Cryptic castes,social context and colony defence in a social bee,Tetragonula carbonaria

Division of labour in social insect colonies is facilitated in two ways: through temporal sharing of tasks or by morphologically specialised castes. In casteless species, colony defence is maintained by morphologically indistinct workers, who lack the obvious defensive specialisation of polymorphic species. Discrimination of intruders is carried out via antenna, which also detects defensive social cues such as alarm pheromones. Despite their functional importance however, antennal morphology is rarely considered in studies of nestmate recognition. We investigated antennal morphology and the necessity of social cues in mediating defensive behaviour across differentially tasked workers of a casteless social bee, Tetragonula carbonaria. Our results suggest that the current understanding of division of labour in casteless worker species remains poorly understood, with differences in antennal morphology and aggression creating morphologically and behaviourally distinct ‘cryptic castes’. Further, we found that defensive behaviour was only elicited near nest odours, highlighting the importance of mediating aggression among workers.     

Peripheral modulation of olfaction by physiological state in the Egyptian leaf worm Spodoptera littoralis (Lepidoptera: Noctuidae)

Insects show behavioural plasticity based on their physiological state. Deprivation from a resource will normally make them more responsive to it or to perform behaviour increasing the probability of encountering such a resource. Modulation of the olfactory system has been shown mainly in the central nervous system, but also in the periphery. In this study, antennal sensitivity of females of the Egyptian cotton leaf worm Spodoptera littoralis to different plant and sex pheromone odours was measured using electroantennography (EAG). Different mating status, age, and feeding status were used to investigate peripheral changes in olfactory responses. Virgin females were found to be more sensitive to plant odours and sex pheromone compared to mated females. Age also had an impact on antennal sensitivity, 2 and 4 days old females being more sensitive than just-emerged ones. Such an increase in sensitivity could be explained by maturation of olfactory receptor neurons after emergence or by an increase in motivation as the available expected life time remaining decreases. Finally, feeding status did not modify the antennal sensitivity of females. A peripheral modulation could thus be demonstrated in the olfactory system of female Spodoptera littoralis at certain life stages.     

Temporal modulation and adaptive control of the behavioural response to odours in Rhodnius prolixus

It has been demonstrated in several insect species that a circadian clock makes the whole of antennal chemoreceptors more sensitive during a particular temporal window every day. This assessment raises the question about how insects exhibiting bimodal activity handle their sensitivity to odours which are relevant at different moments of the day. To shed some light on this problem, we studied in Rhodnius prolixus the daily dynamics of their responsiveness to CO₂ (host-associated cue) and aggregation cues (refuge-associated), which are relevant at dusk and dawn, respectively. We analysed: (1) whether a temporal modulation of the responsiveness to odours does exist in R. prolixus, (2) if this modulation is a general one or it is specific for each type of volatile, and (3) if it is controlled by exogenous or endogenous mechanisms. We found that the responsiveness to CO₂ only occurs at dusk and that to assembling odours is restricted to dawn. Experiments under free-running conditions revealed that only the responsiveness to CO₂ is controlled by a circadian clock, but not that to assembling signals. Thus, by combining endogenous and exogenous mechanisms, sensitivities to different odours are adjusted according to their associated behavioural context and moment of the day.     

Morphological and developmental analysis of peripheral antennal chemosensory sensilla and central olfactory glomeruli in worker castes of Camponotus compressus (Fabricius, 1787)

The antennal lobes of different castes of the ant species Camponotus compressus show a marked diversity in the organization of their olfactory glomeruli. Notably, there is a significant difference in the number and size of glomeruli between the reproductives and the workers and among the different worker castes. In this report, we investigate the notion that these caste-specific differences in glomerular number might be accounted for, at least in part, by the differences in numbers of olfactory sensilla that target the antennal lobe. For this, we examine the number of sensilla on the antennal flagella of all the individual castes of C. compressus. This analysis reveals a striking correlation between sensillar number and the number of antennal glomeruli in a given caste. As a first step in investigating the causal mechanisms that might give raise to this correlation, we carry out an initial characterization of olfactory system development in the minor workers of C. compressus. We analyze the temporal pattern of innervations of the developing antennal lobe by olfactory sensory neuron axons. We document the development of the olfactory glomeruli in the antennal lobe during this process, which occurs during early pupal stages. Our findings provide the basis for future manipulative developmental studies on the role of sensory afferent number in glomerular development of different castes within the same species.     

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.     

A multimodal approach for tracing lateralisation along the olfactory pathway in the honeybee through electrophysiological recordings,morpho-functional imaging,and behavioural studies

Recent studies have revealed asymmetries between the left and right sides of the brain in invertebrate species. Here we present a review of a series of recent studies from our laboratories, aimed at tracing asymmetries at different stages along the honeybee’s (Apis mellifera) olfactory pathway. These include estimates of the number of sensilla present on the two antennae, obtained by scanning electron microscopy, as well as electroantennography recordings of the left and right antennal responses to odorants. We describe investigative studies of the antennal lobes, where multi-photon microscopy was used to search for possible morphological asymmetries between the two brain sides. Moreover, we report on recently published results obtained by two-photon calcium imaging for functional mapping of the antennal lobe aimed at comparing patterns of activity evoked by different odours. Finally, possible links to the results of behavioural tests, measuring asymmetries in single-sided olfactory memory recall, are discussed.     

Asymmetrical generalisation between pheromonal and floral odours in appetitive olfactory conditioning of the honey bee (Apis mellifera L.)

The capacity to generalise between similar but not identical olfactory stimuli is crucial for honey bees, allowing them to find rewarding food sources with varying volatile emissions. We studied bees' generalisation behaviour with odours having different biological values: typical floral odours or alarm compounds. Bees' behavioural and peripheral electrophysiological responses were investigated using a combined proboscis extension response conditioning-electroantennogram assay. Bees were conditioned to pure linalool (floral) or to pure isoamyl acetate (alarm) and were tested with different concentrations of both compounds. Electrophysiological responses were not influenced by conditioning, suggesting that the learning of individual compounds does not rely on modulations of peripheral sensitivity. Behaviourally, generalisation responses of bees conditioned to the alarm compound were much higher than those of bees conditioned to the floral odour. We further demonstrated such asymmetrical generalisation between alarm and floral odours by using differential conditioning procedures. Conditioning to alarm compounds (isoamyl acetate or 2-heptanone) consistently induced more generalisation than conditioning to floral compounds (linalool or phenylacetaldehyde). Interestingly, generalisation between the two alarm compounds, which are otherwise chemically different, was extremely high. These results are discussed in relation to the neural representation of compounds with different biological significance for bees.     

Innervation pattern of suboesophageal ventral unpaired median neurones in the honeybee brain

In honeybees (Apis mellifera), the biogenic amine octopamine has been shown to play a role in associative and non-associative learning and in the division of labour in the hive. Immunohistochemical studies indicate that the ventral unpaired median (VUM) neurones in the suboesophageal ganglion (SOG) are putatively octopaminergic and therefore might be involved in the octopaminergic modulation of behaviour. In contrast to our knowledge about the behavioural effects of octopamine, only one neurone (VUMmx1) has been related to a behavioural effect (the reward function during olfactory learning). In this study, we have investigated suboesophageal VUM neurones with fluorescent dye-tracing techniques and intracellular recordings combined with intracellular staining. Ten different VUM neurones have been found including six VUM neurones innervating neuropile regions of the brain and the SOG exclusively (central VUM neurones) and four VUM neurones with axons in peripheral nerves (peripheral VUM neurones). The central VUM neurones innervate the antennal lobes, the protocerebral lobes (including the lateral horn) and the mushroom body calyces. Of these, a novel mandibular VUM neurone, VUMmd1, exhibits the same branching pattern in the brain as VUMmx1 and responds to sucrose and odours in a similar way. The peripheral VUM neurones innervate the antennal and the mandibular nerves. In addition, we describe one labial unpaired median neurone with a dorsal cell body, DUMlb1. The possible homology between the honeybee VUM neurones and the unpaired median neurones in other insects is discussed. This work was supported by the DFG ME 365/24-2.     

Anatomical and physiological characteristics of individual neurones in the central antennal pathway of insects

Signals of tens up to hundreds of thousands of (mostly olfactory) receptor cells on an insect antenna are switched to a comparatively low number of neurones in the antennal lobe of the deutocerebrum in circumscribed units of neuropile, the glomeruli. Each glomerulus is connected via its output neurone to two separate neuropiles (calyces of mushroom body, and lateral lobe) of the protocerebrum. Local interneurones interconnect between the glomeruli. Certain modes of convergence between receptors and central neurones provide for a very high sensitivity of the latter to certain odours and their sensitivity for complex odour stimuli, and in many cases for a marked multimodality. Anatomical and physiological data are given especially for pheromone sensitive neurones and their projections.     

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 × 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.     

Dynamic representation of odours by oscillating neural assemblies

Stimulus evoked oscillatory synchronization of neural assemblies has been most clearly documented in the olfactory and visual systems. Recent results with the olfactory system of locusts show that information about odour identity is contained in spatial and temporal aspects of an oscillatory population response. This suggests that brain oscillations may reflect a common reference for messages encoded in time. Although stimulus-evoked oscillatory phenomena are reliable, their roles in perception, memory and pattern recognition remain to be demonstrated. Using honey bees, we demonstrated that odour encoding involves, as in locusts, the oscillatory synchronization of assemblies of neurons, and that this synchronization is, here also, selectively abolished by the GABA receptor antagonist picrotoxin. In collaboration with Dr Brian Smith's laboratory, we showed, using a behavioural learning paradigm, that picrotoxin-induced desynchronization impairs the discrimination of molecularly similar odourants, but not that of dissimilar odours. It appears, therefore, that oscillatory synchronization of neuronal assemblies is relevant, and essential for fine odour discrimination. Finally, experiments with locust mushroom body neurons, two synapses downstream from the antennal lobe, indicate that their responses to odours become less specific when antennal lobe neurons are desynchronized by picrotoxin injection. These results suggest that oscillatory synchronization and the kind of temporal encoding it affords provide an additional dimension by which the brain can segment spatially overlapping stimulus representations.     

Alarm pheromones and chemical communication in nymphs of the tropical bed bug Cimex hemipterus (Hemiptera: Cimicidae)

The recent resurge of bed bug infestations (Cimex spp.; Cimicidae) and their resistance to commonly used pesticides calls for alternative methods of control. Pheromones play an important role in environmentally sustainable methods for the management of many pest insects and may therefore be applicable for the control of bed bugs. The tropical bed bug, Cimex hemipterus, is a temporary ectoparasite on humans and causes severe discomfort. Compared to the common bed bug, Cimex lectularius, little is known about the chemical signalling and pheromone-based behaviour of the tropical species. Here, we show that the antennal morphology and volatile emission of C. hemipterus closely resembles those of C. lectularius and we test their behavioural responses to conspecific odour emissions. Two major volatiles are emitted by male, female and nymph C. hemipterus under stress, (E)-2-hexenal and (E)-2-octenal. Notably, nymph emissions show contrasting ratios of these compounds to adults and are further characterized by the addition of 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal. The discovery of this nymph pheromone in C. hemipterus is potentially the cause of a repellent effect observed in the bio-tests, where nymph odours induce a significantly stronger repellent reaction in conspecifics than adult odours. Our results suggest that pheromone-based pest control methods developed for C. lectularius could be applicable to C. hemipterus, with the unique nymph blend showing promising practical properties.     

Extreme sensitivity in an olfactory system

We recorded olfactory-induced cardiac responses to evaluate olfactory response thresholds to behaviourally relevant odours in a moth. Specific antennal receptor neurons enable insects to detect biologically meaningful odours such as sex pheromones and host-plant volatiles. The response threshold values demonstrated here are well below anything earlier reported in any organism. A heart response was triggered by less than six molecules of the most efficient odours hitting the antennae of the insect. The behavioural significance of this extreme sensitivity most likely lies in the creation of awareness and readiness to respond behaviourally at higher concentration levels.     

Concurrent modulation of neuronal and behavioural olfactory responses to sex and host plant cues in a male moth

Mating has profound effects on animal physiology and behaviour, not only in females but also in males, which we show here for olfactory responses. In cotton leafworm moths, Spodoptera littoralis, odour-mediated attraction to sex pheromone and plant volatiles are modulated after mating, producing a behavioural response that matches the physiological condition of the male insect. Unmated males are attracted by upwind flight to sex pheromone released by calling females, as well as to volatiles of lilac flowers and green leaves of the host plant cotton, signalling adult food and mating sites, respectively. Mating temporarily abolishes male attraction to females and host plant odour, but does not diminish attraction to flowers. This behavioural modulation is correlated with a response modulation in the olfactory system, as shown by electro-physiological recordings from antennae and by functional imaging of the antennal lobe, using natural odours and synthetic compounds. An effect of mating on the olfactory responses to pheromone and cotton plant volatiles but not to lilac flowers indicates the presence of functionally independent neural circuits within the olfactory system. Our results indicate that these circuits interconnect and weigh perception of social and habitat odour signals to generate appropriate behavioural responses according to mating state.     

Caste and sex specific olfactory glomerular organization and brain architecture in two sympatric ant species Camponotus sericeus and Camponotus compressus (Fabricius, 1798)

We use monoclonal antibodies against synaptic proteins and anterograde tracing with neurobiotin to describe the architecture of the antennal lobes in different castes of two ant species – Camponotus sericeus and Camponotus compressus. The reproductives and worker classes are readily categorized based on size and external morphology. The overall organization of brain neuropile is comparable between castes with differences only in the visual ganglia. Males have a larger fraction of neuropile occupied by the medulla and lobula than females. In the diurnal species, C. sericeus these regions are more highly represented, than in the nocturnal species C. compressus. The most striking differences are in the antennal lobe where males possess a macroglomerulus, which is about ten times larger in volume than the other glomeruli; such a specialization is absent in females. Minor workers possess a significantly larger number of glomeruli than the majors despite the smaller overall volume of the lobe. These caste-specific differences occur mainly within glomerular clusters that receive input from sensory neurons that project in tracts – T4 and T5 – within the antennal nerve. The comparative anatomy of different castes of ants provides an entry point into a future systematic analysis of how divergent brain architectures can arise within a single species.     

Polyethism in social interactions in ants

Worker ants, which perform various tasks in their society (division of labour or polyethism), also demonstrate different types of antennal activity in trophallactic interactions. These differences concern the variability in successions of behavioural units performed by the worker in the course of substance transmission as well as in the sequential organization of these units. The antennal activity of an aged worker varies according to whether the ant gives food to another aged worker or to a callow worker. In the latter case, the donor's behaviour does not differ according to its social function (brood-tending worker or forager). A parallel can be established between these results and those obtained in a previous study of aggressive behaviour. This behaviour is liable to appear when a callow worker extracted from its society before hatching and therefore deprived of relations with aged workers of the society, is therefore placed with an aged worker. Polyethism is then manifested in social interactions, and not only in the division of labour between members of the society.     

Responses of olfactory receptor neurones to behaviourally important odours in gregarious and solitarious desert locust, Schistocerca gregaria

Abstract.Recordings from antennal olfactory receptor neurones in young adult Schistocerca gregaria Forskål (Orthoptera: Acrididae) showed that behaviourally important odours are detected by receptor neurones present in morphologically identifiable sensillum types. Both nymph- and adult-produced aggregation pheromones activate receptor neurones housed in sensilla basiconica. The receptor neurones in this sensillum type in solitary-reared locusts display a higher sensitivity to aggregation pheromones and to some other behaviourally relevant odours than the same type of neurones in gregarious locusts. Receptor neurones present in sensilla coeloconica respond to green leaf odours, organic acids, and nymphal odours but are inhibited by mature adult-produced aggregation pheromones. Receptor neurones housed in sensilla trichodea respond to a possible sex pheromone. No phase differences were found in the response of coeloconic- or trichoid-associated receptor neurones.     

The evolutionary implications of qualitative variation in the grooming behaviour of the Hymenoptera (Insecta)

The grooming behaviour of 115 species of Hymenoptera (including castes and sexes) was recorded on an event recorder. Twenty-eight primary and secondary grooming movements are described, and eighteen are illustrated. An analysis of the grooming behaviour revealed consistent qualitative behavioural differences between virtually every super-family. The pattern of these differences parallels an existing hymenopteran phylogeny. Limited data suggest possible qualitative differences at the family and sub-family level in some instances. Possible use of grooming data in phylogenetic interpretatons is suggested.     

雌性根田鼠断奶后对亲本尿气味的记忆

为研究亲子分开后雌性根田鼠对亲本气味的记忆持续时间,分别在未分开(20日龄)、分开10d(30日龄)、20d(40日龄)、30d(50日龄)、40d(60日龄)时,以新鲜尿作气味源,在行为观察箱中记录雌性根田鼠对不同气味源的行为响应模式,结果表明(1)未分开时,雌鼠遭遇父本气味时自我修饰的频次极显著高于陌生雄鼠气味,在分开10d时,雌鼠接近父本气味的频次显著多于接近陌生雄鼠气味的频次,其对前者反标记显著少于后者;(2)分开20d后,雌鼠对父本和陌生雄鼠气味的行为响应无明显差异;(3)未分窝时,雌性根田鼠幼仔对母本和陌生雌鼠气味的行为响应无差异;(4)分开10—40d时,雌性根田鼠对母本和陌生雌鼠气味表现出不同的行为响应模式。以上结果表明,在亲子分开10d时,雌鼠仍能识别父本和陌生雄鼠的气味;分开20d后,雌鼠不再能够识别父本和陌生雄鼠的气味;在亲子分开40d时,雌鼠仍能识别母本和陌生雌鼠的气味。因此,雌鼠对父本气味的嗅觉记忆时间可以持续到亲子分开10—20d之间;而对母本气味的嗅觉记忆时间则可以持续到亲子分开40d以上。     

Brief exposure to sensory cues elicits stimulus-nonspecific general sensitization in an insect

The effect of repeated exposure to sensory stimuli, with or without reward is well known to induce stimulus-specific modifications of behaviour, described as different forms of learning. In recent studies we showed that a brief single pre-exposure to the female-produced sex pheromone or even a predator sound can increase the behavioural and central nervous responses to this pheromone in males of the noctuid moth Spodoptera littoralis. To investigate if this increase in sensitivity might be restricted to the pheromone system or is a form of general sensitization, we studied here if a brief pre-exposure to stimuli of different modalities can reciprocally change behavioural and physiological responses to olfactory and gustatory stimuli. Olfactory and gustatory pre-exposure and subsequent behavioural tests were carried out to reveal possible intra- and cross-modal effects. Attraction to pheromone, monitored with a locomotion compensator, increased after exposure to olfactory and gustatory stimuli. Behavioural responses to sucrose, investigated using the proboscis extension reflex, increased equally after pre-exposure to olfactory and gustatory cues. Pheromone-specific neurons in the brain and antennal gustatory neurons did, however, not change their sensitivity after sucrose exposure. The observed intra- and reciprocal cross-modal effects of pre-exposure may represent a new form of stimulus-nonspecific general sensitization originating from modifications at higher sensory processing levels.