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Este trabalho descreve o desenvolvimento e a validação de um gerador de estímulos visuais naturalísticos, GEN, utilizado em experimentos com o sistema visual de moscas. Tal gerador projeta a imagem em um anteparo que abrange todo o campo visual da mosca. Um espelho acoplado a um motor linear movimenta a imagem horizontalmente de acordo com velocidades predefinidas pelo experimentador. Ao contrário dos tradicionais geradores com monitores de raios catódicos - que apresentam imagens através de uma sequencia de quadros entre 60 e 200Hz - o GEN apresenta a imagem de forma contínua. Além de eliminar o problema das altas taxas de quadros por segundo exigidas nestes experimentos, o novo sistema também gera imagens com uma maior resolução, brilho e contraste. Durante a apresentação dos estímulos visuais foram registrados os potenciais de ação do neurônio H1 da mosca Chrysomya megacephala, localizado na placa lobular e responsável pela detecção de movimentos horizontais. Os resultados deste trabalho mostram como a resposta do neurônio estudado depende de parâmetros do estímulo tais como: velocidade, luminância, campo visual estimulado e frequência espacial da imagem utilizada. A influência destes parâmetros na resposta neural demonstram a importância de possuir geradores capazes de simular em laboratório estímulos que a mosca encontra em seu habitat natural. / This work presents the development and the validation of a naturalistic visual stimuli generator, NSG, to be used in neuroscience experiment with the flys visual system. To satisfy the requirement of naturalistic stimuli, the new generator projects a slide onto a screen covering the whole visual field of the fly. A mirror controlled by a linear electrical motor moves the image horizontally according to a predefined velocity profile. In addition, the NSG shows the image continuously as opposed to usual stimuli generators with cathode ray monitors having a frame rate between 60 and 200Hz. A further advantage of the NSG is its high luminance, brightness and contrast. During the stimulus presentations, the activity of the H1 neuron of the blowfly Chrysomya megacephala was recorded, this neuron is sensitive to horizontal image displacements. The results show how the response of the studied neuron depends on stimulus parameters such as speed, luminance, visual field stimulated and the spatial frequency of the image used. The influence of these parameters on neural response demonstrates the importance of generators that reproduces in laboratory the flys natural habitat.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

Este trabalho descreve o desenvolvimento e a validação de um gerador de estímulos visuais naturalísticos, GEN, utilizado em experimentos com o sistema visual de moscas. Tal gerador projeta a imagem em um anteparo que abrange todo o campo visual da mosca. Um espelho acoplado a um motor linear movimenta a imagem horizontalmente de acordo com velocidades predefinidas pelo experimentador. Ao contrário dos tradicionais geradores com monitores de raios catódicos - que apresentam imagens através de uma sequencia de quadros entre 60 e 200Hz - o GEN apresenta a imagem de forma contínua. Além de eliminar o problema das altas taxas de quadros por segundo exigidas nestes experimentos, o novo sistema também gera imagens com uma maior resolução, brilho e contraste. Durante a apresentação dos estímulos visuais foram registrados os potenciais de ação do neurônio H1 da mosca Chrysomya megacephala, localizado na placa lobular e responsável pela detecção de movimentos horizontais. Os resultados deste trabalho mostram como a resposta do neurônio estudado depende de parâmetros do estímulo tais como: velocidade, luminância, campo visual estimulado e frequência espacial da imagem utilizada. A influência destes parâmetros na resposta neural demonstram a importância de possuir geradores capazes de simular em laboratório estímulos que a mosca encontra em seu habitat natural. / This work presents the development and the validation of a naturalistic visual stimuli generator, NSG, to be used in neuroscience experiment with the flys visual system. To satisfy the requirement of naturalistic stimuli, the new generator projects a slide onto a screen covering the whole visual field of the fly. A mirror controlled by a linear electrical motor moves the image horizontally according to a predefined velocity profile. In addition, the NSG shows the image continuously as opposed to usual stimuli generators with cathode ray monitors having a frame rate between 60 and 200Hz. A further advantage of the NSG is its high luminance, brightness and contrast. During the stimulus presentations, the activity of the H1 neuron of the blowfly Chrysomya megacephala was recorded, this neuron is sensitive to horizontal image displacements. The results show how the response of the studied neuron depends on stimulus parameters such as speed, luminance, visual field stimulated and the spatial frequency of the image used. The influence of these parameters on neural response demonstrates the importance of generators that reproduces in laboratory the flys natural habitat.

Made available in DSpace on 2016-04-28T15:01:30Z (GMT). No. of bitstreams: 1 2006-Roney Rodrigues Guimaraes.pdf: 1366984 bytes, checksum: a3849d32ef332f39a6a2b06635783317 (MD5) Previous issue date: 2006-03-17 / Among the dipterans of medic-sanitary interest the blowfly families Calliphoridae and Sarcophagidae are distinguished. Its occurrence, distribution and predominance in metropolitans areas are factors of great importance to the Public Health, because the adults are vectors of pathogens for the man and its larvae attack the man in such a way as animal domestic servants. The capture of the calliphorid fauna with traps, bait with viscera cool of chicken, in three distinct ecotypes (agricultural, urban and forest). 31.480 califor?deos dipterans distributed in the following species had been captured: Chrysomya megacephala (54,45%), C. putoria (11,18%), C. albiceps (13,63%), Phaenicia eximia (5,90%), P. cuprina (1,07%), Cochliomyia macellaria would (9,06%), C. hominivorax (0,53%), Hemilucilia segmentaria would (0,80%), H. semidiaphana (1,40%), Mesembrinella bellardiana (1,24%), Eumesembrinella sp. (0,51%), Chloroprocta idioidea (0,09%) and P. sericata (0,13%). The Sinanthropy Index and Seasonal Fluctuation and the Coefficient of Constancy of the species between the autumn of the 2002 and summer of 2004 had been determined. C. albiceps had the highest Sinanthropy Index (IS = + 41,7) conferring to this species was has Preference for Inhabited Areas. Also C. megacephala (IS = +33.01), C. putoria (IS = +25.1), C. hominivorax (IS = +24.1), C. macellaria would (IS = +21,14), P. cuprina (IS = +18,78), P. eximia (-9,67) species with Preference for Inhabited Areas and finally H. segmentaria, H. semidiaphana, M. bellardiana, Eumesembrinella sp., C. idioidea, P. sericata all pertaining to the group, according to data, that possess Complete Absence in Inhabited Areas (IS = -100). The microhimenopteran parasitoids are important tools in the biological control of flies of economic importance that cause damages to the health of human being and other animals. The captures had occurred between january and december of 2004 in urban, rural and forest area. 1.528 larvae of C. hominivorax (Coquerel, 1858) had been used as bait, 505 in the urban one, 556 in agricultural and the 467 in the forested one. The Sinanthropy Index, Coefficient of Constancy, the risk of parasitism (Odds Ratio) between the areas had been calculated, prevalence and parasitic intensity. The percentage was 46,6%. Aphaereta laeviuscula (Spinola, 1851) was captured only in agricultural environment, its indices had been: Sinanthropy I. = +50, C. constancy =25%, prevalence =0,72% and I. parasitoid =44,5; on the other hand Nasonia vitripennis (Walker, 1836) was captured in the areas agricultural and urban and the indices had been: Sinanthropy I. = +98, C. constance =58.3%, Odds Ratio = IC95% = 0,025 < > 0,27, p<0,05, prevalence= 3,2% and I. parasitoid =7,35. The risk of parasitism for N. vitripennis in urban areas is high. It was registered the occurrence of A. laeviuscula as parasite of C. hominivorax in the State of Rio de Janeiro. Blowfly, Parasitoids, Sinanthropy, Seasonal Variation. / Entre os d?pteros de interesse m?dico-sanit?rio destacam-se as moscas varejeiras das fam?lias Calliphoridae e Sarcophagidae. Sua ocorr?ncia, distribui??o e predomin?ncia em ?reas metropolitanas s?o fatores de grande import?ncia na Sa?de P?blica, pois os adultos s?o vetores de pat?genos para o homem e suas larvas atacam tanto o homem como animais dom?sticos. A captura da fauna califor?dica foi realizada atrav?s de armadilhas, iscadas com v?sceras frescas de frango, em tr?s ec?topos distintos (rural, urbano e de floresta). Foram capturados 31.480 d?pteros califor?deos distribu?dos nas seguintes esp?cies: Chrysomya megacephala (54,45%), Chrysomya putoria (11,18%), Chrysomya albiceps (13,63%), Phaenicia eximia (5,90%), Phaenicia cuprina (1,07%), Cochliomyia macellaria (9,06%), Cochliomyia hominivorax (0,53%), Hemilucilia segmentaria (0,80%), Hemilucilia semidiaphana (1,40%), Mesembrinella bellardiana (1,24%), Eumesembrinella sp. (0,51%), Chloroprocta idioidea (0,09%) e Phaenicia sericata (0,13%). Foram determinados os ?ndices de Sinantropia e Flutua??o Sazonal e o Coeficiente de Const?ncia das esp?cies entre o outono de 2002 e o ver?o de 2004. C. albiceps teve o mais alto ?ndice de Sinantropia (IS= + 41,7) conferindo a esta esp?cie, a seguinte classifica??o de acordo com Ferreira (1978 e 1983): esp?cie que tem Prefer?ncia por ?reas Habitadas. Tamb?m C. megacephala (IS= +33,01), C. putoria (IS= +25,1), C. hominivorax (IS= +24,1), C. macellaria (IS= +21,14), P. cuprina (IS= +18,78), P. eximia (-9,67) esp?cie com Prefer?ncia por ?reas Habitadas e finalmente H. segmentaria, H. semidiaphana, M. bellardiana, Eumesembrinella sp. C. idioidea, P. sericata todas pertencentes aos grupos, segundo os dados obtidos, que possuem Completa Aus?ncia em ?reas Habitadas (IS= -100). Os microhimen?pteros parasit?ides s?o ferramentas importantes no controle biol?gico de moscas de import?ncia econ?mica e causadora de danos ? sa?de humana e de outros animais. As capturas ocorreram entre janeiro e dezembro de 2004 em ?rea urbana, rural e florestal. 1.528 larvas de Cochliomyia hominivorax (Coquerel, 1858) foram usadas como iscas, 505 na urbana, 556 na rural e 467 na florestada. Foram calculados os ?ndices de Sinantropia, Coeficiente de Const?ncia, o risco (Odds Ratio) de parasitismo entre as ?reas, preval?ncia e intensidade parasit?ria. O percentual de emers?o foi de 46,6%. Aphaereta laeviuscula (Spinola, 1851), foi capturada apenas em ambiente rural, seus ?ndices foram: I. sinantropia= +50, C. const?ncia=25%, preval?ncia=0,72% e I. parasit?ria=44,5; j? Nasonia vitripennis (Walker, 1836) foi capturada nas ?reas rural e urbana e os ?ndices foram: I. sinantropia= +98, C. const?ncia=58,3%, Odds Ratio= IC95%= 0,025 <?> 0,27, p<0,05, preval?ncia, =3,2% e I. parasit?ria=7,35. O risco de parasitismo por N. vitripennis em ?reas urbanas ? alto. Registra-se a ocorr?ncia de A. laeviuscula como parasito de C. hominivorax no Estado do Rio de Janeiro.

Massively parallel high throughput sequencers are transforming the scientific research by reducing the cost and time necessary to sequence entire genomes. The goal of this project is to produce preliminary genome assemblies of calliphorid flies using Life Technologies’ Ion Torrent sequencing and Illumina’s MiSeq sequencing. I located, assembled, and annotated a novel mitochondrial genome for one such fly, the little studied Chrysomya pacifica that is central to one hypothesis about blow fly evolution. With sequencing data from Chrysomya megacephala, its forensically relevant sister species, much insight can be gained by alignments, sequence and protein analysis, and many more tools within the CLC Genomics Workbench software program. I present these analyses here of these recently diverged species.

No / The resurgence of larval biotherapy as a debridement tool in wound management has been accompanied by several clinical reports highlighting concomitant tissue regeneration. Studies employing in vitro cell motility assays have found that purified excretory/secretory (ES) products from Greenbottle larvae (blowfly, Lucilia sericata) are motogenic for human dermal fibroblasts when used as a supplement in culture media. The objective of the present study was to determine whether ES delivered using a prototype hydrogel wound dressing induced similar motogenic effects on fibroblastic (3T3) and epithelial cells (HaCaTs) comprising a scratched-monolayer wound model. Quantitative analysis by MTT assay failed to detect significant mitogenic effects of ES on either cell type. Quantitative image analysis revealed that ES exposure markedly accelerated wound closure through a motogenic effect on both fibroblasts and keratinocytes. Quantitative histochemical analysis detected significantly higher phosphotyrosine (pTyr) expression in ES-exposed cell cultures than in controls; moreover immunocytochemistry revealed conspicuously raised levels of pTyr expression in cells located at the wound margin. By attenuation with a panel of enzyme inhibitors these effects were attributed to the protease components of ES. The present results suggest that controlled delivery of ES as a follow-up to maggot debridement therapy may be an effective therapeutic option for stimulation of tissue regeneration in wound management.

Flüssigkeitssekretion und Proteinsekretion werden in Speicheldrüsen von Insekten über Hormone und Neurotransmitter gesteuert. Diese entfalten ihre physiologische Wirkung in den sekretorischen Drüsenzellen hauptsächlich über den zyklischen Adenosinmonophosphat (cAMP)-Signalweg und den Inositoltrisphosphat (IP<SUB>3</SUB>) / Ca²⁺-Signalweg. Die Mechanismen möglicher Wechselwirkungen zwischen diesen Signalwegen und ihre physiologischen Auswirkungen sind unzureichend bekannt.<p> Im Mittelpunkt dieser Arbeit stand die Frage, ob und wie sich der Ca²⁺-Signalweg und der cAMP-Signalweg in der Speicheldrüse der Diptere <I>Calliphora vicina</I> beeinflussen. Substanzen wie 5-Fluoro-α-Methyltryptamin und Histamin wurden in früheren Arbei-ten als Agonisten genutzt, um in den Speicheldrüsen von <I>C. vicina</I> selektiv den cAMP-Signalweg (getrennt vom IP<SUB>3</SUB>/Ca²⁺-Signalweg) zu aktivieren. Es zeigte sich in transepithelialen Potentialmessungen und mikrofluorometrischen Ca²⁺-Untersuchungen, dass beide Substanzen sowohl den cAMP-Weg als auch den Ca²⁺-Signalweg aktivierten. Die physiologischen Ursachen der Histamin-induzierten Ca²⁺-Erhöhung wurden genauer untersucht. <p> Zusammengefasst zeigten diese Untersuchungen, dass Histamin wie 5-HT den cAMP-Weg und die Phosphoinositidkaskade aktivierte. Im Gegensatz zu den 5-HT-induzierten Ca²⁺-Oszillationen, welche durch interzelluläre Ca²⁺-Wellen synchronisiert werden, verursachte Histamin bei niedrigen Konzentrationen lokale Ca²⁺-Oszillationen in einzelnen Zellen (keine Wellen). Bei höheren Histamin-Konzentrationen war eine anhaltende Ca²⁺-Erhöhung oder ein synchrones <quote>Ca²⁺-beating</quote> in der gesamten Drüse zu beobachten. <p> Des Weiteren wurde die Frage untersucht, ob eine Erhöhung der intrazellulären cAMP-Konzentration den IP<SUB>3</SUB> Ca²⁺-Signalweg in den Epithelzellen der Speicheldrüse beeinflussen kann. Es zeigte sich, dass cAMP den durch schwellennahe 5-HT-Konzentrationen induzierten Ca²⁺-Anstieg verstärkte. Diese Verstärkung wurde durch eine PKA-vermittelte Sensitivierung des IP<SUB>3</SUB>-Rezeptor/Ca²⁺-Kanals für IP<SUB>3</SUB> verursacht. Immunzytochemische Untersuchungen deuten dar-auf hin, dass die Proteinkinase A eng mit dem IP<SUB>3</SUB>-Rezeptor/Ca²⁺-Kanal assoziiert ist. Diese Messungen zeigen erstmals, dass auch bei Invertebraten der Botenstoff cAMP, PKA-vermittelt, den IP<SUB>3</SUB>-Rezeptor/Ca²⁺-Kanal des ER für IP<SUB>3</SUB> sensitiviert. / Fluid- and protein-secretion in the salivary glands of insects are controlled by hormones or neurotransmitters. These agonists activate two signalling cascades: the cAMP-pathway and the IP>sub>3</sub>/Ca-pathway. The functional crosstalk between these two signalling pathways is poorly understood. <p> Functional crosstalk between cAMP-pathway and IP₃/Ca²⁺-pathway was investigated in the salivary glands of the blowfly, <I>Calliphora vicina</I>. Histamine and 5-alpha-methyltryptamine were used in an attempt to activate the cAMP-pathway selectively, as suggested previously. By using transepithelial potential-measurements and microfluorometric Ca²⁺-imaging it was demonstrated that both substances activate the cAMP- and the IP₃/Ca²⁺-pathway. The physiological effects of histamine were investigated in detail. These experiments show that histamine causes an intracellular Ca²⁺-elevation that, in some preparations exhibits oscillations with concentration-dependent frequencies. In contrast to 5-HT induced intracellular Ca²⁺-oscillations and propagating intercellular Ca²⁺-waves histamine produces local Ca²⁺-oscillations in single cells or synchronous <quote>Ca²⁺-beating</quote> in the whole gland.<p> In addition the effects of increasing cAMP on the IP₃/Ca²⁺-pathway in the salivary glands of the blowfly were studied. It could be demonstrated that cAMP augments the 5-HT-induced Ca²⁺-increase in glands stimulated with low doses of 5-HT. This potentiation is the result of a PKA-mediated sensitisation of the IP₃-receptor/Ca²⁺-channel for IP₃. Results of immunocytochemical analyses show that the PKA is spatially associated with the ER.<p> These results show for the first time that in invertebrates as well as in vertebrates the second messenger cAMP sensitises the IP₃-receptor/Ca²⁺-channel for IP₃ by the action of a PKA.