INTRODUCTION:Whirligig beetles (Coleoptera: Gyrinidae) are aquatic insects living on the water surface. They are equipped with four compound eyes, an upper pair viewing above the water surface and a lower submerged pair viewing beneath the water surface, but little is known about how their visual brain centers (optic lobes) are organized to serve such unusual eyes. We show here, for the first time, the peculiar optic lobe organization of the larval and adult whirligig beetle Dineutus sublineatus.RESULTS:The divided compound eyes of adult whirligig beetles supply optic lobes that are split into two halves, an upper half and lower half, comprising an upper and lower lamina, an upper and lower medulla and a bilobed partially split lobula. However, the lobula plate, a neuropil that in flies is known to be involved in mediating stabilized flight, exists only in conjunction with the lower lobe of the lobula. We show that, as in another group of predatory beetle larvae, in the whirligig beetle the aquatic larva precociously develops a lobula plate equipped with wide-field neurons. It is supplied by three larval laminas serving the three dorsal larval stemmata, which are adjacent to the developing upper compound eye.CONCLUSIONS:In adult whirligig beetles, dual optic neuropils serve the upper aerial eyes and the lower subaquatic eyes. The exception is the lobula plate. A lobula plate develops precociously in the larva where it is supplied by inputs from three larval stemmata that have a frontal-upper field of view, in which contrasting objects such as prey items trigger a body lunge and mandibular grasp. This precocious lobula plate is lost during pupal metamorphosis, whereas another lobula plate develops normally during metamorphosis and in the adult is associated with the lower eye. The different roles of the upper and lower lobula plates in supporting, respectively, larval predation and adult optokinetic balance are discussed. Precocious development of the upper lobula plate represents convergent evolution of an ambush hunting lifestyle, as exemplified by the terrestrial larvae of tiger beetles (Cicindelinae), in which activation of neurons in their precocious lobula plates, each serving two large larval stemmata, releases reflex body extension and mandibular grasp.

Signal processing in the visual system is mediated by classic neurotransmission and neuropeptidergic modulatory pathways. In Dipteran insects, especially in the fruitfly Drosophila melanogaster, the morphology of the visual system is very well described. However neurotransmitter and neuropeptidergic circuits within the optic lobe neuropil are only partially known. Using several transgenic fly lines and antibodies we determined the localization of the classical neurotransmitters GABA, acetylcholine and glutamate in the visual system, and their putative targets via detecting several neurotransmitter receptors. We paid particular attention to the peripheral neuropil layer called the lamina, where the light signals are filtered, channeled and amplified (Paper I). We discovered four new types of efferent tangential neurons branching distally to the lamina. Among them was the first neuropeptidergic neuron (LMIo) in this region of Drosophila. The LMIo expresses myoinhibitory peptide (MIP) and has its cell body located close to the main lateral clock neurons that express the neuropeptide pigment-dispersing factor (PDF)(Paper II). Since in other Dipteran species PDF is expressed in processes distally to the lamina, we performed comparative anatomical studies of the MIP, PDF, Ion Transport Peptide (ITP) and serotonin (5-HT) distribution in the visual system of the flies Drosophila and Calliphora. Our data suggest that PDF signaling distal to the lamina of the blowfly might be replaced by MIP signaling in the fruitfly, while ITP and 5-HT expression is conserved in the two species (Paper III). Serotonin is crucial in light adaptation during the daily light-dark cycles. We analyzed putative serotonergic circuits in the lamina. We found that LMIo neurons express the inhibitory receptor 5-HT1A, while 5-HT1B and 5-HT2 are both expressed in the epithelial glia of the lamina. Another novel wide-field neuron with lamina branches expresses the excitatory serotonin receptor 5-HT7. Our studies have identified a fairly complex neuronal circuitry in the tangential plexus above the lamina. (Paper IV). Finally we tested circadian locomotor activity rhythms in flies with the GABAB receptor knocked down on the lateral PDF-expressing clock neurons. We observed significant changes in the activity periods and diminished strength of rhythmicity during DD suggesting a modulatory role of GABA in clock function (Paper V). / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.

Os processos que regem o desenvolvimento dos olhos compostos em insetos têm sido amplamente estudados em Drosophila melanogaster onde estes se originam a partir de discos imaginais. Pouco se sabe, porém, sobre o desenvolvimento do lóbulo óptico e da retina em outros insetos que, na sua grande maioria, não possuem discos imaginais de olhos separados do sistema nervoso central. Neste sentido, a análise comparada do desenvolvimento dos olhos de Apis mellifera pode contribuir não somente para aspectos evo-devo entre as grandes famílias dos insetos holometábolos, quanto pode elucidar questões de plasticidade de desenvolvimento pois os olhos compostos apresentam fortes características sexo e casta-específicas. Com o objetivo primário de elucidar os padrões de divisão e diferenciação celular durante o desenvolvimento do olho em A. mellifera realizamos análises histológicas e de imunomarcação durante o desenvolvimento pós-embrionário, juntamente com análise de expressão do gene roughest em tempo real. Para imunomarcação utilizamos o anticorpo anti-fosfo-histona H3 fosforilada que marca células em fase M do ciclo celular. Foram analisadas larvas operárias entre o terceiro instar larval (L3) até pupas de olho branco, rosa e marrom, com foco sobre o quinto instar larval que fica subdividida em fase de alimentação e crescimento (L5F), fases de tecelagem de casulo (L5S) e prepupa (PP). O desenvolvimento do lóbulo óptico em Apis mellifera ocorre por dobramento neuroepitelial, a partir de um centro de diferenciação, seqüencialmente gerando as camadas neurais do lóbulo óptico (lóbula, medula e lâmina). A lâmina (última a surgir) 6 apresentou-se com desenvolvimento mais lento e em duas fases antes da metamorfose: a primeira fase é o seu surgimento no começo do quinto instar larval acompanhando o primeiro pico de expressão de roughest e a segunda fase ocorre durante a tecelagem de casulo com o desenvolvimento do córtex acompanhando o segundo pico de expressão de roughest. Ainda durante o segundo pico de expressão de roughest os rabdômeros da retina começam a ficar visíveis, assim como os feixes axonais. Estes porém estarão completamente formados somente após a metamorfose.. O desenvolvimento completo da lâmina, lóbula e medula e da retina ocorre somente após a metamorfose. Durante a fase pupal as estruturas do lóbulo óptico estão prontas, porém na retina observa-se ainda gradual pigmentação, encurtamento dos feixes axonais e alongamento dos rabdômeros até atingirem o seu comprimento final logo antes da emergência. / The processes that drive compound eye development in insects have been broadly studied in Drosophila melanogaster in which they arise from imaginal discs. Little is known about optic lobe and retina development in other insects, most of which do not have imaginal eye discs attached to the nervous system. For this reason, a comparative analysis of eye development in the honey bee, Apis mellifera, not only contributes to evo-devo aspects comparing the major families of holometabolous insects, but also may elucidate questions about developmental plasticity because the compound eyes of the honeybee show strong sex and caste-specific differences. Since our primary objective was to elucidate the pattern of cellular differentiation and division during eye development we performed histological and immunolabelling analyses during the postembrionic stages of development, concomitant with a realtime analysis of roughest gene expression. For the immunolabelling experiments we used an anti-phospho-histone H3 antibody that labels cells in M phase. We analyzed eye development in worker larvae starting with the third instar until white, pink and browneyed pupae, paying special attention to the fifth instar which was subdivided into feeding phase (L5F), cocoon spinning phase (L5S) and prepupae (PP). Optic Lobe development in Apis mellifera occurs by neuroepithelial folding initiating from a differentiation center, in the larval brain. This center sequentially produces the neural layers of the optic lobe (medulla, lobula and lamina). Development of the lamina, which is the last layer to be formed, takes more time and happens in two steps before metamorphosis. The first step is emergence at the beginning of the fifth larval instar coinciding with the first peak of roughest gene expression. The second step 8 occurs during the cocoon spinning phase and is marked by its inner differentiation, again accompanied by a second peak of roughest expression. During this second peak of roughest expression the rabdomers in the retina become visible. These, however, cplete thir development only during the pupal stage. The development of the lamina, lobula and medulla is not complete until after metamorphosis, even though these optic lobe structures are structurally defined already at the beginning of the pupal phase. Retinal development in this phase is marked by gradual pigmentation, axonal bundle shortening and rabdomer elongation, which reach their final size just prior to emergence of the bees from their brood cells.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [106]-128).

Os processos que regem o desenvolvimento dos olhos compostos em insetos têm sido amplamente estudados em Drosophila melanogaster onde estes se originam a partir de discos imaginais. Pouco se sabe, porém, sobre o desenvolvimento do lóbulo óptico e da retina em outros insetos que, na sua grande maioria, não possuem discos imaginais de olhos separados do sistema nervoso central. Neste sentido, a análise comparada do desenvolvimento dos olhos de Apis mellifera pode contribuir não somente para aspectos evo-devo entre as grandes famílias dos insetos holometábolos, quanto pode elucidar questões de plasticidade de desenvolvimento pois os olhos compostos apresentam fortes características sexo e casta-específicas. Com o objetivo primário de elucidar os padrões de divisão e diferenciação celular durante o desenvolvimento do olho em A. mellifera realizamos análises histológicas e de imunomarcação durante o desenvolvimento pós-embrionário, juntamente com análise de expressão do gene roughest em tempo real. Para imunomarcação utilizamos o anticorpo anti-fosfo-histona H3 fosforilada que marca células em fase M do ciclo celular. Foram analisadas larvas operárias entre o terceiro instar larval (L3) até pupas de olho branco, rosa e marrom, com foco sobre o quinto instar larval que fica subdividida em fase de alimentação e crescimento (L5F), fases de tecelagem de casulo (L5S) e prepupa (PP). O desenvolvimento do lóbulo óptico em Apis mellifera ocorre por dobramento neuroepitelial, a partir de um centro de diferenciação, seqüencialmente gerando as camadas neurais do lóbulo óptico (lóbula, medula e lâmina). A lâmina (última a surgir) 6 apresentou-se com desenvolvimento mais lento e em duas fases antes da metamorfose: a primeira fase é o seu surgimento no começo do quinto instar larval acompanhando o primeiro pico de expressão de roughest e a segunda fase ocorre durante a tecelagem de casulo com o desenvolvimento do córtex acompanhando o segundo pico de expressão de roughest. Ainda durante o segundo pico de expressão de roughest os rabdômeros da retina começam a ficar visíveis, assim como os feixes axonais. Estes porém estarão completamente formados somente após a metamorfose.. O desenvolvimento completo da lâmina, lóbula e medula e da retina ocorre somente após a metamorfose. Durante a fase pupal as estruturas do lóbulo óptico estão prontas, porém na retina observa-se ainda gradual pigmentação, encurtamento dos feixes axonais e alongamento dos rabdômeros até atingirem o seu comprimento final logo antes da emergência. / The processes that drive compound eye development in insects have been broadly studied in Drosophila melanogaster in which they arise from imaginal discs. Little is known about optic lobe and retina development in other insects, most of which do not have imaginal eye discs attached to the nervous system. For this reason, a comparative analysis of eye development in the honey bee, Apis mellifera, not only contributes to evo-devo aspects comparing the major families of holometabolous insects, but also may elucidate questions about developmental plasticity because the compound eyes of the honeybee show strong sex and caste-specific differences. Since our primary objective was to elucidate the pattern of cellular differentiation and division during eye development we performed histological and immunolabelling analyses during the postembrionic stages of development, concomitant with a realtime analysis of roughest gene expression. For the immunolabelling experiments we used an anti-phospho-histone H3 antibody that labels cells in M phase. We analyzed eye development in worker larvae starting with the third instar until white, pink and browneyed pupae, paying special attention to the fifth instar which was subdivided into feeding phase (L5F), cocoon spinning phase (L5S) and prepupae (PP). Optic Lobe development in Apis mellifera occurs by neuroepithelial folding initiating from a differentiation center, in the larval brain. This center sequentially produces the neural layers of the optic lobe (medulla, lobula and lamina). Development of the lamina, which is the last layer to be formed, takes more time and happens in two steps before metamorphosis. The first step is emergence at the beginning of the fifth larval instar coinciding with the first peak of roughest gene expression. The second step 8 occurs during the cocoon spinning phase and is marked by its inner differentiation, again accompanied by a second peak of roughest expression. During this second peak of roughest expression the rabdomers in the retina become visible. These, however, cplete thir development only during the pupal stage. The development of the lamina, lobula and medulla is not complete until after metamorphosis, even though these optic lobe structures are structurally defined already at the beginning of the pupal phase. Retinal development in this phase is marked by gradual pigmentation, axonal bundle shortening and rabdomer elongation, which reach their final size just prior to emergence of the bees from their brood cells.

Mecanismos que regem o desenvolvimento do olho composto e lóbulo óptico tem sido amplamente estudados em Drosophila melanogaster onde a retina é formada a partir de um disco imaginal anexado com o cérebro e os lóbulos opticos a partir do primórdio óptico externo. Através de histologia comparativa e análise de expressão gênica no desenvolvimento do sistema visual em Apis mellifera nós procuramos elucidar questões sobre plasticidade do desenvolvimento subjacente a fortes diferenças sexo- e casta-específico no olho assim como contribuir com aspectos evo-devo. O desenvolvimento dos lóbulos ópticos ocorre por dobramento neuroepitelial a partir de um centro de diferenciação no cérebro larval. Deste centro, a medula, lamina e lóbula surgem ao mesmo tempo em operárias e zangões. Dois passos marcam a diferenciação da lâmina (i) sua origem a partir da diferenciação de neuroblastos da camada mais externa da medula, isso coincidindo com o primeiro pico de expressão de roughest, e (ii) 24 horas mais tarde o aparecimento dos omatideos hexagonais coincidindo com o segundo pico de expressão de roughest. Com a inclusão de genes candidatos relacionados com o desenvolvimento do olho e lóbulos ópticos em insetos [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) e epidermal growth factor receptor (egfr)] nós encontramos distintos picos de expressão para sol, eya, mnb e so em níveis de transcritos e tempo de aparição do pico diferindo entre operárias e zangões. Enquanto estes quatro genes mostraram relativa sincronia durante o desenvolvimento em zangões, o mesmo não ocorreu em operárias. Além disso, em operárias sol é muito mais expresso na pré-pupa do que em zangões. Ambos os sexo mostraram padrões muito similares de expressão de elav, exceto por um atraso em zangões. Em contraste, a expressão de egfr ocorre antes em zangões. Durante a phase chave no desenvolvimento do sistema visual, uma análise global do transcriptoma, por meio de micro-arranjos mostrou vários genes relacionados com ciclo celular entre os diferencialmente expressos. Em conclusão, a relação entre tempo e eventos morfológicos com os padrões de expressão gênica revelou diferenças possivelmente relacionadas com mecanismos subjacentes ao desenvolvimento do sistema visual altamente dimorfico de Apis mellifera. / Developmental mechanisms governing compound eye development in insects have been broadly studied in Drosophila melanogaster, where the retina is formed from an imaginal disc attached to the larval brain. However little is known about eye development in other insects, most of which do not have such imaginal eye discs. Through a comparative histological and gene expression analysis of eye development in the honey bee, Apis mellifera, we intended to elucidate questions about developmental plasticity underlying the marked sex and castespecific differences in eye size, as well as to contribute to evo-devo aspects. Optic lobe development occurs by neuroepithelial folding initiating from a differentiation center in the larval brain. From this center, the medula, lamina and lobula arise at the same time in drones and workers. Two steps mark the differentiation of the lamina (i) its origin from neuroblasts differentiating in the outer layer of the medula, this coinciding with the first peak of roughest expression during the feeding stage of the fifth larval instar, and (ii) 24 hours later, the appearance of hexagonal ommatidia, coinciding with a second peak in roughest expression. Upon including further candidate genes related to insect eye development [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) and epidermal growth factor receptor (egfr)] we found distinct expression peaks for sol, eya, mnb and so, with timing and relative transcript levels differing between drones and workers. Whereas these four genes showed a relatively synchronous pattern of expression in drones in the fifth larval instar, this was not so in workers. Furthermore, in prepupae sol was higher expressed in workers than the other three genes, and also in comparison to drones. Both sexes showed a strikingly similar expression pattern for elav, except for some delay in drones. In contrast, egfr expression was found to occur earlier in drones. Through a global transcriptom analysis, done at a key step of larval development, several genes were reveled as diffetentially expressed, many of these regulating cell cycle steps. In conclusion, the relationship in the timing of morphological events with gene expression patterns revealed differences possibly related to mechanisms underlying development of the highly dimorphic compound eye in the honey bee.

Mecanismos que regem o desenvolvimento do olho composto e lóbulo óptico tem sido amplamente estudados em Drosophila melanogaster onde a retina é formada a partir de um disco imaginal anexado com o cérebro e os lóbulos opticos a partir do primórdio óptico externo. Através de histologia comparativa e análise de expressão gênica no desenvolvimento do sistema visual em Apis mellifera nós procuramos elucidar questões sobre plasticidade do desenvolvimento subjacente a fortes diferenças sexo- e casta-específico no olho assim como contribuir com aspectos evo-devo. O desenvolvimento dos lóbulos ópticos ocorre por dobramento neuroepitelial a partir de um centro de diferenciação no cérebro larval. Deste centro, a medula, lamina e lóbula surgem ao mesmo tempo em operárias e zangões. Dois passos marcam a diferenciação da lâmina (i) sua origem a partir da diferenciação de neuroblastos da camada mais externa da medula, isso coincidindo com o primeiro pico de expressão de roughest, e (ii) 24 horas mais tarde o aparecimento dos omatideos hexagonais coincidindo com o segundo pico de expressão de roughest. Com a inclusão de genes candidatos relacionados com o desenvolvimento do olho e lóbulos ópticos em insetos [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) e epidermal growth factor receptor (egfr)] nós encontramos distintos picos de expressão para sol, eya, mnb e so em níveis de transcritos e tempo de aparição do pico diferindo entre operárias e zangões. Enquanto estes quatro genes mostraram relativa sincronia durante o desenvolvimento em zangões, o mesmo não ocorreu em operárias. Além disso, em operárias sol é muito mais expresso na pré-pupa do que em zangões. Ambos os sexo mostraram padrões muito similares de expressão de elav, exceto por um atraso em zangões. Em contraste, a expressão de egfr ocorre antes em zangões. Durante a phase chave no desenvolvimento do sistema visual, uma análise global do transcriptoma, por meio de micro-arranjos mostrou vários genes relacionados com ciclo celular entre os diferencialmente expressos. Em conclusão, a relação entre tempo e eventos morfológicos com os padrões de expressão gênica revelou diferenças possivelmente relacionadas com mecanismos subjacentes ao desenvolvimento do sistema visual altamente dimorfico de Apis mellifera. / Developmental mechanisms governing compound eye development in insects have been broadly studied in Drosophila melanogaster, where the retina is formed from an imaginal disc attached to the larval brain. However little is known about eye development in other insects, most of which do not have such imaginal eye discs. Through a comparative histological and gene expression analysis of eye development in the honey bee, Apis mellifera, we intended to elucidate questions about developmental plasticity underlying the marked sex and castespecific differences in eye size, as well as to contribute to evo-devo aspects. Optic lobe development occurs by neuroepithelial folding initiating from a differentiation center in the larval brain. From this center, the medula, lamina and lobula arise at the same time in drones and workers. Two steps mark the differentiation of the lamina (i) its origin from neuroblasts differentiating in the outer layer of the medula, this coinciding with the first peak of roughest expression during the feeding stage of the fifth larval instar, and (ii) 24 hours later, the appearance of hexagonal ommatidia, coinciding with a second peak in roughest expression. Upon including further candidate genes related to insect eye development [small optic lobe (sol), eyes absent (eya), minibrain (mnb), sine oculis (so), embryonic lethal, abnormal vision (elav) and epidermal growth factor receptor (egfr)] we found distinct expression peaks for sol, eya, mnb and so, with timing and relative transcript levels differing between drones and workers. Whereas these four genes showed a relatively synchronous pattern of expression in drones in the fifth larval instar, this was not so in workers. Furthermore, in prepupae sol was higher expressed in workers than the other three genes, and also in comparison to drones. Both sexes showed a strikingly similar expression pattern for elav, except for some delay in drones. In contrast, egfr expression was found to occur earlier in drones. Through a global transcriptom analysis, done at a key step of larval development, several genes were reveled as diffetentially expressed, many of these regulating cell cycle steps. In conclusion, the relationship in the timing of morphological events with gene expression patterns revealed differences possibly related to mechanisms underlying development of the highly dimorphic compound eye in the honey bee.

Retinal ganglion cell (RGC) axon arbors within the optic tectum are refined in development through a dynamic process of activity-dependent remodeling. The synaptic adhesion molecule N-cadherin is a candidate for mediating selective stabilization and elaboration of RGC axons due to its localization to perisynaptic sites and its modifiability by neural activity. RGCs of Xenopus tadpoles were co-transfected with plasmids encoding a dominant negative N-cadherin (N-cadDeltaE) and eGFP or eYFP. Using two-photon in vivo time-lapse imaging, we found that axons expressing N-cadDeltaE became less elaborate than controls over three days of daily live imaging. Shorter interval time-lapse imaging of axons expressing synaptophysin-GFP to visualize putative synaptic sites revealed that N-cadDeltaE expressing axons form fewer stable branches than controls and that stabilization of axonal branches at synaptic sites is altered. We conclude that N-cadherin participates in the stabilization of axonal branches in the Xenopus retinotectal system.

No description available.

No description available.