Habituation leads to short but not long term memory formation in mosquito larvae.

In animals, memory allows to remember important locations and conserve energy by not responding to irrelevant stimuli. However, memory formation and maintenance are metabolically costly, making it worthwhile to understand the mechanisms underlying different types of memory and their adaptive value. In this study, we investigated the memory persistence of Aedes aegypti mosquito larvae, after habituation to a visual stimulus. We used an automated tracking system for quantifying the response of mosquito larvae to the passage of a shadow, simulating an approaching predator. First, we compared different retention times, from 4 min to 24 h, and found that mosquito larvae only exhibited memory capabilities less than 3 h after training. Secondly, we investigated the role of inter-trial intervals in memory formation. In contrast to other aquatic invertebrates, mosquito larvae showed no long-term memory even at long inter-trial intervals (i.e., 5 min and 10 min). Our results are discussed in relation to the ecological constraints.

Assessing learning in mosquito larvae using video-tracking.

Mosquito larvae display a stereotyped escape response when they rest attached to the water surface. It consists in detaching from the surface and diving, to return to the surface after a brief time. It has been shown that this response can be evoked several times, by repeatedly presenting a moving shadow. Diving triggered by a potential danger revealed as a simple bioassay for investigating behavioural responses in mosquito larvae, in particular their ability to learn. In the present work, we describe an automated system, based on video-tracking individuals, and extracting quantitative data of their movements. We validated our system, by reinvestigating the habituation response of larvae of Aedes aegypti reared in the laboratory, and providing original data on field-collected larvae of genera Culex and Anopheles. Habituation could be demonstrated to occur in all the species, even though it was not possible to induce dishabituation in Culex and Anopheles mosquitoes. In addition to non-associative learning, we characterised motor activity in the studied species, thanks to the possibility offered by the tracking system to extract multiple variables. The here-described system and algorithms can be easily adapted to multiple experimental situations and variables of interest.

Habituation in Aedes aegypti mosquito larvae is context specific.

Mosquito larvae live in water and perform a stereotyped escape response when a moving object projects its shadow on the water surface, indicating potential risk of predation. Repeated presentations of the shadow induce a decrease in the response as a result of habituation, a form of non-associative learning defined as the progressive and reversible decrease in response to a specific reiterative innocuous stimulus. Nevertheless, habituation can be context specific, which indicates an association between the context and the stimulus. The aim of this work was to study context specificity in habituation in mosquito larvae Aedes aegypti. Larvae were individually placed in Petri dishes positioned over black, white or black-white striped cardboard as background (visual context). Larvae were presented with a shadow produced by a cardboard square (training) over the course of 15 trials. After the 15th trial, the background was changed and the stimulus was presented once again (test). To analyse habituation in different contexts, we developed a series of learning curve models. We performed a Bayesian model selection procedure using those models and the data from the experiments to find which model best described the results. The selected model was a power law learning curve with six parameters (habituation rate; context-specific asymptotic habituation response, with one parameter per context, i.e. 3 parameters in total; response increase; and autocorrelation) describing the whole experimental setup with a generalised r2 of 0.96. According to the model, a single habituation rate would indicate that habituation was independent of the context, whilst asymptotic habituation would be context specific. If the background was changed after training, there was an increase in response in the test, evincing context specificity in habituation.

Honeybees generalize among pollen scents from plants flowering in the same seasonal period.

When honey bees (Apis mellifera) feed on flowers, they extend their proboscis to absorb the nectar, i.e. they perform the proboscis extension response (PER). The presence of pollen and/or nectar can be associated with odors, colors or visual patterns, which allows honey bees to recognize food sources in the environment. Honey bees can associate similar, though different, stimuli with the presence of food; i.e. honey bees discriminate and generalize among stimuli. Here, we evaluated generalization among pollen scents from six different plant species. Experiments were based on the PER conditioning protocol over two phases: (1) conditioning, in which honey bees associated the scent of each pollen type with sucrose, and (2) test, in which honey bees were presented with a novel scent, to evaluate generalization. Generalization was evinced by honey bees extending their proboscis to a novel scent. The level of PER increased over the course of the conditioning phase for all pollen scents. Honey bees generalized pollen from Pyracantha coccinea and from Hypochaeris radicata These two plants have different amounts of protein and are not taxonomically related. We observed that the flowering period influences the olfactory perceptual similarity and we suggest that both pollen types may share volatile compounds that play key roles in perception. Our results highlight the importance of analyzing the implications of the generalization between pollen types of different nutritional quality. Such studies could provide valuable information for beekeepers and agricultural producers, as the generalization of a higher quality pollen can benefit hive development, and increase pollination and honey production.

Glyphosate impairs learning in Aedes aegypti mosquito larvae at field-realistic doses.

Glyphosate is the most widely used herbicide in the world. Over the past few years, the number of studies revealing deleterious effects of glyphosate on non-target species has been increasing. Here, we studied the impact of glyphosate at field-realistic doses on learning in mosquito larvae (Aedes aegypti). Larvae of A. aegypti live in small bodies of water and perform a stereotyped escape response when a moving object projects its shadow on the water surface. Repeated presentations of an innocuous visual stimulus induce a decrease in response due to habituation, a non-associative form of learning. In this study, different groups of larvae were reared in water containing different concentrations of glyphosate that are commonly found in the field (50 µg l-1, 100 µg l-1, 210 µg l-1 and 2 mg l-1). Larvae reared in a glyphosate solution of 2 mg l-1 (application dose) could complete their development. However, glyphosate at a concentration of 100 µg l-1 impaired habituation. A dose-dependent deleterious effect on learning ability was observed. This protocol opens new avenues to further studies aimed at understanding how glyphosate affects non-target organisms, such as insects. Habituation in mosquito larvae could serve as a parameter for testing the impact of pollutants in the water.

Learning in mosquito larvae (Aedes aegypti): Habituation to a visual danger signal.

In spite of the mosquito Aedes aegypti being a vector of several infectious diseases, a limited number of studies has been undertaken on learning in this species. Moreover, larval stages have been neglected as model organisms, although they are active, aquatic and perform stereotyped behavioural responses, e.g. the escape response when disturbed. To study the learning abilities of mosquito larvae, we focused on habituation, a form of non-associative learning widely studied in vertebrates and invertebrates. Habituation was defined as the progressive and reversible decrease in response to a reiterative stimulus. We first aimed at confirming habituation of the escape response in mosquito larvae (4th instar). Then, we determined whether a mnesic trace was established. Larvae were individually stimulated with a visual danger stimulus inducing the escape response. We set up a protocol for testing larvae individually, allowing the control of different parameters that are crucial for the study of cognitive abilities. After 15 trials, the escape response of mosquitoes was significantly lower. A disturbance stimulus presented after the 15th trial, induced the escape response and reversed habituation. Retention was confirmed up to 1h after the last habituation trial. This original bioassay can be adapted for studying the physiology of learning and memory in mosquito larvae, for analysing the effects of chemicals in the water, the characterisation of the cognitive abilities related to the life history of different mosquito species across preimaginal stages.

Chemical structure of odorants and perceptual similarity in ants.

Animals are often immersed in a chemical world consisting of mixtures of many compounds rather than of single substances, and they constantly face the challenge of extracting relevant information out of the chemical landscape. To this purpose, the ability to discriminate among different stimuli with different valence is essential, but it is also important to be able to generalise, i.e. to treat different but similar stimuli as equivalent, as natural variation does not necessarily affect stimulus valence. Animals can thus extract regularities in their environment and make predictions, for instance about distribution of food resources. We studied perceptual similarity of different plant odours by conditioning individual carpenter ants to one odour, and subsequently testing their response to another, structurally different odour. We found that asymmetry in generalisation, where ants generalise from odour A to B, but not from B to A, is dependent on both chain length and functional group. By conditioning ants to a binary mixture, and testing their reaction to the individual components of the mixture, we show that overshadowing, where parts of a mixture are learned better than others, is rare. Additionally, generalisation is dependent not only on the structural similarity of odorants, but also on their functional value, which might play a crucial role. Our results provide insight into how ants make sense of the complex chemical world around them, for example in a foraging context, and provide a basis with which to investigate the neural mechanisms behind perceptual similarity.

Experience-dependent modulation of antennal sensitivity and input to antennal lobes in male moths (Spodoptera littoralis) pre-exposed to sex pheromone.

Sex pheromones are intraspecific olfactory signals emitted by one sex to attract a potential mating partner. Behavioural responses to sex pheromones are generally highly stereotyped. However, they can be modulated by experience, as male moths previously exposed to female sex pheromone respond with a lower threshold upon further detection, even after long delays. Here, we address the question of the neural mechanisms underlying such long-term modulation. As previous work has shown increased responses to pheromone in central olfactory neurons, we asked whether brief exposure to the pheromone increases input activity from olfactory receptor neurons. Males pre-exposed to sex pheromone exhibited increased peripheral sensitivity to the main pheromone component. Among nine antennal genes targeted as putatively involved in pheromone reception, one encoding a pheromone-binding protein showed significant upregulation upon exposure. In the primary olfactory centre (antennal lobe), the neural compartment processing the main pheromone component was enlarged after a brief pheromone exposure, thus suggesting enduring structural changes. We hypothesise that higher peripheral sensitivity following pre-exposure leads to increased input to the antennal lobe, thus contributing to the structural and functional reorganization underlying a stable change in behaviour.

Learning and perceptual similarity among cuticular hydrocarbons in ants.

Nestmate recognition in ants is based on perceived differences in a multi-component blend of hydrocarbons that are present on the insect cuticle. Although supplementation experiments have shown that some classes of hydrocarbons, such as methyl branched alkanes and alkenes, have a salient role in nestmate recognition, there was basically no information available on how ants detect and perceive these molecules. We used a new conditioning procedure to investigate whether individual carpenter ants could associate a given hydrocarbon (linear or methyl-branched alkane) to sugar reward. We then studied perceptual similarity between a hydrocarbon previously associated with sugar and a novel hydrocarbon. Ants learnt all hydrocarbon-reward associations rapidly and with the same efficiency, regardless of the structure of the molecules. Ants could discriminate among a large number of pairs of hydrocarbons, but also generalised. Generalisation depended both on the structure of the molecule and the animal's experience. For linear alkanes, generalisation was observed when the novel molecule was smaller than the conditioned one. Generalisation between pairs of methyl-alkanes was high, while generalisation between hydrocarbons that differed in the presence or absence of a methyl group was low, suggesting that chain length and functional group might be coded independently by the ant olfactory system. Understanding variations in perception of recognition cues in ants is necessary for the general understanding of the mechanisms involved in social recognition processes based on chemical cues.

Long-term olfactory memories are stabilised via protein synthesis in Camponotus fellah ants.

Ants exhibit impressive olfactory learning abilities. Operant protocols in which ants freely choose between rewarded and non-rewarded odours have been used to characterise associative olfactory learning and memory. Yet, this approach precludes the use of invasive methods allowing the dissection of molecular bases of learning and memory. An open question is whether the memories formed upon olfactory learning that are retrievable several days after training are indeed based on de novo protein synthesis. Here, we addressed this question in the ant Camponotus fellah using a conditioning protocol in which individually harnessed ants learn an association between odour and reward. When the antennae of an ant are stimulated with sucrose solution, the insect extends its maxilla-labium to absorb the solution (maxilla-labium extension response). We differentially conditioned ants to discriminate between two long-chain hydrocarbons, one paired with sucrose and the other with quinine solution. Differential conditioning leads to the formation of a long-term memory retrievable at least 72 h after training. Long-term memory consolidation was impaired by the ingestion of cycloheximide, a protein synthesis blocker, prior to conditioning. Cycloheximide did not impair acquisition of either short-term memory (10 min) or early and late mid-term memories (1 or 12 h). These results show that, upon olfactory learning, ants form different memories with variable molecular bases. While short- and mid-term memories do not require protein synthesis, long-term memories are stabilised via protein synthesis. Our behavioural protocol opens interesting research avenues to explore the cellular and molecular bases of olfactory learning and memory in ants.

Rapid decision-making with side-specific perceptual discrimination in ants.

BACKGROUND: Timely decision making is crucial for survival and reproduction. Organisms often face a speed-accuracy trade-off, as fully informed, accurate decisions require time-consuming gathering and treatment of information. Optimal strategies for decision-making should therefore vary depending on the context. In mammals, there is mounting evidence that multiple systems of perceptual discrimination based on different neural circuits emphasize either fast responses or accurate treatment of stimuli depending on the context. METHODOLOGY/PRINCIPAL FINDINGS: We used the ant Camponotus aethiops to test the prediction that fast information processing achieved through direct neural pathways should be favored in situations where quick reactions are adaptive. Social insects discriminate readily between harmless group-members and dangerous strangers using easily accessible cuticular hydrocarbons as nestmate recognition cues. We show that i) tethered ants display rapid aggressive reactions upon presentation of non-nestmate odor (120 to 160 ms); ii) ants' aggressiveness towards non-nestmates can be specifically reduced by exposure to non-nestmate odor only, showing that social interactions are not required to alter responses towards non-nestmates; iii) decision-making by ants does not require information transfer between brain hemispheres, but relies on side-specific decision rules. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest that first-order olfactory processing centers (up to the antennal lobes) are likely to play a key role in ant nestmate recognition. We hypothesize that the coarse level of discrimination achieved in the antennal lobes early in odor processing provides enough information to determine appropriate behavioral responses towards non-nestmates. This asks for a reappraisal of the mechanisms underlying social recognition in insects.

Associative learning in ants: conditioning of the maxilla-labium extension response in Camponotus aethiops.

Associative learning has been studied in many vertebrates and invertebrates. In social insects, the proboscis extension response conditioning of honey bees has been widely used for several decades. However, a similar paradigm has not been developed for ants, which are advanced social insects showing different morphological castes and a plethora of life histories. Here we present a novel conditioning protocol using Camponotus aethiops. When the antennae of a harnessed ant are stimulated with sucrose solution, the ant extends its maxilla-labium to absorb the sucrose. We term this the "maxilla-labium extension response" (MaLER). MaLER could be conditioned by forward pairing an odour (conditioned stimulus) with sucrose (unconditioned stimulus) in the course of six conditioning trials (absolute conditioning). In non-rewarded tests following conditioning, ants gave significantly higher specific responses to the conditioned stimulus than to a novel odour. When trained for differential conditioning, ants discriminated between the odour forward-paired with sucrose and an odour forward-paired with quinine (a putative aversive stimulus). In both absolute and differential conditioning, memory lasted for at least 1h. MaLER conditioning allows full control of the stimulation sequence, inter-stimulus and inter-trial intervals and satiety, which is crucial for any further study on associative learning in ants.

Ants recognize foes and not friends.

Discriminating among individuals and rejecting non-group members is essential for the evolution and stability of animal societies. Ants are good models for studying recognition mechanisms, because they are typically very efficient in discriminating 'friends' (nest-mates) from 'foes' (non-nest-mates). Recognition in ants involves multicomponent cues encoded in cuticular hydrocarbon profiles. Here, we tested whether workers of the carpenter ant Camponotus herculeanus use the presence and/or absence of cuticular hydrocarbons to discriminate between nest-mates and non-nest-mates. We supplemented the cuticular profile with synthetic hydrocarbons mixed to liquid food and then assessed behavioural responses using two different bioassays. Our results show that (i) the presence, but not the absence, of an additional hydrocarbon elicited aggression and that (ii) among the three classes of hydrocarbons tested (unbranched, mono-methylated and dimethylated alkanes; for mono-methylated alkanes, we present a new synthetic pathway), only the dimethylated alkane was effective in eliciting aggression. Our results suggest that carpenter ants use a fundamentally different mechanism for nest-mate recognition than previously thought. They do not specifically recognize nest-mates, but rather recognize and reject non-nest-mates bearing odour cues that are novel to their own colony cuticular hydrocarbon profile. This begs for a reappraisal of the mechanisms underlying recognition systems in social insects.

The mandible opening response: quantifying aggression elicited by chemical cues in ants.

Social insects have evolved efficient recognition systems guaranteeing social cohesion and protection from enemies. To defend their territories and threaten non-nestmate intruders, ants open their mandibles as a first aggressive display. Albeit chemical cues play a major role in discrimination between nestmates and non-nestmates, classical bioassays based on aggressive behaviour were not particularly effective in disentangling chemical perception and behavioural components of nestmate recognition by means of categorical variables. We therefore developed a novel bioassay that accurately isolates chemical perception from other cues. We studied four ant species: Camponotus herculeanus, C. vagus, Formica rufibarbis and F. cunicularia. Chemical analyses of cuticular extracts of workers of these four species showed that they varied in the number and identity of compounds and that species of the same genus have more similar profiles. The antennae of harnessed ants were touched with a glass rod coated with the cuticular extract of (a) nestmates, (b) non-nestmates of the same species, (c) another species of the same genus and (d) a species of a different genus. The mandible opening response (MOR) was recorded as the aggressive response. In all assayed species, MOR significantly differed among stimuli, being weakest towards nestmate odour and strongest towards odours originating from ants of a different genus. We thus introduce here a new procedure suitable for studying the chemical basis of aggression in ants.

Olfactory blocking and odorant similarity in the honeybee.

Blocking occurs when previous training with a stimulus A reduces (blocks) subsequent learning about a stimulus B, when A and B are trained in compound. The question of whether blocking exists in olfactory conditioning of proboscis extension reflex (PER) in honeybees is under debate. The last published accounts on blocking in honeybees state that blocking occurs when odors A and B are similar (the "similarity hypothesis"). We have tested this hypothesis using four odors (1-octanol, 1-nonanol, eugenol, and limonene) chosen on the basis of their chemical and physiological similarity (experiment 1). We established a generalization matrix that measured perceptual similarity. Bees in the "block group" were first trained with an odor A and, in the second phase, with the mixture AB. Bees in the "novel group" (control group) were first trained with an odor N and, in the second phase, with the mixture AB. After conditioning, bees in both groups were tested for their response to B. We assayed all 24 possible combinations for the four odors standing for A, B, and N. We found blocking in four cases, augmentation in two cases, and no difference in 18 cases; odor similarity could not account for these results. We also repeated the experiments with those six odor combinations that gave rise to the similarity hypothesis (experiment 2: 1-hexanol, 1-octanol, geraniol) and found augmentation in one and no effect in five cases. Thus, blocking is not a consistent phenomenon, nor does it depend on odor similarity.

Perceptual and neural olfactory similarity in honeybees.

The question of whether or not neural activity patterns recorded in the olfactory centres of the brain correspond to olfactory perceptual measures remains unanswered. To address this question, we studied olfaction in honeybees Apis mellifera using the olfactory conditioning of the proboscis extension response. We conditioned bees to odours and tested generalisation responses to different odours. Sixteen odours were used, which varied both in their functional group (primary and secondary alcohols, aldehydes and ketones) and in their carbon-chain length (from six to nine carbons). The results obtained by presentation of a total of 16 x 16 odour pairs show that (i) all odorants presented could be learned, although acquisition was lower for short-chain ketones; (ii) generalisation varied depending both on the functional group and the carbon-chain length of odours trained; higher generalisation was found between long-chain than between short-chain molecules and between groups such as primary and secondary alcohols; (iii) for some odour pairs, cross-generalisation between odorants was asymmetric; (iv) a putative olfactory space could be defined for the honeybee with functional group and carbon-chain length as inner dimensions; (v) perceptual distances in such a space correlate well with physiological distances determined from optophysiological recordings of antennal lobe activity. We conclude that functional group and carbon-chain length are inner dimensions of the honeybee olfactory space and that neural activity in the antennal lobe reflects the perceptual quality of odours.