The Loss of Sociality Is Accompanied by Reduced Neural Investment in Mushroom Body Volume in the Sweat Bee Augochlora Pura (Hymenoptera: Halictidae).

Social behavior has been predicted to select for increased neural investment (the social brain hypothesis) and also to select for decreased neural investment (the distributed cognition hypothesis). Here, we use two related bees, the social Augochlorella aurata (Smith) (Hymenoptera: Halictidae) and the related Augochlora pura (Say), which has lost social behavior, to test the contrasting predictions of these two hypotheses in these taxa. We measured the volumes of the mushroom body (MB) calyces, a brain area shown to be important for cognition in previous studies, as well as the optic lobes and antennal lobes. We compared females at the nest foundress stage when both species are solitary so that brain development would not be influenced by social interactions. We show that the loss of sociality was accompanied by a loss in relative neural investment in the MB calyces. This is consistent with the predictions of the social brain hypothesis. Ovary size did not correlate with MB calyx volume. This is the first study to demonstrate changes in mosaic brain evolution in response to the loss of sociality.

Stimulus-dependent learning and memory in the neotropical ant Ectatomma ruidum.

Learning and memory are major cognitive processes strongly tied to the life histories of animals. In ants, chemotactile information generally plays a central role in social interaction, navigation and resource exploitation. However, in hunters, visual information should take special relevance during foraging, thus leading to differential use of information from different sensory modalities. Here, we aimed to test whether a hunter, the neotropical ant Ectatomma ruidum, differentially learns stimuli acquired through multiple sensory channels. We evaluated the performance of E. ruidum workers when trained using olfactory, mechanical, chemotactile and visual stimuli under a restrained protocol of appetitive learning. Conditioning of the maxilla labium extension response enabled control of the stimuli provided. Our results show that ants learn faster and remember for longer when trained using chemotactile or visual stimuli than when trained using olfactory and mechanical stimuli separately. These results agree with the life history of E. ruidum, characterized by a high relevance of chemotactile information acquired through antennation as well as the role of vision during hunting.

Experience, but not age, is associated with volumetric mushroom body expansion in solitary alkali bees.

In social insects, changes in behavior are often accompanied by structural changes in the brain. This neuroplasticity may come with experience (experience-dependent) or age (experience-expectant). Yet, the evolutionary relationship between neuroplasticity and sociality is unclear, because we know little about neuroplasticity in the solitary relatives of social species. We used confocal microscopy to measure brain changes in response to age and experience in a solitary halictid bee (Nomia melanderi). First, we compared the volume of individual brain regions among newly emerged females, laboratory females deprived of reproductive and foraging experience, and free-flying, nesting females. Experience, but not age, led to significant expansion of the mushroom bodies - higher-order processing centers associated with learning and memory. Next, we investigated how social experience influences neuroplasticity by comparing the brains of females kept in the laboratory either alone or paired with another female. Paired females had significantly larger olfactory regions of the mushroom bodies. Together, these experimental results indicate that experience-dependent neuroplasticity is common to both solitary and social taxa, whereas experience-expectant neuroplasticity may be an adaptation to life in a social colony. Further, neuroplasticity in response to social chemical signals may have facilitated the evolution of sociality.

Feeding specialization and longer generation time are associated with relatively larger brains in bees.

Despite their miniature brains, insects exhibit substantial variation in brain size. Although the functional significance of this variation is increasingly recognized, research on whether differences in insect brain sizes are mainly the result of constraints or selective pressures has hardly been performed. Here, we address this gap by combining prospective and retrospective phylogenetic-based analyses of brain size for a major insect group, bees (superfamily Apoidea). Using a brain dataset of 93 species from North America and Europe, we found that body size was the single best predictor of brain size in bees. However, the analyses also revealed that substantial variation in brain size remained even when adjusting for body size. We consequently asked whether such variation in relative brain size might be explained by adaptive hypotheses. We found that ecologically specialized species with single generations have larger brains-relative to their body size-than generalist or multi-generation species, but we did not find an effect of sociality on relative brain size. Phylogenetic reconstruction further supported the existence of different adaptive optima for relative brain size in lineages differing in feeding specialization and reproductive strategy. Our findings shed new light on the evolution of the insect brain, highlighting the importance of ecological pressures over social factors and suggesting that these pressures are different from those previously found to influence brain evolution in other taxa.

Brain differences between social castes precede group formation in a primitively eusocial bee.

Social interactions may shape brain development. In primitively eusocial insects, the mushroom body (MB), an area of the brain associated with sensory integration and learning, is larger in queens than in workers. This may reflect a strategy of neural investment in queens or it may be a plastic response to social interactions in the nest. Here, we show that nest foundresses-the reproductive females who will become queens but are solitary until their first workers are born-have larger MBs than workers in the primitively eusocial sweat bee Augochlorella aurata. Whole brain size and optic lobe size do not differ between the two groups, but foundresses also have larger antennal lobes than workers. This shows that increased neural investment in MBs precedes social group formation. Larger MBs among foundresses may reflect the increased larval nutrition provisioned to future queens and the lack of social aggression from a dominant queen upon adult emergence.

Queen Dominance May Reduce Worker Mushroom Body Size in a Social Bee.

The mushroom body (MB) is an area of the insect brain involved in learning, memory, and sensory integration. Here, we used the sweat bee Megalopta genalis (Halictidae) to test for differences between queens and workers in the volume of the MB calyces. We used confocal microscopy to measure the volume of the whole brain, MB calyces, optic lobes, and antennal lobes of queens and workers. Queens had larger brains, larger MB calyces, and a larger MB calyces:whole brain ratio than workers, suggesting an effect of social dominance in brain development. This could result from social interactions leading to smaller worker MBs, or larger queen MBs. It could also result from other factors, such as differences in age or sensory experience. To test these explanations, we next compared queens and workers to other groups. We compared newly emerged bees, bees reared in isolation for 10 days, bees initiating new observation nests, and bees initiating new natural nests collected from the field to queens and workers. Queens did not differ from these other groups. We suggest that the effects of queen dominance over workers, rather than differences in age, experience, or reproductive status, are responsible for the queen-worker differences we observed. Worker MB development may be affected by queen aggression directly and/or manipulation of larval nutrition, which is provisioned by the queen. We found no consistent differences in the size of antennal lobes or optic lobes associated with differences in age, experience, reproductive status, or social caste.

Differential investment in brain regions for a diurnal and nocturnal lifestyle in Australian Myrmecia ants.

Animals are active at different times of the day. Each temporal niche offers a unique light environment, which affects the quality of the available visual information. To access reliable visual signals in dim-light environments, insects have evolved several visual adaptations to enhance their optical sensitivity. The extent to which these adaptations reflect on the sensory processing and integration capabilities within the brain of a nocturnal insect is unknown. To address this, we analyzed brain organization in congeneric species of the Australian bull ant, Myrmecia, that rely predominantly on visual information and range from being strictly diurnal to strictly nocturnal. Weighing brains and optic lobes of seven Myrmecia species, showed that after controlling for body mass, the brain mass was not significantly different between diurnal and nocturnal ants. However, the optic lobe mass, after controlling for central brain mass, differed between day- and night-active ants. Detailed volumetric analyses showed that the nocturnal ants invested relatively less in the primary visual processing regions but relatively more in both the primary olfactory processing regions and in the integration centers of visual and olfactory sensory information. We discuss how the temporal niche occupied by each species may affect cognitive demands, thus shaping brain organization among insects active in dim-light conditions.

Camponotusfloridanus Ants Incur a Trade-Off between Phenotypic Development and Pathogen Susceptibility from Their Mutualistic Endosymbiont Blochmannia.

Various insects engage in microbial mutualisms in which the reciprocal benefits exceed the costs. Ants of the genus Camponotus benefit from nutrient supplementation by their mutualistic endosymbiotic bacteria, Blochmannia, but suffer a cost in tolerating and regulating the symbiont. This cost suggests that the ants face secondary consequences such as susceptibility to pathogenic infection and transmission. In order to elucidate the symbiont's effects on development and disease defence, Blochmannia floridanus was reduced in colonies of Camponotus floridanus using antibiotics. Colonies with reduced symbiont levels exhibited workers of smaller body size, smaller colony size, and a lower major-to-minor worker caste ratio, indicating the symbiont's crucial role in development. Moreover, these ants had decreased cuticular melanisation, yet higher resistance to the entomopathogen Metarhizium brunneum, suggesting that the symbiont reduces the ants' ability to fight infection, despite the availability of melanin to aid in mounting an immune response. While the benefits of improved growth and development likely drive the mutualism, the symbiont imposes a critical trade-off. The ants' increased susceptibility to infection exacerbates the danger of pathogen transmission, a significant risk given ants' social lifestyle. Thus, the results warrant research into potential adaptations of the ants and pathogens that remedy and exploit the described disease vulnerability.

Morphine addiction in ants: a new model for self-administration and neurochemical analysis.

Conventional definitions of drug addiction are focused on characterizing the neurophysiological and behavioral responses of mammals. Although mammalian models have been invaluable in studying specific and complex aspects of addiction, invertebrate systems have proven advantageous in investigating how drugs of abuse corrupt the most basic motivational and neurochemical systems. It has recently been shown that invertebrates and mammals have remarkable similarities in their behavioral and neurochemical responses to drugs of abuse. However, until now only mammals have demonstrated drug seeking and self-administration without the concurrent presence of a natural reward, e.g. sucrose. Using a sucrose-fading paradigm, followed by a two-dish choice test, we establish ants as an invertebrate model of opioid addiction. The ant species Camponotus floridanus actively seeks and self-administers morphine even in the absence of caloric value or additional natural reward. Using HPLC equipped with electrochemical detection, the neurochemicals serotonin, octopamine and dopamine were identified and subsequently quantified, establishing the concurrent neurochemical response to the opioid morphine within the invertebrate brain. With this study, we demonstrate dopamine to be governing opioid addiction in the brains of ants. Thus, this study establishes ants as the first non-mammalian model of self-administration that is truly analogous to mammals.

How embryos escape from danger: the mechanism of rapid, plastic hatching in red-eyed treefrogs.

Environmentally cued hatching allows embryos to escape dangers and exploit new opportunities. Such adaptive responses require a flexibly regulated hatching mechanism sufficiently fast to meet relevant challenges. Anurans show widespread, diverse cued hatching responses, but their described hatching mechanisms are slow, and regulation of timing is unknown. Arboreal embryos of red-eyed treefrogs, Agalychnis callidryas, escape from snake attacks and other threats by very rapid premature hatching. We used videography, manipulation of hatching embryos and electron microscopy to investigate their hatching mechanism. High-speed video revealed three stages of the hatching process: pre-rupture shaking and gaping, vitelline membrane rupture near the snout, and muscular thrashing to exit through the hole. Hatching took 6.5-49 s. We hypothesized membrane rupture to be enzymatic, with hatching enzyme released from the snout during shaking. To test this, we displaced hatching embryos to move their snout from its location during shaking. The membrane ruptured at the original snout position and embryos became trapped in collapsed capsules; they either moved repeatedly to relocate the hole or shook again and made a second hole to exit. Electron microscopy revealed that hatching glands are densely concentrated on the snout and absent elsewhere. They are full of vesicles in embryos and release most of their contents rapidly at hatching. Agalychnis callidryas' hatching mechanism contrasts with the slow process described in anurans to date and exemplifies one way in which embryos can achieve rapid, flexibly timed hatching to escape from acute threats. Other amphibians with cued hatching may also have novel hatching mechanisms.

Social isolation and brain development in the ant Camponotus floridanus.

Social interactions play a key role in the healthy development of social animals and are most pronounced in species with complex social networks. When developing offspring do not receive proper social interaction, they show developmental impairments. This effect is well documented in mammalian species but controversial in social insects. It has been hypothesized that the enlargement of the mushroom bodies, responsible for learning and memory, observed in social insects is needed for maintaining the large social networks and/or task allocation. This study examines the impact of social isolation on the development of mushroom bodies of the ant Camponotus floridanus. Ants raised in isolation were shown to exhibit impairment in the growth of the mushroom bodies as well as behavioral differences when compared to ants raised in social groups. These results indicate that social interaction is necessary for the proper development of C. floridanus mushroom bodies.

Biogenic amines are associated with worker task but not patriline in the leaf-cutting ant Acromyrmex echinatior.

Division of labor among eusocial insect workers is a hallmark of advanced social organization, but its underlying neural mechanisms are not well understood. We investigated whether differences in whole-brain levels of the biogenic amines dopamine (DA), serotonin (5HT), and octopamine (OA) are associated with task specialization and genotype in similarly sized and aged workers of the leaf-cutting ant Acromyrmex echinatior, a polyandrous species in which genotype correlates with worker task specialization. We compared amine levels of foragers and waste management workers to test for an association with worker task, and young in-nest workers across patrilines to test for a genetic influence on brain amine levels. Foragers had higher levels of DA and OA and a higher OA:5HT ratio than waste management workers. Patrilines did not significantly differ in amine levels or their ratios, although patriline affected worker body size, which correlated with amine levels despite the small size range sampled. Levels of all three amines were correlated within individuals in both studies. Among patrilines, mean levels of DA and OA, and OA and 5HT were also correlated. Our results suggest that differences in biogenic amines could regulate worker task specialization, but may be not be significantly affected by genotype.

The allometry of CNS size and consequences of miniaturization in orb-weaving and cleptoparasitic spiders.

Allometric studies of the gross neuroanatomy of adults from nine species of spiders from six web-weaving families (Orbicularia), and nymphs from six of these species, show that very small spiders resemble other small animals in having disproportionately larger central nervous systems (CNSs) relative to body mass when compared with large-bodied forms. Small spiderlings and minute adult spiders have similar relative CNS volumes. The relatively large CNS of a very small spider occupies up to 78% of the cephalothorax volume. The CNSs of very small spiders extend into their coxae, occupying as much as 26% of the profile area of the coxae of an Anapisona simoni spiderling (body mass < 0.005 mg). Such modifications occur both in species with minute adults, and in tiny spiderlings of species with large-bodied adults. In at least one such species, Leucauge mariana, the CNS of the spiderling extends into a prominent ventral bulge of the sternum. Tiny spiders also have reduced neuronal cell body diameters. The adults of nearly all orbicularian spiders weave prey capture webs, as do the spiderlings, beginning with second instar nymphs. Comparable allometric relations occur in adults of both orb-weaving and cleptoparasitic species, indicating that this behavioral difference is not reflected in differences in gross CNS allometry.

The allometry of brain miniaturization in ants.

Extensive studies of vertebrates have shown that brain size scales to body size following power law functions. Most animals are substantially smaller than vertebrates, and extremely small animals face significant challenges relating to nervous system design and function, yet little is known about their brain allometry. Within a well-defined monophyletic taxon, Formicidae (ants), we analyzed how brain size scales to body size. An analysis of brain allometry for individuals of a highly polymorphic leaf-cutter ant, Atta colombica, shows that allometric coefficients differ significantly for small (<1.4 mg body mass) versus large individuals (b = 0.6003 and 0.2919, respectively). Interspecifically, allometric patterns differ for small (<0.9 mg body mass) versus large species (n = 70 species). Using mean values for species, the allometric coefficient for smaller species (b = 0.7961) is significantly greater than that for larger ones (b = 0.669). The smallest ants had brains that constitute ∼15% of their body mass, yet their brains were relatively smaller than predicted by an overall allometric coefficient of brain to body size. Our comparative and intraspecific studies show the extent to which nervous systems can be miniaturized in taxa exhibiting behavior that is apparently comparable to that of larger species or individuals.

Socially induced brain development in a facultatively eusocial sweat bee Megalopta genalis (Halictidae).

Changes in the relative size of brain regions are often dependent on experience and environmental stimulation, which includes an animal's social environment. Some studies suggest that social interactions are cognitively demanding, and have examined predictions that the evolution of sociality led to the evolution of larger brains. Previous studies have compared species with different social organizations or different groups within obligately social species. Here, we report the first intraspecific study to examine how social experience shapes brain volume using a species with facultatively eusocial or solitary behaviour, the sweat bee Megalopta genalis. Serial histological sections were used to reconstruct and measure the volume of brain areas of bees behaving as social reproductives, social workers, solitary reproductives or 1-day-old bees that are undifferentiated with respect to the social phenotype. Social reproductives showed increased development of the mushroom body (an area of the insect brain associated with sensory integration and learning) relative to social workers and solitary reproductives. The gross neuroanatomy of young bees is developmentally similar to the advanced eusocial species previously studied, despite vast differences in colony size and social organization. Our results suggest that the transition from solitary to social behaviour is associated with modified brain development, and that maintaining dominance, rather than sociality per se, leads to increased mushroom body development, even in the smallest social groups possible (i.e. groups with two bees). Such results suggest that capabilities to navigate the complexities of social life may be a factor shaping brain evolution in some social insects, as for some vertebrates.

Delayed axonal pruning in the ant brain: a study of developmental trajectories.

The coordination of neuronal maturation and behavioral development is a vital component of survival. The degradation of excessive axonal processes and neuronal networks is a ubiquitous developmental process. In Drosophila, a great portion of axonal pruning occurs during metamorphosis and transpires within hours after pupation. In contrast, we show, using EM-serial sectioning and 3D-reconstructions, that axonal pruning occurs after eclosion and over the course of 60 days in Cataglyphis albicans. Using the mushroom bodies of the brains of Cataglyphis, which have well-developed lip (olfactory integrator) and collar (visual integrator) regions, we show that axonal pruning is dependent upon the differences in the developmental trajectory of the lip and the collar brain regions and happens after eclosion. The elimination of the axonal boutons is most delayed in the collar region, where it is postponed until the ant has had extensive visual experience. We found that individual brain components within a single neuropil can develop at different rates that correlate with the behavioral ecology of these ants and suggest that glia may be mediating the axonal pruning. Our study provides evidence that adult ants may have relatively neotenous brains, and thus more flexibility, allowing them to neuronally adapt to the environment. This neoteny may, in part, explain the neural basis for age-dependent division of labor and the amazing behavioral flexibility exhibited by ants.

Age- and subcaste-related patterns of serotonergic immunoreactivity in the optic lobes of the ant Pheidole dentata.

Serotonin, a biogenic amine known to be a neuromodulator of insect behavior, has recently been associated with age-related patterns of task performance in the ant Pheidole dentata. We identified worker age- and subcaste-related patterns of serotonergic activity within the optic lobes of the P. dentata brain to further examine its relationship to polyethism. We found strong immunoreactivity in the optic lobes of the brains of both minor and major workers. Serotonergic cell bodies in the optic lobes increased significantly in number as major and minor workers matured. Old major workers had greater numbers of serotonergic cell bodies than minors of a similar age. This age-related increase in serotonergic immunoreactivity, as well as the presence of diffuse serotonin networks in the mushroom bodies, antennal lobes, and central complex, occurs concomitantly with an increase in the size of worker task repertoires. Our results suggest that serotonin is associated with the development of the visual system, enabling the detection of task-related stimuli outside the nest, thus playing a significant role in worker behavioral development and colony-wide division of labor.

Ultrastructure and synaptic differences of the boutons of the projection neurons between the lip and collar regions of the mushroom bodies in the ant, Cataglyphis albicans.

The mushroom bodies of insects are viewed as key neuropils for sensory integration and perhaps learning and memory. In Hymenoptera, particularly ants, the calyx of the mushroom bodies is divided into two main regions, the lip and the collar. Although most ants are highly dependent on olfaction and have enlarged calyces comprised mostly of lip, some ant groups are also highly visual and have well-developed collars. The desert ant Cataglyphis albicans, known for its navigational abilities, shifts from the dark olfactory demanding nest interior to the visually demanding desert environment, and unlike many other ants their mushroom bodies are comprised of both a well-developed lip and collar. In this study, using electron microscope serial-sectioning and 3D-reconstructions, we show that axonal processes that innervate the lip and collar are inherently different in structure and synaptic connectivity. The boutons of the lip are larger, with more synaptic vesicles and larger synapses than the collar, while boutons of the collar have more postsynaptic partners per synapse. Our morphological findings suggest that the signals originating from olfactory projection neurons that innervate the lip appear stronger and more likely to propagate than signals that innervate the collar, while the signals entering the collar appear relatively weaker and are further integrated between more postsynaptic partners. We discuss the differences of the signaling properties between the lip and collar projection neurons and suggest that the greater postsynaptic integration in the collar is presumably for spatial processing for visual navigation in Cataglyphis.

Age-related changes in the number and structure of synapses in the lip region of the mushroom bodies in the ant Pheidole dentata.

Behavioral development in the worker caste of many adult ants follows a pattern of task transitions that contribute to the division of labor within colonies. In the ant Pheidole dentata, the number of tasks that minor workers attend to increases as they progress from brood-care activities within the nest to acts outside the nest such as foraging and defense. In this study we investigated synapse maturation in the lip region of mushroom bodies in young and old minor workers because of its potentially crucial role in behavioral development, task performance, and repertoire expansion. As minor workers aged, individual presynaptic boutons enlarged and acquired more synapses and vesicles, but the total number of synapses in the lip region did not change significantly. Glial cell processes occupied less of the synaptic neuropil as ants matured. These findings indicate an expansion and enhancement of efficacy at specific sets of synaptic connections between the projection interneurons and Kenyon cell dendrites and a commensurate loss of other connections as minor workers age and expand their behavioral repertoire.