Multimodal interaction in the insect brain.

BACKGROUND: The magnitude of multimodal enhancement in the brain is believed to depend on the stimulus intensity and timing. Such an effect has been found in many species, but has not been previously investigated in insects. RESULTS: We investigated the responses to multimodal stimuli consisting of an odour and a colour in the antennal lobe and mushroom body of the moth Manduca sexta. The mushroom body shows enhanced responses for multimodal stimuli consisting of a general flower odour and a blue colour. No such effect was seen for a bergamot odour. The enhancement shows an inverse effectiveness where the responses to weaker multimodal stimuli are amplified more than those to stronger stimuli. Furthermore, the enhancement depends on the precise timing of the two stimulus components. CONCLUSIONS: Insect multimodal processing show both the principle of inverse effectiveness and the existence of an optimal temporal window.

Comparing Analysis Methods in Functional Calcium Imaging of the Insect Brain.

We investigate four different methods for background estimation in calcium imaging of the insect brain and evaluate their performance on six data sets consisting of data recorded from two sites in two species of moths. The calcium fluorescence decay curve outside the potential response is estimated using either a low-pass filter or constant, linear or polynomial regression, and is subsequently used to calculate the magnitude, latency and duration of the response. The magnitude and variance of the responses that are obtained by the different methods are compared, and, by computing the receiver operating characteristics of a classifier based on response magnitude, we evaluate the ability of each method to detect the stimulus type and conclude that a polynomial approximation of the background gives the overall best result.

Learning of multi-modal stimuli in hawkmoths.

The hawkmoth, Manduca sexta, uses both colour and odour to find flowers when foraging for nectar. In the present study we investigated how vision and olfaction interact during learning. Manduca sexta were equally attracted to a scented blue coloured feeding target (multimodal stimulus) as to one that does not carry any scent (unimodal stimulus; visual) or to an invisible scented target (unimodal stimulus; odour). This naive attraction to multimodal as well as to unimodal stimuli could be manipulated through training. Moths trained to feed from a blue, scented multimodal feeding target will, when tested in a set-up containing all three feeding targets, select the multimodal target as well as the scented, unimodal target, but ignore the visual target. Interestingly, moths trained to feed from a blue, unimodal visual feeding target will select the visual target as well as the scented, multimodal target, but ignore the unimodal odour target. Our results indicate that a multimodal target is perceived as two separate modalities, colour and odour, rather than as a unique fused target. These findings differ from earlier studies of desert ants that perceive combined visual and odour signals as a unique fused stimulus following learning trials.

Neural coding merges sex and habitat chemosensory signals in an insect herbivore.

Understanding the processing of odour mixtures is a focus in olfaction research. Through a neuroethological approach, we demonstrate that different odour types, sex and habitat cues are coded together in an insect herbivore. Stronger flight attraction of codling moth males, Cydia pomonella, to blends of female sex pheromone and plant odour, compared with single compounds, was corroborated by functional imaging of the olfactory centres in the insect brain, the antennal lobes (ALs). The macroglomerular complex (MGC) in the AL, which is dedicated to pheromone perception, showed an enhanced response to blends of pheromone and plant signals, whereas the response in glomeruli surrounding the MGC was suppressed. Intracellular recordings from AL projection neurons that transmit odour information to higher brain centres, confirmed this synergistic interaction in the MGC. These findings underscore that, in nature, sex pheromone and plant odours are perceived as an ensemble. That mating and habitat cues are coded as blends in the MGC of the AL highlights the dual role of plant signals in habitat selection and in premating sexual communication. It suggests that the MGC is a common target for sexual and natural selection in moths, facilitating ecological speciation.

Discrimination training with multimodal stimuli changes activity in the mushroom body of the hawkmoth Manduca sexta.

BACKGROUND: The mushroom bodies of the insect brain play an important role in olfactory processing, associative learning and memory. The mushroom bodies show odor-specific spatial patterns of activity and are also influenced by visual stimuli. METHODOLOGY/PRINCIPAL FINDINGS: Functional imaging was used to investigate changes in the in vivo responses of the mushroom body of the hawkmoth Manduca sexta during multimodal discrimination training. A visual and an odour stimulus were presented either together or individually. Initially, mushroom body activation patterns were identical to the odour stimulus and the multimodal stimulus. After training, however, the mushroom body response to the rewarded multimodal stimulus was significantly lower than the response to the unrewarded unimodal odour stimulus, indicating that the coding of the stimuli had changed as a result of training. The opposite pattern was seen when only the unimodal odour stimulus was rewarded. In this case, the mushroom body was more strongly activated by the multimodal stimuli after training. When no stimuli were rewarded, the mushroom body activity decreased for both the multimodal and unimodal odour stimuli. There was no measurable response to the unimodal visual stimulus in any of the experiments. These results can be explained using a connectionist model where the mushroom body is assumed to be excited by olfactory stimulus components, and suppressed by multimodal configurations. CONCLUSIONS: Discrimination training with multimodal stimuli consisting of visual and odour cues leads to stimulus specific changes in the in vivo responses of the mushroom body of the hawkmoth.

Floral to green: mating switches moth olfactory coding and preference.

Mating induces profound physiological changes in a wide range of insects, leading to behavioural adjustments to match the internal state of the animal. Here, we show for the first time, to our knowledge, that a noctuid moth switches its olfactory response from food to egg-laying cues following mating. Unmated females of the cotton leafworm (Spodoptera littoralis) are strongly attracted to lilac flowers (Syringa vulgaris). After mating, attraction to floral odour is abolished and the females fly instead to green-leaf odour of the larval host plant cotton, Gossypium hirsutum. This behavioural switch is owing to a marked change in the olfactory representation of floral and green odours in the primary olfactory centre, the antennal lobe (AL). Calcium imaging, using authentic and synthetic odours, shows that the ensemble of AL glomeruli dedicated to either lilac or cotton odour is selectively up- and downregulated in response to mating. A clear-cut behavioural modulation as a function of mating is a useful substrate for studies of the neural mechanisms underlying behavioural decisions. Modulation of odour-driven behaviour through concerted regulation of odour maps contributes to our understanding of state-dependent choice and host shifts in insect herbivores.

Flight behaviour of the hawkmoth Manduca sexta towards unimodal and multimodal targets.

Here, we analyse the flight behaviour of the hawkmoth Manduca sexta while it approaches three different artificial flower stimuli: a clearly visible blue flower, an invisible scented flower and a flower that is both visible and scented. By tracking the moths in fine temporal detail, we find that flight towards an artificial flower differs depending on whether the stimulus is unimodal (either visual or olfactory) or multimodal (both visual and olfactory). In all three cases, the moth reduces its speed as it nears the target but the speed is higher overall when the visual stimulus is not present. Visual feedback, as well as the concentration gradient of the odour, is used to guide the moths towards the stimulus. The main difference in flight behaviour between an approach towards a visual and a multimodal stimulus is that the olfactory information makes the moths turn more rapidly towards the multimodal stimulus. We also find that moths extend their proboscises in front of a clearly visible feeder independent of whether an odour is present. In contrast, a scented transparent artificial flower only occasionally triggers this response.

Behaviour towards an unpreferred colour: can green flowers attract foraging hawkmoths?

Naïve hawkmoths (Manduca sexta) learn from a single trial to approach and attempt to feed from an artificial flower of an innately unpreferred green colour even when a distractor flower with a preferred yellow colour is present. In some of the animals, the choice of the innately unpreferred colour during free-flight testing persists for several days despite not being rewarded and eventually leads to starvation. The results show that moths exhibit a very strong flower constancy that is not limited to the colours of nectar flowers.

Interaction of visual and odour cues in the mushroom body of the hawkmoth Manduca sexta.

The responses to bimodal stimuli consisting of odour and colour were recorded using calcium-sensitive optical imaging in the mushroom bodies of the hawkmoth Manduca sexta. The results show that the activity in the mushroom bodies is influenced by both olfaction and vision. The interaction between the two modalities depends on the odour and the colour of the visual stimulus. A blue stimulus suppressed the response to a general flower scent (phenylacetaldehyde). By contrast, the response to a green leaf scent (1-octanol) was enhanced by the presence of the blue stimulus. A green colour had no influence on these odours but caused a marked increase in the response to an odour component (benzaldehyde) of the hawkmoth-pollinated Petunia axillaris.

The absolute threshold of colour vision in the horse.

Arrhythmic mammals are active both during day and night if they are allowed. The arrhythmic horses are in possession of one of the largest terrestrial animal eyes and the purpose of this study is to reveal whether their eye is sensitive enough to see colours at night. During the day horses are known to have dichromatic colour vision. To disclose whether they can discriminate colours in dim light a behavioural dual choice experiment was performed. We started the training and testing at daylight intensities and the horses continued to choose correctly at a high frequency down to light intensities corresponding to moonlight. One Shetland pony mare, was able to discriminate colours at 0.08 cd/m(2), while a half blood gelding, still discriminated colours at 0.02 cd/m(2). For comparison, the colour vision limit for several human subjects tested in the very same experiment was also 0.02 cd/m(2). Hence, the threshold of colour vision for the horse that performed best was similar to that of the humans. The behavioural results are in line with calculations of the sensitivity of cone vision where the horse eye and human eye again are similar. The advantage of the large eye of the horse lies not in colour vision at night, but probably instead in achromatic tasks where presumably signal summation enhances sensitivity.

Colour perception in a dichromat.

Most mammals have dichromatic colour vision based on two different types of cones: a short-wavelength-sensitive cone and a long-wavelength-sensitive cone. Comparing the signal from two cone types gives rise to a one-dimensional chromatic space when brightness is excluded. The so-called ;neutral point' refers to the wavelength that the animal cannot distinguish from achromatic light such as white or grey because it stimulates both cone types equally. The question is: how do dichromats perceive their chromatic space? Do they experience a continuous scale of colours or does the neutral point divide their chromatic space into two colour categories, i.e. into colours of either short or long wavelengths? We trained horses to different colour combinations in a two-choice behavioural experiment and tested their responses to the training and test colours. The horses chose colours according to their similarity/relationship to rewarded and unrewarded training colours. There was no evidence for a categorical boundary at the neutral point or elsewhere. This study suggests that dichromats perceive their chromatic space as a continuous scale of colours, treating the colour at the neutral point as any other colour they can distinguish.

Colour preferences influences odour learning in the hawkmoth, Macroglossum stellatarum.

The hummingbird hawkmoth, Macroglossum stellatarum, learns colour fast and reliably. It has earlier been shown to spontaneously feed from odourless artificial flowers. Now, we have studied odour learning. The moths were trained to discriminate feeders of the same colour but marked with different odours. They did not learn to discriminate two natural flower odours when they were presented with the innately preferred colour blue, but they did learn this discrimination combined with yellow or green colours that are less attractive to the moth. The yellow colour could be trained to become as attractive as the innately preferred blue colour and the blue colour could be trained to become less attractive. This is the first proof of odour learning in a diurnal moth. The results show that M. stellatarum can use more than one modality in their foraging behaviour and that the system is plastic. By manipulating the preferences for the different colours, their influence on odour learning could be changed.

The relative importance of olfaction and vision in a diurnal and a nocturnal hawkmoth.

Nectar-feeding animals can use vision and olfaction to find rewarding flowers and different species may give different weight to the two sensory modalities. We have studied how a diurnal or nocturnal lifestyle affects the weight given to vision and olfaction. We tested naïve hawkmoths of two species in a wind tunnel, presenting an odour source and a visual stimulus. Although the two species belong to the same subfamily of sphingids, the Macroglossinae, their behaviour was quite different. The nocturnal Deilephila elpenor responded preferably to the odour while the diurnal Macroglossum stellatarum strongly favoured the visual stimulus. Since a nocturnal lifestyle is ancestral for sphingids, the diurnal species, M. stellatarum, has evolved from nocturnal moths that primarily used olfaction. During bright daylight visual cues may have became more important than odour.

Colour constancy in diurnal and nocturnal hawkmoths.

Diurnal and nocturnal hawkmoths have been shown to use colour vision for flower discrimination. Here, we present evidence that the nocturnal hawkmoth Deilephila elpenor and the diurnal hawkmoth Macroglossum stellatarum also have colour constancy. Colour constancy was shown in D. elpenor in two multiple-choice experiments with five different bluish colour patches under white and blue illumination and with five yellowish colour patches under white, blue and yellow illumination. The mechanism underlying colour constancy in both species was investigated in two dual-choice experiments. The choice behaviour is consistent with the use of the von Kries coefficient law. Although the moths have colour constancy, they react to the colour of the illumination. They make fewer choices when tested under the changed illumination, where they never receive a reward, compared with the training illumination. Even if colour constancy can be explained by a von Kries adaptation mechanism, the fact that the animals discriminate between different illuminations indicates that some additional process must be involved.

Colour vision in diurnal and nocturnal hawkmoths.

Diurnal and nocturnal hawkmoths (Sphingidae, Lepidoptera) have three spectral types of receptor sensitive to ultraviolet, blue and green light. As avid flower visitors and pollinators, they use olfactory and visual cues to find and recognise flowers. Moths of the diurnal species Macroglossum stellatarum and the nocturnal species Deilephila elpenor, Hyles lineata and Hyles gallii use and learn the colour of flowers. Nocturnal species can discriminate flowers at starlight intensities when humans and honeybees are colour-blind. M. stellatarum can use achromatic, intensity-related cues if colour cues are absent, and this is probably also true for D. elpenor. Both species can recognise colours even under a changed illumination colour.

Scotopic colour vision in nocturnal hawkmoths.

Humans are colour-blind at night, and it has been assumed that this is true of all animals. But colour vision is as useful for discriminating objects at night as it is during the day. Here we show, through behavioural experiments, that the nocturnal hawkmoth Deilephila elpenor uses colour vision to discriminate coloured stimuli at intensities corresponding to dim starlight (0.0001 cd x m(-2)). It can do this even if the illumination colour changes, thereby showing colour constancy-a property of true colour vision systems. In identical conditions humans are completely colour-blind. Our calculations show that the possession of three photoreceptor classes reduces the absolute sensitivity of the eye, which indicates that colour vision has a high ecological relevance in nocturnal moths. In addition, the photoreceptors of a single ommatidium absorb too few photons for reliable discrimination, indicating that spatial and/or temporal summation must occur for colour vision to be possible. Taken together, our results show that colour vision occurs at nocturnal intensities in a biologically relevant context.