders Diptera [38,74], Blattoptera [13,65], Orthoptera [14,60], Lepidoptera [23,24], and Hymenoptera [17,47], have provided evidence that the ability to perform simple types of learning is present to a greater or lesser extent in all insects, while the most complex individual forms of behavior are seen in members of the order Hymenoptera, which lead a social lifestyle. Among these, the honeybee occupies a special position due to the characteristics of its food-procuring behavior and communicative activity; the honeybee has learning ability comparable to that of vertebrates. ...
This review summarizes studies directed to seeking the neuroanatomical basis of associative learning (conditioned olfactory proboscis-extension feeding reflex) in honeybees. Data on the structure of the bee brain are presented. Parallel pathways involved in responses to conditioned and unconditioned stimuli are demonstrated. The contributions of various brain structures and specific neurons (VUMmx1, PE1) to forming the conditioned reflex are addressed.
... The insects can also be released after the experiment. Classical reward-based first-order conditioning (as defined by Carew and Sahley [51]) has been described in several insect species, including the honeybee Apis mellifera [52], the fruit fly Drosophila melanogaster [53,54], the tobacco budworm Heliothis virescens [55] and its parasite the braconid wasp Microplitis croceipes [56], the African cotton leafworm Spodoptera littoralis [57], the American cockroach Periplaneta americana [58], and the seven-spotted ladybird Coccinella septempunctata [59]. Target substances can be associated with measurable behavior. ...
Insect antennae are among the most sensitive and selective chemical-sensing organs in the animal kingdom. Insects can perceive picograms of specific volatile organic compounds per cubic meter of air in milliseconds, which is far below the detection thresholds of current analytical devices. These exceptional sensing abilities have many uses in the context of insect biotechnology. Living specimens or parts of them, such as isolated antennae or individual proteins, can serve as biosensors in the field. As volatiles occur in a crude mixture in the environment, knowing which trigger-volatiles are crucial for the insects’ perception of specific incidents is of great value. This knowledge promotes the development of selective sensors for applications, such as fire detection. In this chapter, we discuss the different technical procedures for the preparation and use of insect-based biosensors for the detection of organic volatiles, including those based on insect behavior, insect olfactory proteins, and biomimetic sensing units. We also consider the use of these applications in portable devices outside the laboratory under field conditions.
Graphical Abstract
... We used 14 monomolecular odorants (Sigma-Aldrich, St Louis, MO, USA) from five chemical classes: methyl salicylate (CAS number: 119-36-8), phenyl acetaldehyde (122-78-1), hexanol (111-27-3), octanol (111-87-5), nonanol (143- 08-8), hexanal (66-25-1), octanal (124-13-0), nonanal (124-19-6), 2-hexanone (591-78-6), 2-octanone (111-13-7), 2-nonanone (821- 55-6), geraniol (106-24-1), (±)-linalool (78-70-6) and caryophyllene (87-44-5). Most of these odorants have been found to evoke responses in the antennae of female M. sexta and S. littoralis (Anderson et al., 1995; Fraser et al., 2003; Shields and Hildebrand, 2000) and to be behaviourally active in these two species (Daly et al., 2007; Fan and Hansson, 2001; Riffell et al., 2009). The main larval host-plant families of our sphingid species and most of the known hosts of the two Spodoptera species emit at least one of the tested odorants (The Pherobase, www.pherobase.com). ...
The aim of the present study was to determine what impact phylogeny and life history might have on the coding of odours in the brain. Using three species of hawk moths (Sphingidae) and two species of owlet moths (Noctuidae), we visualized neural activity patterns in the antennal lobe, the first olfactory neuropil in insects, evoked by a set of ecologically relevant plant volatiles. Our results suggest that even between the two phylogenetically distant moth families, basic olfactory coding features are similar. But we also found different coding strategies in the moths' antennal lobe; namely, more specific patterns for chemically similar odorants in the two noctuid species than in the three sphingid species tested. This difference demonstrates the impact of the phylogenetic distance between species from different families despite some parallel life history traits found in both families. Furthermore, pronounced differences in larval and adult diet among the sphingids did not translate into differences in the olfactory code; instead, the three species had almost identical coding patterns.
... Olfactory PER conditioning has become a versatile tool for the study of questions, not only in the field of comparative experimental psychology (e.g., Chandra and Smith 1998; Hellstern et al. 1998; Deisig et al. 2001 Deisig et al. , 2002 Deisig et al. , 2003), as originally planned by Takeda, but also in diversified fields such as olfactory perception (e.g., Vareschi 1971; Guerrieri et al. 2005b; Reinhard et al. 2010), neurobiology of olfaction and olfactory learning (e.g., Hammer 1993; Stopfer et al. 1997; Faber et al. 1999; Sandoz et al. 2003; Rath et al. 2011), molecular bases of memory (for review, see Menzel 1999; Schwärzel and Müller 2006), social bases of behavior in bees (e.g., Chaline et al. 2005; Arenas and Farina 2008), and floral ecology (Wright et al. 2002Wright et al. , 2005), to cite only a few examples. The basic premises of olfactory PER conditioning have also been adapted in other species, such as bumblebees (Laloi et al. 1999; Riveros and Gronenberg 2009), stingless bees (McCabe et al. 2007; Farina 2009, 2010), moths (Fan et al. 1997; Fan and Hansson 2001; Daly et al. 2004), and even ants, which do not have a proboscis but whose mouthpart movements can also be conditioned (Guerrieri and d'Ettorre 2010; Guerrieri et al. 2011). The choice of examples provided in this article is necessarily incomplete, as it would be impossible to cite all the extensive literature on PER conditioning produced since Takeda's original work. ...
The honeybee Apis mellifera has emerged as a robust and influential model for the study of classical conditioning, thanks to the existence of a powerful Pavlovian conditioning protocol, the olfactory conditioning of the proboscis extension response (PER). In 2011, the olfactory PER conditioning protocol celebrates 50 years since it was first introduced by Kimihisa Takeda in 1961. Here, we review its origins, developments, and perspectives in order to define future research avenues and necessary methodological and conceptual evolutions. We show that olfactory PER conditioning has become a versatile tool for the study of questions in extremely diverse fields in addition to the study of learning and memory and that it has allowed behavioral characterizations, not only of honeybees, but also of other insect species, for which the protocol was adapted. We celebrate, therefore, Takeda's original work and prompt colleagues to conceive and establish further robust behavioral tools for an accurate characterization of insect learning and memory at multiple levels of analysis.
... For example, butterflies and moths can rapidly undergo reverse conditioning when rewarding and unrewarding flower colours or odours are exchanged (e.g. Fan and Hansson, 2001;Goulson and Cory, 1993;Kelber, 1996;Weiss, 1997). With increased experience, bees and butterflies can improve their flower handling skills and/or shorten the time required to find a nectar or pollen source in flowers (e.g. ...
Learning plays an important role in food acquisition for a wide range of insects and has been demonstrated to be essential during flower foraging in taxa such as bees, parasitoid wasps, butterflies and moths. However, little attention has been focused on differences in floral cue learning abilities among species and sexes. We examined the associative learning of flower colour with nectar in four butterfly species: Idea leuconoe, Argyreus hyperbius, Pieris rapae and Lycaena phlaeas. All butterflies that were trained learned the flower colours associated with food. The flower colour learning rates were significantly higher in I. leuconoe and A. hyperbius than in P. rapae and L. phlaeas. Among the four species examined, the larger and longer-lived species exhibited higher learning rates. Furthermore, female butterflies showed a significantly higher learning rate than males. This study provides the first evidence that learning abilities related to floral cues differ among butterfly species. The adaptive significance of superior learning abilities in the larger and longer-lived butterfly species and in females is discussed.
... (Daly and Smith, 2000; Fan and Hansson, 2001; Fan et al., 1997; Hartlieb, 1996; Laloi et al., 1999). For a variety of phytophagous insects, field experiments have underlined the importance of olfactory learning in host selection (Papaj and Prokopy, 1989), including in the heliothine moth Helicoverpa armigera (Cunningham et al., 1998aCunningham et al., ,b,1999). ...
The importance of olfactory learning in host plant selection is well demonstrated in insects, including the heliothine moths. In the present study olfactory conditioning of the proboscis extension response was performed to determine the moths' ability to learn and discriminate three plant odorants: beta-ocimene and beta-myrcene (activating the same receptor neurone type), and racemic linalool (activating two different types). The conditioned stimulus (CS) was an air puff with each odorant blown into a constant air stream and over the antennae, and the unconditioned stimulus (US) was sucrose solution applied first to the antennal taste sensilla, then to the proboscis. Conditioning with increasing odorant concentrations induced increased learning performance. The concentration threshold for learning was 100 times lower for racemic linalool than for the two other odorants, a fact that can be correlated with a higher sensitivity of the moths' antennae to racemic linalool as shown in electroantennogram recordings. After correcting for the different odour sensitivities, the moths' ability to discriminate the odorants was studied. Differential conditioning experiments were carried out, in which moths had to distinguish between a rewarded (CS+) odorant and an explicitly unrewarded odorant (CS-), choosing odour concentrations giving the same learning rate in previous experiments. The best discrimination was found with beta-myrcene as the rewarded odorant and racemic linalool as the unrewarded. The opposite combination gave lower discrimination, indicating a higher salience for beta-myrcene than for racemic linalool. The moths could also discriminate between beta-ocimene and beta-myrcene, which was surprising, since they activate the same receptor neurone type. No difference in salience was found between these two odorants.
... For example, OA modulates the activity of olfactory receptor neurons (Pophof, 2000;Dolzer et al., 2001;Grosmaitre et al., 2001;Pophof, 2002;Stelinski et al., 2003), it increases the sensitivity of male moths to female sex pheromone (Linn and Roelofs, 1986;Linn et al., 1992), and it modulates the activity of flight motor neurons (Claassen and Kammer, 1986;Fitch and Kammer, 1986;Klaassen et al., 1986). More recently, it has been demonstrated that moths are also excellent models for studies of olfactory-based appetitive learning (Hartlieb, 1996;Fan et al., 1997;Hartlieb et al., 1999;Daly and Smith, 2000;Daly et al., 2001aDaly et al., , b, 2004Fan and Hansson, 2001;Cunningham et al., 2004), but the possible role of OA in moth olfactory learning remains unclear. ...
Octopamine is a neuroactive monoamine that functions as a neurohormone, a neuromodulator, and a neurotransmitter in many invertebrate nervous systems, but little is known about the distribution of octopamine in the brain. We therefore used a monoclonal antibody to study the distribution of octopamine-like immunoreactivity in the brain of the hawkmoth Manduca sexta. Immunoreactive processes were observed in many regions of the brain, with the distinct exception of the upper division of the central body. We focused our analysis on nine ventral unpaired median (VUM) neurons with cell bodies in the labial neuromere of the subesophageal ganglion. Seven of these neurons projected caudally through the ventral nerve cord. Two neurons projected rostrally into the brain (supraesophageal ganglion), and one of these was a bilateral neuron that sent projections to the gamma-lobe of the mushroom body and the lateral protocerebrum. Octopamine-immunoreactive processes from one or more cells originating in the subesophageal ganglion also form direct connections between the antennal lobes and the calyces of the mushroom bodies.
... Previous electrophysiological and optophysiological studies have shown that these odorants evoke strong responses in ORNs and glomeruli (Anderson et al. 1995; Carlsson and Hansson 2003). In addition, both larvae and adults could be trained to respond behaviorally to these compounds (Carlsson et al. 1999; Fan and Hansson 2001). Odorants were dissolved in paraffin oil, which does not evoke a detectable response in the AL (Carlsson and Hansson 2003). ...
Natural odors are often complex mixtures of different compounds. These mixtures can be perceived to have qualities that are
different from their components. Moreover, components can be difficult to distinguish within a blend, even if those components
are identifiable when presented individually. Thus, odor components can interact along the olfactory pathway in a nonlinear
fashion such that the mixture is not perceived simply as the sum of its components. Here we investigated odor-evoked changes
in Ca2+ concentration to binary blends of plant-related substances in individually identified glomeruli in the moth Spodoptera littoralis. We used a wide range of blend ratios and a range of concentrations below the level at which glomerular responses become
saturated. We found no statistically significant cases where the mixture response was greater than both component responses
at the same total concentration (synergistic interactions) and no statistically significant cases where the mixture response
was less than either component presented individually (suppressive interactions). Therefore, we conclude that, for the plant
mixtures studied, information of their components is preserved in the neural representations encoded at the first stage of
olfactory processing in this moth species.
... Behavioral studies of olfactory function establish that the ability of animals to discriminate across a broad array of odorants, blends, and concentrations is remarkable. Insects such as moths (Hartlieb et al. 1999;Daly and Smith 2000;Fan and Hansson 2001;Skiri et al. 2005) and honeybees (Smith et al. 1991;Smith and Cobey 1994;Chandra and Smith 1998;Hosler and Smith 2000;Wright et al. 2002;Wright and Smith 2004;Wright, Lutmerding et al. 2005;Wright, Thomson et al. 2005) appear to be no exception in this regard. To account for this seemingly limitless ability with relatively few receptor types, it has been argued that the initial spatial pattern of input is locally transformed within the insect antennal lobe (AL) or vertebrate olfactory bulb (OB) into a spatiotemporal code (Laurent 1999). ...
What is the spatial and temporal nature of odor representations within primary olfactory networks at the threshold of an animal's ability to discriminate? Although this question is of central importance to olfactory neuroscience, it can only be answered in model systems where neural representations can be measured and discrimination thresholds between odors can be characterized. Here, we establish these thresholds for a panel of odors using a Pavlovian paradigm in the moth Manduca sexta. Moths were differentially conditioned to respond to one odor (CS+) but not another (CS-) using undiluted odorants to minimize salience-dependent learning effects. At 24 and 48 h postconditioning, moths were tested for the presence of a conditioned response (CR) with a blank, then the CS+ and CS- (pseudorandomly) across a 5-log step series of increasing concentration. Results identified discrimination thresholds and established that differential CRs to the CS+ and CS- increased with stimulus concentration. Next, 3 separate groups of moths were differentially conditioned at either one-log step below, at, or one log step above the identified discrimination threshold. At 24 and 48 h postconditioning, moths were tested sequentially with a blank, the concentration used for conditioning, and then undiluted odor. Conditioning at one log step below the discrimination threshold established a CR, indicating both stimulus detection and learning, but was insufficient to establish evidence of discrimination. Moths conditioned at the discrimination threshold were able to discriminate but only when stimulated with undiluted odors, indicating learning, but discrimination measures were hampered. When conditioned above the discrimination threshold, moths had no difficulty in discriminating. These results establish methods for psychophysical characterization of discrimination and indicate that differential conditioning at lowered concentrations biases threshold measures.
... This behavior is, however, abruptly interrupted by escape maneuvers in response to the nearby echolocation sounds emitted by a hunting bat, suggesting crossmodal sensory integration between olfactory and auditory cues (Roeder, 1962; Miller and Surlykke, 2001; Skals et al., 2005). Both males and females have shown to be able to robustly associate an odor with a reward, although males were unable to learn the female sex pheromone (Fan and Hansson, 2001; Hartlieb et al., 1999 ). This suggests that olfactory context may be perceived differently in the two sexes but this difference is likely to have its cause in the primary olfactory neuropil, the antennal lobe, which is differently organized in the two sexes. ...
The aim of this study was to further reveal the organization of Kenyon cells in the mushroom body calyx and lobes of the male moth Spodoptera littoralis, by using immunocytochemical labeling. Subdivisions of the mushroom bodies were identified employing antisera raised against the amino acids taurine and aspartate, the neuropeptides FMRF-amide and Mas-allatotropin, and against the protein kinase A catalytic subunit DC0. These antisera have previously been shown to label subsets of Kenyon cells in other species. The present results show that the organization of the mushroom body lobes into discrete divisions, described from standard neuroanatomical methods, is confirmed by immunocytology and shown to be further elaborated. Anti-taurine labels the accessory Y-tract, the gamma division of the lobes, and a thin subdivision of the most posterior component of the lobes. Aspartate antiserum labels the entire mushroom body. FMRF-amide-like immunolabeling is pronounced in the gamma division and in the anterior perimeter of the alpha/beta and alpha'/beta' divisions. Mas-allatotropin-like immunolabeling shows the opposite of FMRF-amide-like and taurine-like immunolabeling: the gamma division and the accessory Y-system is immunonegative whereas strong labeling is seen in both the alpha/beta and alpha'/beta' divisions. The present results agree with findings from other insects that mushroom bodies are anatomically divided into discrete parallel units. Functional and developmental implications of this organization are discussed.
... Behavioral studies of insects have revealed that their olfactory systems can readily discriminate among a wide variety of odors and odor blends (Laska et al. 1999;Daly and Smith 2000;Fan and Hansson 2001;Wright et al. 2002;Daly, Wright, et al. 2004;Skiri et al. 2005). These studies typically use stimulus generalization and differential conditioning protocols, which are important behavioral paradigms for investigating the perceptual relatedness of stimuli. ...
Studies of olfactory function show that disruption of GABA A receptors within the insect antennal lobe (AL) disrupts discrimination of closely related odors, suggesting that local processing within the AL specifically enhances fine odor discrimination. It remains unclear, however, how extensively AL function has been disrupted in these circumstances. Here we psychophysically characterize the effect of GABA A blockade in the AL of the moth Manduca sexta. We used 2 GABA A antagonists and 3 Pavlovian-based behavioral assays of olfactory function. In all cases, we used matched saline-injected controls in a blind study. Using a stimulus generalization assay, we found that GABA A disruption abolished the differential response to related odors, suggesting that local processing mediates fine odor discrimination. We then assessed the effect of GABA A antagonist on discrimination thresholds. Moths were differentially conditioned to respond to one odor (reinforced conditioned stimulus [CS+]) but not a second (unreinforced conditioning stimulus [CS-]) then tested for a significant differential conditioned response between them across a series of increasing concentrations. Here, GABA A blockade disrupted discrimination of both similar and dissimilar odor pairs as indicated by generally increased discrimination thresholds. Finally, using a detection threshold assay, we established that GABA A blockade also increases detection thresholds. Because detection is a prerequisite of discrimination, this later finding suggests that disrupted discrimination may be due to impairment of the ability to detect. We conclude that the loss of ability to detect and subsequently discriminate is attributable to a loss of ability of the AL to provide a clear neural signal from background.
Warning coloration deters predators from attacking unpalatable prey. Warning colors are often characterized as long-wavelength, such as orange and red. However, many warning colors exist in nature, including short-wavelength colors, like blue. Blue has evolved as a primary defense in some animals but is not common. One hypothesis for the maintenance of this diversity is interspecific variation in predator response to signals. We tested this hypothesis with galliform birds: Gambel’s quail (Callipepla gambelii) and two domestic chicken breeds (Gallus gallus domesticus; Plymouth Rocks, Cochin Bantams). We measured innate avoidance and learning responses to only blue prey, only orange prey, and orange-and-blue prey, where the blue was iridescent to represent the natural coloration of the pipevine swallowtail butterfly (Battus philenor). We predicted birds would have similar responses to orange, but vary in response to blue. Upon first encounter, Cochin Bantams did not attack blue and Gambel’s quail readily attacked, indicating innate avoidance by Cochin Bantams. Plymouth Rocks had no innate aversion to any color, lower attack latencies and attacked most prey items. Cochin Bantams and Gambel’s quail both learned orange and orange-and-blue quicker than blue. Our results support the hypothesis that interspecific variation in predator response could maintain warning color diversity.
Warning colours can deter predators from attacking unpalatable or toxic prey. These colours are avoided by predators, either innately or by learned response through trial and error. Predators that have learned these signals can also generalize their responses to similar colours. Much of the previous research on warning colours has examined long-wavelength colours (e.g. orange and red) and little is known about short-wavelength coloration (e.g. blue) in warning signals, despite its presence in unpalatable animals in many different taxa. We determined whether prey with only blue coloration, only orange coloration, and with both colours elicited innate avoidance from a naïve predator (Gambel's quail chicks, Callipepla gambelii), as well as how effectively the signals were learned and generalized. Prey were composed of pieces of mealworm (palatable or unpalatable) beneath a coloured paper tent. Blue did not elicit any innate avoidance, whereas orange and a combination of both colours (orange-and-blue) were effective at deterring naïve predators. Predators were able to learn the association between colour and unpalatability most effectively for orange prey, but also learned this association for blue and for orange-and-blue prey. Predators showed asymmetrical generalization, in which birds were more likely to generalize from the colour they learned as unpalatable (conditioned stimulus) to orange prey and orange-and-blue prey than to blue prey. We conclude that orange is likely a more effective warning colour to these birds, but blue can still be learned alone and when displayed adjacent to orange.
The mushroom bodies are paired, high-order neuropils in the insect brain involved in complex functions such as learning and memory, sensory integration, context recognition and olfactory processing. This thesis explores the structure of the mushroom bodies in the noctuid moth Spodoptera littoralis using neuroanatomical staining methods, immunocytochemistry and electron microscopy, and investigates how the intrinsic neurons of the mushroom body, the Kenyon cells, respond to olfactory stimulation of the antennae using whole-cell patch clamp technique.
The mushroom body in S. littoralis contains about 4,000 Kenyon cells, and consists of a calyx, pedunculus and two lobes, one medial and one vertical. The calyx houses dendritic branches of Kenyon cells and the pedunculus and lobes contain the axons and terminals of these neurons respectively. The calyx is doubled and concentrically divided into a broad peripheral zone, which receives input from antennal lobe projection neurons, and a narrow inner zone, which receives yet unidentified input. The lobes are parsed into three longitudinal divisions, which contain a separate subset of Kenyon cells each. The Kenyon cells are divided into three morphological classes, I-III. Class I Kenyon cells have widely branching spiny dendritic arborisations in both zones of the calyx and occupy the two most posterior subdivisions of the lobes called α/β and α´/β´. Class II Kenyon cells have narrow clawed dendritic trees in the calyx and invade the most anterior division in the lobes, called γ. Class III Kenyon cells have clawed, diffusely branching dendrites in the calyx and provide a separate system of axons and terminal branches, partly detached from the rest of the mushroom body, called the Y tract and lobelets. Kenyon cells within the classes display differential labeling with antisera against neuroactive substances. Kenyon cells make synaptic contact with one another and with other neuron types in the mushroom body. Extrinsic inhibitory and putative modulatory neurons were identified.
Whole-cell patch clamp recordings revealed that Kenyon cells exhibit broadly tuned subthreshold activation by odor stimulation and a few cells responded with action potentials to specific biologically relevant odor combinations.
Taste is one of the fundamental senses by which animals can detect food sources (sugars, salts, lipids, amino acids) but also noxious compounds dissolved in aqueous solution or adsorbed on surfaces (leaf, cuticle). Unlike olfaction, where only cephalic organs are involved in the detection of volatile compounds, in insects, gustatory sensilla are located on different parts of the insect body (mouthparts, legs, wings, ovipositor) which results in the precise spatial location of the stimuli which excite them. These sensilla are involved in different behaviours and might therefore be tuned to different types of contact chemosensory stimuli. These functional constraints imply a different organisation of the nervous centres processing the information received from gustatory receptor neurons. Whereas projections from olfactory receptor neurons are clearly chemotopic, comparatively little is known on how gustatory neurons project to the central nervous system and how signals are encoded and processed by central neurons. In different insect species, including Lepidoptera, responses of gustatory receptor neurons situated on the tarsae and the abdomen have been described. However, physiological characteristics of antennal gustatory sensilla and the behavioural context in which they are used are only starting to be investigated. The objectives of this thesis were to study the gustatory neurons of contact chemosensory sensilla present on the antennae of adult Spodoptera littoralis using two different approaches: an electrophysiological approach of testing soluble chemicals and recording the firing pattern of these neurons; and a neuroanatomical approach of staining their pathways and target regions in the brain. Our electrophysiological observations show that taste sensilla possess neurons that respond to sugars like sucrose, fructose and glucose and to NaCl. We could not identify a gustatory receptor neuron responding to bitter compounds or amino acids, but the range of tested substances was limited and nothing is known on the behavioural significance of such compounds. We were able to test the sensitivity along the antenna of the sensilla located on the lateral side of the antenna but no differences were noticed. However, sensilla in males and females differed in sensitivity. In females, the intensity of responses was found to be weaker for the sensilla on the dorsal side of the antennae than for those on the ventral side. Antennation is a behaviour frequently described before mating or egg laying. The precise role of contact chemoreceptors in this kind of behaviours is however, unknown. For a conclusive interpretation of our data on the neuronal coding and central representation of taste information from the antennae, the involvement of antennal gustatory receptors in mating behaviour, host-plant detection and oviposition and their possible interactions with olfactory receptor neurons remains to be investigated. A scanning electron microscopic study showed no sexual dimorphism in the distribution of taste sensilla on the antennae. Mass fills of antennal afferents and backfills of individual contact chemoreceptive sensilla using Neurobiotin revealed 4 distinct projection areas of antennal gustatory sensilla. Two areas are within the deutocerebrum: the antennal motor and mechanosensory centre (AMMC) and a region situated posteriorly to the antennal lobes. The two other areas are in the tritocerebrum/suboesophageal ganglion complex. As our electrophysiological investigations showed that different neurons in the same sensillum respond to different stimuli, including mechanical stimuli for one of the neurons, it can be hypothesized that the projection areas are functionally distinct. No evidence for somatotopy of sensillar afferents originating from different parts of the antenna was found, with the methods used. A more detailed analysis of branching patterns within each target zone might reveal some form of somatotopy, however. CHAPTER 1 THE INSECT CHEMOSENSORY SYSTEM 11 Host plant selection in insects 11 Chemical detection and integration 12 Chemical detection at the antennal level 12 Identification of chemosensory receptor genes 20 Transduction mechanisms 22 Functional characterisation of olfactory and gustatory neurons 23 The insect brain and central chemosensory processing 27 Anatomy of the insect central nervous system 27 References 33 CHAPTER 2 CONTRIBUTION TO THE STUDY OF THE ANTENNAL GUSTATORY SYSTEM OF SPODOPTERA LITTORALIS 39 The model insect 39 Objectives 39 Results 40 References 43 CHAPTER 3 SUGAR AND SALT DETECTION BY SENSILLA CHAETICA ON THE ANTENNAE OF THE EGYPTIAN COTTON LEAFWORM, SPODOPTERA LITTORALIS (LEPIDOPTERA: NOCTUIDAE) 45 Abstract 46 Introduction 46 Materials and methods 48 Insects 48 Chemicals 48 Scanning electron microscopic study 49 Electrophysiology 49 Results 50 Distribution of contact chemoreceptive sensilla 50 Electrophysiology 50 Sensitivity of taste sensilla from the same flagellomere 51 Sensitivity of taste sensilla across the antenna 52 Differences between males and females 53 Discussion 54 Figures and legends for Chapter 3 58 References 67 CHAPTER 4 ELECTROPHYSIOLOGICAL RESPONSES OF ANTENNAL GUSTATORY RECEPTOR NEURONS TO APPETITIVE AND AVERSIVE COMPOUNDS 71 Abstract 71 Introduction 71 Materials and methods 73 Insects 73 Chemicals 74 Electrophysiology 74 Cross-adaptation test 75 Results 75 Responses to single amino acids and alkaloids 75 Responses to mixtures of salts and sugars 76 Responses to sodium chloride after adaptation to sucrose 76 Discussion 76 Responsiveness of antennal taste neurons to amino acids and bitter compounds 76 Interpretation of the inhibitory effects 77 Responses from individual neurons within a sensillum 78 Figures and legends for Chapter 4 80 References 89 CHAPTER 5 ANTENNAL PROJECTIONS FROM TASTE SENSILLA IN A MOTH BRAIN 91 Abstract 92 Introduction 93 Materials and methods 95 Insects 95 Staining of gustatory receptor neurons 95 Confocal laser scanning microscopy 96 Reconstructions 96 Results 97 Mass-staining of receptor neuron projections 97 Neurons originating from individual sensilla 98 Discussion 99 Segregation of mechanosensory and gustatory fibres 99 Separate target areas of gustatory receptor neurons 99 Conclusions 100 Figures and legends for Chapter 5 102 References 107 CHAPTER 6 FINAL DISCUSSION AND CONCLUSION 111 Function: Responsiveness of antennal taste sensilla to various compounds 111 Sugars and salts 111 Amino acids and bitter compounds 112 Structure: Projection areas of gustatory receptor neurons 112 Future developments 113 References 115
Glomeruli within the antennal lobe (AL) of moths are convergence sites for a large number of olfactory receptor neurons (ORNs). The ORNs target single glomeruli. In the male-specific cluster of glomeruli, the macroglomerular complex (MGC), the input is chemotypic in that each glomerulus of the MGC receives information about a specific component of the conspecific female sex pheromone. Little is known about how neurons that detect other odorants arborize in and amongst glomeruli. The present study focuses on how sex pheromones and biologically relevant semiochemicals are represented in the ALs of both sexes of the moth Spodoptera littoralis. To assess this, we optically measured odour-evoked changes of calcium concentration in the ALs. Foci of calcium increase corresponded in size and shape with anatomical glomeruli. More than one glomerulus was normally activated by a specific non-pheromonal odorant and the same glomerulus was activated by several odorants. All odorants and pheromone components tested evoked unique patterns of glomerular activity that were highly reproducible at repeated stimulations within an individual. Odour-evoked patterns were similar between individuals for a given odorant, implicating a spatial olfactory code. In addition, we demonstrated that activity patterns evoked by host-plant related volatiles are similar between males and females.
Conditioned extension of the proboscis in restrained honeybees with odor as the CS and sucrose solution––delivered to the antenna (to elicit extension of the proboscis) and then to the proboscis itself––as the UCS. In a 1st series of experiments, acquisition was found to be rapid, both in massed and in spaced trials; its associative basis was established by differential conditioning and by an explicitly unpaired control procedure. Both extinction and spontaneous recovery in massed trials were demonstrated. In experiments on the nature of the UCS, eliminating the proboscis component lowered the asymptotic level of performance, whereas eliminating the antennal component was without effect; reducing the concentration of sucrose from 20% to 7% slowed acquisition but did not lower the asymptotic level of performance; and second-order conditioning was demonstrated. In experiments on the role of the UCS, an omission contingency designed to eliminate adventitious response-reinforcer contiguity was found to have no adverse effect on acquisition. In experiments designed to analyze the resistance to acquisition found after explicitly unpaired training in the 1st experiments, no significant effect was found of prior exposure either to the CS alone or to the UCS alone, although the unpaired procedure again produced resistance that was shown to be due to inhibition rather than to inattention; extinction after paired training was found to be facilitated by unpaired presentations of the UCS. (33 ref)
In Experiments 1, 2, and 3, pigeons were trained with an ABC+ BCo discrimination, in which three stimuli, A, B, and C, were presented together and paired with food, and the compound BC was followed by nothing; they were also trained with a DEF+ Eo Fo discrimination in which stimuli E and F were presented separately and followed by nothing, whereas the compound DEF was paired with food. On completion of discrimination training, test trials with the feature A consistently revealed a higher rate of responding than with D. In Experiment 4, reinforced presentations of D were intermixed with the DEF+ Eo Fo discrimination. Test trials revealed that E enhanced responding when it was paired with F, but it had the opposite effect when paired with D. The results are seen as being more consistent with a configural than an elemental model of conditioning. (PsycINFO Database Record (c) 2012 APA, all rights reserved)
In 4 experiments, rats were initially trained with an A+ AXo discrimination in which Stimulus A by itself signaled the delivery of food, A+, whereas the simultaneous presentation of A and X was followed by nothing, AXo. In each experiment X was then paired with food prior to a test phase in which A and X were again presented for a discrimination. The discrimination was of the form A+ AXo in Experiments 1 and 2, whereas it was of the form X+ AXo for Experiments 3 and 4. In all 4 experiments the test discrimination was acquired more rapidly than a control discrimination. The results are interpreted in terms of the original A+ AXo discrimination resulting in the growth of an association between a representation of the entire AX compound and the effects of nonreinforcement.
Proboscis extension conditioning of honeybee workers was used to test the ability of bees to respond to appetitive and aversive stimuli while restrained in a harness that allows subjects to move their antennae and mouthparts (Kuwabara, 1957; Menzel, Erber, & Masuhr, 1974). Subjects were conditioned to discriminate between two odors, one associated with sucrose feeding and the other associated with a 10 V AC shock if they responded to the sucrose unconditioned stimulus (US) in the context of that odor. Most Ss readily learned to respond to the odor followed by sucrose feeding and not to the odor associated with sucrose stimulation plus shock. Furthermore, in the context of the odor associated with shock, significantly more subjects withheld or delayed proboscis extension on stimulation with the sucrose US than they did in the context of the odor associated with feeding. Thus, restrained honeybees can readily learn to avoid shock according to an odor context by withholding proboscis extension to a normally powerful releaser. Analysis of individual learning curves revealed that subjects differed markedly in performance on this task. Some learn the discrimination quickly, whereas others show different kinds of response patterns.
Animals that are primarily dependent on olfaction must obtain a description of the spatial location and the individual odor quality of environmental odor sources through olfaction alone. The variable nature of turbulent air flow makes such a remote sensing problem solvable if the animal can make use of the information conveyed by the fluctuation with time of the mixture of odor sources. Behavioral evidence suggests that such analysis takes place. An adaptive network can solve the essential problem, isolating the quality and intensity of the components within a mixture of several individual unknown odor sources. The network structure is an idealization of olfactory bulb circuitry. The dynamics of synapse change is essential to the computation. The synaptic variables themselves contain information needed by higher processing centers. The use of the same axons to convey intensity information and quality information requires time-coding of information. Covariation defines an individual odor source (object), and this may have a parallel in vision.
The odor-guided behaviors selected for presentation in this paper encompass the major areas of animal behavior, and illustrate the important principal that complex relations exist between odor-guided behaviors, hormonal state, and experiential factors. Clearly, experiences with odors at several life stages results in profound influences upon later behaviors, including those related to eating, mating, fighting, and nesting. Interestingly, only brief social encounters are needed in rats to induce such phenomena as ultrasonic calling to conspecific estrous females or their odors, preferences for estrous over non-estrous odors, and the short-term modification of feeding behaviors. Although the mechanisms behind these intriguing phenomena are poorly understood, it is noteworthy that rats can learn relatively complex concepts on the basis of odors, rivaling even the ability of our own species to learn analogous tasks by visual cues. Despite the fact that close relationships can be demonstrated between odor-guided behaviors and variables such as endocrine state and sexual experience, caution is warranted in assuming that simple causal relations exist between such variables. In normally cycling women, for example, the correlation between olfactory sensitivity and plasma levels of estradiol during the menstrual cycle is relatively high; however, attenuation of the cyclical estradiol fluctuations by oral contraceptives does not eliminate the olfactory fluctuations, suggesting the relation is not causal. In house mice, social experience can override hormonal factors in their odor-guided urine marking and submissive behaviors. Thus, even though androgen titer usually correlates with such measures, a mouse made subordinant in a social encounter will not exhibit scent marking even when its circulating testosterone is maintained at a high level by a silastic implant. Further reason for caution comes from studies that suggest olfactory input influences the endocrine systems of sexually experienced and sexually inexperienced animals in different ways. For example, in sexually experienced male rats, anosmia decreases testosterone and estradiol levels and increases corticosterone levels, whereas in sexually inexperienced ones it has no significant influence on the levels of these steroids. Taken together, such observations suggest that the causal bases of a number of the odor-guided behaviors described in this paper are complex.(ABSTRACT TRUNCATED AT 400 WORDS)
Extension of the proboscis was conditioned in restrained honeybees with odor as the conditioned stimulus (CS) and sucrose solution--delivered to the antenna (to elicit extension of the proboscis) and then to the proboscis itself--as the unconditioned stimulus (US). In a first series of experiments, acquisition was found to be very rapid, both in massed and in spaced trials; its associative basis was established by differential conditioning and by an explicitly unpaired control procedure (which produced marked resistance to acquisition in subsequent paired training); and both extinction and spontaneous recovery in massed trials were demonstrated. In a series of experiments on the nature of the US, eliminating the proboscis component was found to lower the asymptotic level of performance, whereas eliminating the antennal component was without effect; reducing the concentration of sucrose from 20% to 7% slowed acquisition but did not lower the asymptotic level of performance; and second-order conditioning was demonstrated. In a series of experiments on the role of the US, an omission contingency designed to eliminate adventitious response-reinforcer contiguity was found to have no adverse effect on acquisition. In a series of experiments designed to analyze the resistance to acquisition found after explicitly unpaired training in the first experiments, no significant effect was found of prior exposure either to the CS alone or to the US alone, although the unpaired procedure again produced substantial resistance that was shown to be due to inhibition rather than to inattention; extinction after paired training was found to be facilitated by unpaired presentations of the US. The relation between these results for honeybees and those of analogous experiments with vertebrates is considered.
Olfactory learning in the honeybee was investigated using the conditioned proboscis extension reflex on restrained individuals. We compared, under the same experimental conditions, the most commonly used conditioning procedures, i.e. 1 trial, 3 massed trials (1 min inter-trial intervals), and 3 spaced trials (10 min inter-trial intervals) procedures, using linalool as the conditioned stimulus. Two experiments were performed in which worker bees were subjected to: (1) a single test at different times (30 s to 14 days) after the conditioning procedure; (2) a first test within 3 h after the conditioning procedure, and were then retested daily (up to 5 tests). The memory trace of a learnt odorant stimulus could last for the lifetime of the bee, even after a single association with sugar. Repeated tests with 1 day inter-test duration induced a strong decrease of the response level, this effect being more pronounced after a 1-trial conditioning.
Insects are favorable subjects for neuroethological studies. Their nervous systems are relatively small and contain many individually identifiable cells. The CNS is highly compartmentalized with clear separations between multisensory higher order neuropiles in the brain and neuropiles serving sensory-motor routines in the ventral cord (Huber, 1974). The rich behavior of insects includes orientation in space and time, visual, chemical, and mechanical communication, and complex motor routines for flying, walking, swimming, nest building, defense, and attack. Learning and memory, though, are not usually considered to be a strong point of insects. Rather, insect behavior is often regarded as highly stereotyped and under tight control of genetically programmed neural circuits. This view, however, does not do justice to the insect order of Hymenoptera (bees, wasps, ants). Most Hymenopteran species care for their brood either as individual females or as a social group of females. Consequently, they regularly return to their nest site to feed, protect, and nurse the larvae, store food, and hide from adverse environmental conditions. Since they search for food (prey; nectar and pollen on flowers) at unpredictable sites, they have to learn the celestial and terrestrial cues that guide their foraging trips over long distances and allow them to find their nest sites (central place foraging; von Frisch, 1967; Seeley, 1985). They learn to relate the sun's position and sky pattern of polarized light to the time of the day (Lindauer, 1959), and landmarks are learned in relationship to the nest site within the framework of the time-compensated sun compass. The honeybee communicates direction and distance of a feeding place to hive mates by performing a ritualized body movement, the waggle dance (von Frisch, 1967). Associative learning is an essential component of the bee's central place foraging behavior and dance communication. Hive mates attending a dance performance learn the odor emanating from the dancing bee and seek it at the indicated food site. The odor, color, and shape of flowers are learned when the bee experiences these stimuli shortly before it finds food (nectar, pollen). This appetitive learning in bees has many characteristics of associative learning well known from mammalian learning studies (Menzel, 1985, 1990; Bitterman, 1988). It follows the rules of classical and operant conditioning, respectively, so that stimuli or behavioral acts are associated with evaluating stimuli. Since associative learning, especially of the classical type, is well described at the phenomenological and operational level (Rescorla, 1988), it provides a favorable approach in the search for the neural substrate underlying learning and memory.(ABSTRACT TRUNCATED AT 400 WORDS)
The 1st part of this article evaluates the extent to which 2 elemental theories of conditioning, stimulus sampling theory and the Rescorla-Wagner (1972) theory, are able to account for the influence of similarity on discrimination learning. A number of findings are reviewed that are inconsistent with predictions derived from these theories, either in their present form or in various modified forms. The 2nd part of the article is concerned with developing an alternative, configural account for discrimination learning. In contrast to previous configural theories, the present version is set within the framework of a connectionist network.
We studied the associative learning capabilities for behaviourally relevant cues in the moth Spodoptera littoralis. The moths were trained to associate a conditioned stimulus (CS), geraniol odour, with an unconditioned stimulus (US), a sucrose solution. The occurrence of a proboscis extension reflex (PER) was tested. The PER performance during acquisition increased steadily with the number of training trials. Non-associative control procedures did not result in learning. PER conditioning was achieved when the CS was presented 1-3 s before the US. A wide range of inter-trial intervals was able to support conditioning. Males and females learned equally well. Moths could to some degree learn the CS-US association after a single trial. These results demonstrate that S. littoralis females and males have a good capability to associate an odour with a reward. The neural basis of olfactory coding in moths has been well studied; thus, the moth provides a powerful system in which to examine the neurobiology of olfactory learning.
In 4 experiments, rats were initially trained with an A+ AXo discrimination in which Stimulus A by itself signaled the delivery of food, A+, whereas the simultaneous presentation of A and X was followed by nothing, AXo. In each experiment X was then paired with food prior to a test phase in which A and X were again presented for a discrimination. The discrimination was of the form A+ AXo in Experiments 1 and 2, whereas it was of the form X+ AXo for Experiments 3 and 4. In all 4 experiments the test discrimination was acquired more rapidly than a control discrimination. The results are interpreted in terms of the original A+ AXo discrimination resulting in the growth of an association between a representation of the entire AX compound and the effects of nonreinforcement.
A rearing method, based upon an artificial diet, has proved successful in establishing laboratory colonies of 36 species of the genus Euxoa . The diet is described, along with methods for controlling disease, particularly by the use of antimicrobial agents.
Many of the 36 species of Euxoa have not been reared before and their habits and life histories are little known. The data derived from their development under laboratory conditions and which can be related to seasonal distribution are recorded. Particular emphasis is given to the overwintering stage and to the occurrence of an aestivating stage.
Oviposition in Spodoptera littoralis was strongly deterred by a mixture of six compounds, benzaldehyde and the five terpenes carvacrol, eugenol, nerolidol, phytol and thymol, identified from conspecific larval frass. If one of the compounds was excluded from the mixture the deterrent effect was lost. An electroantennogram-screening was performed on 15 compounds identified from the larval frass. Of the behaviourally active compounds, dose-response relationships were obtained for benzaldehyde, eugenol and nerolidol. Seven types of receptor cells specifically tuned to compounds from the larval frass were identified by single-sensillum recordings from olfactory sensilla on the female antenna. The most abundant of these was a receptor cell type responding equally well to the three aromatic terpenes carvacrol, eugenol and thymol. Receptor cells responding to only a single terpene were found for carvacrol, eugenol, nerolidol and phytol. Receptor cells responding to other frass compounds, green leaf odours and sex pheromone components were also found. No receptor cell responding to benzaldehyde was identified.
Presentation series have not previously been rigidly tested for probable chance score, and many have allowed high scores to be made purely through chance. The author furnishes 44 series of 10 presentations of R's and L's, derived from the 1024 possible combinations of the two, by eliminating all that violate any of five described criteria. (PsycINFO Database Record (c) 2012 APA, all rights reserved)
A history of the changes in nomenclature within the genus Spodoptera is outlined. A key to the imagines of the eight species occurring in Africa and the Near East is presented, together with keys to the larvae and pupae of most of these species, including all those of economic importance in this region, as well as brief notes for identifying the eggs of six species. For each species, a separate account is given of its taxonomic features and affinities, general world and more detailed African distribution, as well as host-plants, including those of economic importance, and its vernacular names. The subspecies Spodoptera leucophlebia malagasy Viette, recorded only from the Malagasy Republic, has been raised to full specific status, and should now be known as Spodoptera malagasy Viette, stat. n.
Receptor neurones with high selectivity and sensitivity to plant odours were found within short sensilla trichodea on the antenna of both female and male Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) by using single-sensillum recording techniques. In 112 sensilla from females and forty-one from males, twenty-four different receptor neurone types were characterized according to their specificity. Altogether, twenty-six plant and three sex pheromone compounds were tested. Receptor neurones responding with high specificity to flower odours, green leaf volatiles, oviposition deterrents and other general host plant odours were identified. In twenty-one receptor neurone types, responses were elicited by one or several plant compounds, and in three types responses were elicited by sex pheromone compounds. The majority of the receptor neurones responded to only one or two of the tested compounds. In general, only one of the two receptor neurones in a sensillum responded to any of the compounds tested. An exception was a receptor neurone responding to plant odours (green leaf volatiles) and another receptor neurone responding to a sex pheromone compound ([Z]-7-dodecenyl (acetate), which occurred in the same sensillum. The majority of the receptor neurones displayed a high sensitivity to plant odours. No morphological difference was identified the different sensillum types.
Selection of a suitable site for oviposition by phytophagous insects is critical for successful development of the offspring. The behavioral events leading to oviposition are mediated to a large extent by chemical cues associated with potential host plants. Orientation and landing are primarily guided by volatile constituents of a plant, whereas assessment of a leaf surface depends on contact stimuli. Chemical mechanisms that ensure adequate spacing of progeny on limited resources include the production of oviposition-deterring pheromones as well as recognition of plant constituents released as a result of previous damage. Perception of chemical cues that affects oviposition involves receptors on antennae, tarsi, mouthparts or the ovipositor. Complex behavior such as tarsal drumming or stem runs may serve to provide increased receptor contact with chemical stimuli. Abiotic and biotic environmental factors often influence the production or release of behavior-modifying chemicals by a plant, and therefore affect oviposition preferences. Plant chemistry may be involved in associative learning, but may also lead to mistakes. Thus a clear correlation between oviposition preference and offspring success does not always exist.
Significant progress has been made recently in research on lepidopterous sex pheromones. Advances in understanding the biochemical, neurobiological, and behavioral events that results in both successful and unsuccessful pheromone communication have allowed researchers to gain new insights into the genetic control and evolution of phermone systems.
The physiology, morphology and distribution of pheromone receptors on the antennae of both sexes of Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) were investigated by electroantennogram screening, single sensillum recordings and scanning electron microscopy. The electroantennogram recordings showed maximal responses to (Z,E)-9,11-tetradecadienyl acetate in males and to (Z)-7-dodecenyl acetate in females. In subsequent single sensillum recordings, receptor cells specific for the two known pheromone components, (Z,E)-9,11-tetradecadienyl acetate and (Z,E)-9,12-tetradecadienyl acetate, as well as for the behavioural antagonist (Z)-9-tetradecenol, were found in both sexes. The sensitivity of the pheromone receptors to their respective stimuli was the same in the sexes, while the number of pheromone-specific sensilla was much lower in the female. In the male, two physiologically distinct sensillum types were differently distributed over the antennal surface. The most numerous type, containing only one receptor neurone specific for (Z,E)-9,11-tetradecadienyl acetate, was evenly distributed over the ventral antennal surface, while a sensillum type containing one neurone responding to (Z,E)-9,12-tetradecadienyl acetate and one neurone specific for (Z)-9-tetradecenol was only found among more laterally situated sensilla. This distribution was not observed in the female.
The question of which acquisition parameters govern long-term retention is important to an understanding of memory function. We investigate the effects of the time interval between learning trials on mediate (1 day)- and long-term (4 days) retention. In classical conditioning of the proboscis extension reflex, we train honeybees to associate an odorant with a sucrose reward using intertrial intervals of either 30 s, 1 min, 3 min, or 20 min. Intervals of 20 and 1 min result in stable retention but 3-min and 30-s intervals result in reduced retention after 4 days compared to that seen after 1 day. Thus, stability of long-term retention depends nonmonotonously on the intertrial interval. Reduced retention with 3-min intervals might be caused by a disruption of memory consolidation which is known to be especially sensitive to interference 3 min after a conditioning trial. Habituation and backward inhibitory learning are discussed as explanations for reduced retention with 30-s intervals.
Physiological and anatomical characteristics of antennal lobe interneurons in female Spodoptera littoralis (Boisd.) were investigated using intracellular recording and staining techniques. Responses of local interneurons and projection neurons to female sex pheromone components, host plant odours, and behaviourally active oviposition deterrents were recorded. We found local interneurons and projection neurons that responded specifically to only one or two of the tested odours, but we also found less specific cells, and neurons that responded to most of the tested odourants. These findings show that there are not only specific olfactory pathways in female moths up to the protocerebral level, but also that integration can begin in the antennal lobe. No correlation was found between the degree of specificity of either local interneurons or projection neurons and their respective morphological characteristics. Specialized and unspecialized local interneurons arborized throughout the antennal lobe. Specialized and unspecialized projection neurons had uniglomerular arborizations in the antennal lobe and sent their axons to the calyces of the mushroom body, and to the lateral horn of the protocerebrum. One specific projection neuron had multiglomerular arborizations and projected only to the lateral horn of the protocerebrum. Projection neurons arborizing in the glomeruli closest to the entrance of the antennal nerve always responded to pheromone components. No other correlations were found between the arborization pattern of projection neurons in the antennal lobe or in the protocerebrum and their response characteristics. The sensitivity of local interneurons and projection neurons was in the same range as that of receptor neurons in olfactory sensilla on the antennae, suggesting a much lower convergence in the central nervous system in females than in the pheromone-processing pathway in males.
The goal throughout this period was to understand the neural mechanisms mediating chemosensory responses and related behavior. At the same time, it was the behavior that was providing insight to the mechanisms. The behavioral approach was an exciting game of wits [emphasis added]. It proved to be a powerful tactic, and many of its findings were subsequently shown by electrophysiology to be gratifyingly accurate. --Dethier (1990)
Stimulus intensity is an important determinant for perception, learning and behaviour. We studied the effects of odorant concentration on classical conditioning involving odorants and odorant-mechanosensory compounds using the proboscis-extension reflex in the honeybee.
Our results show that high concentrations of odorant (a) support better discrimination in a feature-positive task using rewarded odorant-mechanosensory compounds versus unrewarded mechanosensory stimuli, (b) have a stronger capacity to overshadow learning of a simultaneously trained mechanosensory stimulus, and (c) induce better memory consolidation. Furthermore, honeybees were trained discriminatively to two different concentrations of one odorant. Honeybees are not able to solve this task when presented with rewarded low versus unrewarded high concentrations.
Taken together, our results suggest that high concentrations of odorant support stronger associations (are more ‘salient’) than low concentrations. Our results, however, do not indicate that honeybees can treat two different concentrations of one odorant as qualitatively different stimuli. These findings fill a gap in what is known about honeybee olfactory learning and are a first step in relating behaviour to recent advances in the physiological analysis of coding for odorant concentration in honeybees.
Odor guide behaviour in mammals
- Doty
Doty RL. Odor guide behaviour in mammals. Experientia 1986;42: 257 ± 71.
Trial and intertrial intervals
- J Gibbon
- Baldock
- Cm Locurto
- L Gold
- Terrace
Gibbon J, Baldock MD, Locurto CM, Gold L, Terrace HS. Trial and intertrial intervals. J Exp Psychol: Anim Behav Proc 1977;15:311 ± 28.
Olfactory memory and conditional discrimination in the moth Spodoptera littoralis (Lepidoptera: Noctuidae)
- Fan
Fan R-J, Hansson BS. Olfactory memory and conditional discrimina-tion in the moth Spodoptera littoralis (Lepidoptera: Noctuidae). Soc Neurosci Abstr 1997;23:781.
Insects and flowers: the biology of a partnership
- Fg Barth
Barth FG. Insects and flowers: the biology of a partnership. Princeton,
NJ: Princeton Univ. Press, 1991. (MA, Trans.) Biederman-Thorson.
Trial and intertrial intervals
- Gibbon