Across bilaterian phyla, learning and memory allows animals to benefit from central-place foraging, return to ideal food sources, choose mates and avoid dangerous or harmful external stimuli. Although these behaviors are comparable in both vertebrate and invertebrate animals, it is unknown whether or not they are mediated by homologous brain structures. In insects, paired, lobate forebrain structures called mushroom bodies receive input from primary sensory neuropils and are necessary for learning and memory, whereas in crustaceans, this behavior is mediated by paired, compact forebrain structures called hemiellipsoid bodies. Mammalian learning and memory is mediated by the paired, horn-shaped hippocampi, which also receive sensory input and are likewise situated in the forebrain. Did these structures evolve independently along with the ability for animals to learn and remember associations and places? Alternatively, the hypothesis posited in this dissertation is that the last bilaterian ancestor already possessed the ability to learn and adapt to its environment, behavior mediated by paired forebrain structures that evolved divergently into the elaborated forms we observe in extant, crown-group taxa. This hypothesis is investigated and discussed in the following reports: 1) a review of insect brain anatomy and functional connectivity, including a description of mushroom bodies, in the context of arthropod evolution; 2) a comparison of neuroanatomy, circuitry, and protein expression between insect mushroom bodies and Malacostracan crustacean hemiellipsoid bodies, using cockroaches and Caribbean hermit crabs as representatives of their classes; 3) a deeper investigation of the fine structure of neuronal organization in the hemiellipsoid body of the Caribbean hermit crab, focusing on electron microscopical observations and comparisons to the ultrastructure of the fruit fly mushroom body; 4) a survey of four invertebrate Phyla, employing the strategy of comparing neuroanatomy and protein expression to investigate whether higher order forebrain structures in these animals were inherited from a common ancestor; 5) a comparison of neuroanatomy, connectivity, and protein expression in insect mushroom bodies and mammalian hippocampus, including a survey of PKA-Cα in these and corresponding structures across the Chordata. The total evidence suggests that a common Bilaterian ancestor possessed a center that evolved to become mushroom bodies in invertebrates and hippocampus in vertebrates.

Whirligig beetles are gregarious aquatic insects living on the water surface. They are equipped with two separate pairs of compound eyes, an upper aerial pair and a lower aquatic pair, but little is known about how their brains are organized to serve such an unusual arrangement. In the first study of this dissertation, I describe the neural organization of their primary visual centers (the optic lobes) of the larval and adult whirligig beetle Dineutus sublineatus. I show that the divided compound eyes of adult beetles supply elaborate optic lobes in the brain that are also split into an upper and a lower half, each optic lobe comprising an upper and lower lamina, an upper and lower medulla, and a partly split bilobed lobula. The exception is the fourth neuropil, the lobula plate. Studies of their development show that the lobula plate Anlagen serving the upper and lower eyes develop at different rates and thus different developmental stages. The upper lobula plate develops precociously in the larva and is thought to process information that enables subaquatic ambush hunting. During metamorphosis the upper lobula plate degenerates and is lost as are the larval stemmatal eyes supplying it. The lower lobula plate develops later, during metamorphosis, and is present in the imago where it is supplied by the lower compound retina. By analogy with dipteran lobula plates it is proposed to support subaquatic locomotory balance. In the subsequent study, I describe the neural organization of the whirligig beetle’s mushroom bodies, a pair of prominent brain centers in the forebrain that are best known for their roles in higher olfactory processing and olfactory-based learning and memory. I found that unlike other insects examined so far, the calyces of the whirligig beetle’s mushroom bodies are exclusively supplied by visual neurons from optic lobe neuropils serving the pair of upper aerial compound eyes, thereby showing a complete modality switch from olfaction to vision in this brain center. These findings, along with multiple evidence from hymenopteran insects and cockroaches, suggest that insect mushroom bodies are not merely olfactory-related but may be involved in visual tasks, such as memory of place. In the last study, I describe experiments to demonstrate that a group of D. sublineatus is able to learn their location with respect to visual cues provided from above the water line, and simultaneously establish and maintain their relative positions with each other within the group. These results provide an explanation as to how a collective, such as several hundred whirligig beetles, can maintain cohesion and remember landmarks that "anchor" the collective at a particular location in a pond or stream. Using techniques in comparative neuroanatomy, this dissertation documents visual specializations of an insect brain that has evolved to suit a unique group-living lifestyle on the water surface. In addition, the spatial learning paradigm described in the third study provides an essential assay for future lesion studies to determine if mushroom bodies are indeed required for visually mediated spatial learning and memory.

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Sociality shapes patterns of senescence, evidenced by the remarkable lifespan plasticity of social insect queens and workers. Ants, exemplars of eusociality, provide diverse systems to explore the sociobiology of senescence by examining how sterile workers partition colony labor over their lifespans, and how neurobiological factors affect transitions among social roles and age-related task performance efficacies. Integrating sociobiology, senescence theory, and neurobiology, I examined the relationship of chronological age and social behavior during the ~140-day lifespan of workers of the ant Pheidole dentata. I critically analyzed programmed senescence in respect to the sociobiology of worker longevity and evaluated how large colony size achieved through selection for extended worker lifespan enhances colony fitness. My study found no support for worker programmed senescence. Further testing senescence theory, I determined if workers declined behaviorally as they aged due to increased apoptotic cell death and changes in synaptic complexes associated with higher-order processing in the brain. Using robust behavioral assays I found aging was not correlated with declines in sensory responsiveness or motor functions associated with foraging, nursing, and prey-capture tasks, or activity level and phototaxis. Old minor workers (95 days) followed pheromone trails for greater distances than 20-day old minors and showed higher activity levels, suggesting improvement in behavioral performance. Neural substrates likely underscoring task performance were maintained with age: synaptic complex density was constant and apoptosis was unchanged with age. Sensory and motor control brain regions did not show age-related increases in neurodegeneration. Worker spatial location predicted social role independent of age: foragers exhibited higher activity levels and more aggressive predatory behavior than nurses. Serotonin and dopamine titers increased from 20 to 120 days but showed no clear correlation with social role. Pharmacological manipulations of brain serotonin had no effect on brood care, predatory response, activity, or phototaxis. Finally, I assessed arborization of a serotonergic neuron hypothesized to underscore task performance to determine how aging across subcastes influences neuronal structure. Major workers showed greater branching complexity than minors and an age-related increase in arbor complexity. P. dentata workers appear to show negligible behavioral and neural senescence throughout their lifespans.

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Learning allows animals to adapt their behaviour to changes in the environment. In humans and other mammals, memories are stored in the hippocampus and cerebellum, whereas in insects, they are stored inside the mushroom bodies (MB). Here, MB-intrinsic Kenyon cells (KCs) form plastic synapses to MB output neurons (MBONs) that are modulated by the reinforcing action of dopaminergic neurons (DANs). Despite decades of research on the MB, the main neurotransmitter underlying the plastic KC → MBON synapse has remained a mystery. Here, I show that this synapse is cholinergic in the fruit fly Drosophila melanogaster. MBONs show fast excitatory responses to direct acetylcholine (ACh) application. KCs synthesise ACh-related proteins ChAT and VAChT. MBONs express and require nicotinic ACh receptors (nAChRs) to become fully activated by odour presentation. Lastly, artificial activation of KCs leads to MBON calcium responses that are blocked by nicotinic antagonists and genetic reduction of VAChT in KCs. Short neuropeptide F (sNPF) may play a role as a modulatory co-transmitter that can either excite or inhibit specific MBONs and DANs. The retrieval of memories is state-dependent and known to potentially change the original memory. Fruit flies need to be hungry to express appetitive memories. Hunger state depends on insulin signalling that activates the GABAergic MBON MVP2, while appetitive memory retrieval depends on decreased activity in M4/6 MBONs. Here, I show that optogenetic MVP2 activation acutely inhibits M4/6 odour responses, rendering MVP2 an inhibitory MBON interneuron. I also show that other MBONs are functionally connected to DANs, thus linking memory reinforcement and retrieval pathways in a way that enables the updating of the original memory. These findings show that associative memories in Drosophila are initially formed at cholinergic-MBON synapses, and can be retrieved and modified through an intricate KC-MBON-DAN network.

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The social brain hypothesis predicts that larger group size and greater social complexity select for increased brain size. In ants, social complexity is associated with large colony size, emergent collective action, and division of labor among workers. The great diversity of social organization in ants offers numerous systems to test social brain theory and examine the neurobiology of social behavior. My studies focused on the Australasian weaver ant, Oecophylla smaragdina, a polymorphic species, as a model of advanced social organization. I critically analyzed how biogenic amines modulate social behavior in ants and examined their role in worker subcaste-related territorial aggression. Major workers that naturally engage in territorial defense showed higher levels of brain octopamine in comparison to more docile, smaller minor workers, whose social role is nursing. Through pharmacological manipulations of octopaminergic action in both subcastes, octopamine was found to be both necessary and sufficient for aggression, suggesting subcaste-related task specialization results from neuromodulation. Additionally, I tested social brain theory by contrasting the neurobiological correlates of social organization in a phylogenetically closely related ant species, Formica subsericea, which is more basic in social structure. Specifically, I compared brain neuroanatomy and neurometabolism in respect to the neuroecology and degree of social complexity of O. smaragdina major and minor workers and F. subsericea monomorphic workers. Increased brain production costs were found in both O. smaragdina subcastes, and the collective action of O. smaragdina majors appeared to compensate for these elevated costs through decreased ATP usage, measured from cytochrome oxidase activity, an endogenous marker of neurometabolism. Macroscopic and cellular neuroanatomical analyses of brain development showed that higher-order sensory processing regions in workers of O. smaragdina, but not F. subsericea, had age-related synaptic reorganization and increased volume. Supporting the social brain hypothesis, ecological and social challenges associated with large colony size were found to contribute to increased brain size. I conclude that division of labor and collective action, among other components of social complexity, may drive the evolution of brain structure and function in compensatory ways by generating anatomically and metabolically plastic mosaic brains that adaptively reflect cognitive demands of worker task specialization and colony-level social organization.

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