Dopamine is found in many invertebrate organisms, including insects, however, the mechanisms through which this amine operates remain unclear. We have expressed two dopamine receptors cloned from honey bee (AmDOP1 and AmDOP2) in insect cells (Spodoptera frugiperda), and compared their pharmacology directly using production of cAMP as a functional assay. In each assay, AmDOP1 receptors required lower concentrations of dopamine and 6,7-ADTN for maximal activation than AmDOP2 receptors. Conversely, butaclamol and cis(Z)-flupentixol were more potent at blocking the cAMP response mediated through AmDOP2 than AmDOP1 receptors. Expression of AmDOP1, but not AmDOP2, receptors significantly increased levels of cAMP even in the absence of ligand. This constitutive activity was blocked by cis(Z)-flupentixol. This work provides the first evidence of a constitutively activated dopamine receptor in invertebrates and suggests that although AmDOP1 and AmDOP2 share much less homology than their vertebrate counterparts, they display a number of functional parallels with the mammalian D1-like dopamine receptors.

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera . The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis . Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.

In the honey bee, responsiveness to sucrose correlates with many behavioural parameters such as age of first foraging, foraging role and learning. Sucrose responsiveness can be measured using the proboscis extension response (PER) by applying sucrose solutions of increasing concentrations to the antenna of a bee. We tested whether the biogenic amines octopamine, tyramine and dopamine, and the dopamine receptor agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN) can modulate sucrose responsiveness. The compounds were either injected into the thorax or fed in sucrose solution to compare different methods of application. Injection and feeding of tyramine or octopamine significantly increased sucrose responsiveness. Dopamine decreased sucrose responsiveness when injected into the thorax. Feeding of dopamine had no effect. Injection of 6,7-ADTN into the thorax and feeding of 6,7-ADTN reduced sucrose responsiveness significantly. These data demonstrate that sucrose responsiveness in honey bees can be modulated by biogenic amines, which has far reaching consequences for other types of behaviour in this insect. (C) 2002 Elsevier Science B.V. All rights reserved.

The acinar salivary gland of the cockroach, Periplaneta americana, is innervated by dopaminergic and serotonergic nerve fibers. Stimulation of the glands by serotonin (5-hydroxytryptamine, 5-HT) results in the production of a protein-rich saliva, whereas stimulation by dopamine results in saliva that is protein-free. Thus, dopamine acts selectively on ion-transporting peripheral cells within the acini, and 5-HT acts on protein-producing central cells. We have investigated the pharmacology of the 5-HT-induced secretory activity of isolated salivary glands of P. americana by testing several 5-HT receptor agonists and antagonists. The effects of 5-HT can be mimicked by the non-selective 5-HT receptor agonist 5-methoxytryptamine. All tested agonists that display at least some receptor subtype specificity in mammals, i.e., 5-carboxamidotryptamine, (+/-)-8-OH-DPAT, (+/-)-DOI, and AS 19, were ineffective in stimulating salivary secretion. 5-HT-induced secretion can be blocked by the vertebrate 5-HT receptor antagonists methiothepin, cyproheptadine, and mianserin. Our pharmacological data indicate that the pharmacology of arthropod 5-HT receptors is remarkably different from that of their vertebrate counterparts. (C) 2007 Elsevier Ltd. All rights reserved.

The phenolamines octopamine and tyramine control, regulate, and modulate many physiological and behavioral processes in invertebrates. Vertebrates possess only small amounts of both substances, and thus, octopamine and tyramine, together with other biogenic amines, are referred to as “trace amines.” Biogenic amines evoke cellular responses by activating G-protein-coupled receptors. We have isolated a complementary DNA (cDNA) that encodes a biogenic amine receptor from the American cockroach Periplaneta americana, viz., Peatyr1, which shares high sequence similarity to members of the invertebrate tyramine-receptor family. The PeaTYR1 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with tyramine reduces adenylyl cyclase activity in a dose-dependent manner (EC50 350 nM). The inhibitory effect of tyramine is abolished by co-incubation with either yohimbine or chlorpromazine. Receptor expression has been investigated by reverse transcription polymerase chain reaction and immunocytochemistry. The mRNA is present in various tissues including brain, salivary glands, midgut, Malpighian tubules, and leg muscles. The effect of tyramine on salivary gland acinar cells has been investigated by intracellular recordings, which have revealed excitatory presynaptic actions of tyramine. This study marks the first comprehensive molecular, pharmacological, and functional characterization of a tyramine receptor in the cockroach.

Background and purpose: 5-Hydroxytryptamine (5-HT) has been shown to control and modulate many physiological and behavioural functions in insects. In this study, we report the cloning and pharmacological properties of a 5-HT1 receptor of an insect model for neurobiology, physiology and pharmacology. Experimental approach: A cDNA encoding for the Periplaneta americana 5-HT1 receptor was amplified from brain cDNA. The receptor was stably expressed in HEK 293 cells, and the functional and pharmacological properties were determined in cAMP assays. Receptor distribution was investigated by RT-PCR and by immunocytochemistry using an affinity-purified polyclonal antiserum. Key results: The P. americana 5-HT1 receptor (Pea5-HT1) shares pronounced sequence and functional similarity with mammalian 5-HT1 receptors. Activation with 5-HT reduced adenylyl cyclase activity in a dose-dependent manner. Pea5-HT1 was expressed as a constitutively active receptor with methiothepin acting as a neutral antagonist, and WAY 100635 as an inverse agonist. Receptor mRNA was present in various tissues including brain, salivary glands and midgut. Receptor-specific antibodies showed that the native protein was expressed in a glycosylated form in membrane samples of brain and salivary glands. Conclusions and implications: This study marks the first pharmacological identification of an inverse agonist and a neutral antagonist at an insect 5-HT1 receptor. The results presented here should facilitate further analyses of 5-HT1 receptors in mediating central and peripheral effects of 5-HT in insects.

The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT7 receptor family. Expression of the Am5-HT7 receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT7 is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC50 = 1.1-1.8 nM). The Am5-HT7 receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.

Aerosols and clouds provide the largest uncertainty in the atmospheric radiation budget. The main focus of this thesis was to investigate the ability of organic compounds in aerosol particles to form clouds, and more specifically those emitted by living organisms. The cloud forming properties of the highly water-soluble methyltetrols and polyols, which are compounds produced by plants and fungi that are common in aerosol, were studied. All compounds and their salt mixtures have a moderate potential to serve as cloud condensation nuclei (CCN). They are thus not likely to have a significant global impact on cloudiness. The potential presence of surfactants released by microorganisms was investigated for aerosols sampled at different locations. Very low surface tension values were measured for these aerosol extracts (30 mN/m), which implies that these aerosols have good CCN properties and indicate the presence of biosurfactants. Their occurrence in aerosols still needs to be confirmed directly by chemical identification. Reactions of organic compounds in sulfate salt solutions exposed to UV-light were studied and found to produce surface active compounds. Thus, mixed sulfate/organic aerosol could have more favourable CCN properties after exposure to light than when kept in the dark. The surface active compounds were proposed to be long-chained organosulfates with hydrophilic and hydrophobic parts, similar to other amphiphilic surfactants. Mixtures of salt and strong surfactants formed by bacteria were studied using two different techniques for determining their CCN properties. There were inconsistencies between the two methods which could be accounted for by surface partitioning. The studied mixtures were determined to be good potential CCN material in both techniques. All these aspects require further investigation, but if the impact of strong biogenic surfactants on cloud formation is confirmed, a new link between living organisms and climate would be identified. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript.

The global silicon (Si) cycle is of fundamental importance for the global carbon cycle. Diatom growth in the oceans is a major sequestration pathway for carbon on a global scale (often referred to as the biological pump). Patterns of diatoms preserved in marine sediment records can reveal both natural and anthropogenic driven environmental change, which can be used to understand silicon dynamics and climate change. Si isotopes have been shown to have great potential in order to understand the Si cycle by revealing both past and present patterns of dissolved Si (DSi) utilization, primarily when diatoms form their siliceous frustules (noted as biogenic silica, BSi). However, studies using Si isotopes are still scarce and only a few studies exist where stable Si isotopes are used to investigate the biogeochemical Si cycle in aquatic systems. Therefore, this thesis focuses on developing analytical methods for studying BSi and DSi and also provides tools to understand the observed Si isotope distribution, which may help to understand impacts of climate change and human perturbations on marine ecosystems. The Baltic Sea, one of the biggest estuarine systems in the world, was chosen as the study site. BSi samples from a sediment core in Bothnian Bay, the most northern tip of the Baltic Sea, and diatom samples from the Oder River, draining into the southern Baltic Sea were measured and reported in Paper II and III, after establishing a method for Si isotope measurements (Paper I). Si isotope fractionation during diatom production and dissolution was also investigated in a laboratory-controlled experiment (Paper IV) to validate the observations from the field. The major result is that Si isotope signatures in BSi can be used as an historical archive for diatom growth and also related to changes in climate variables. There is isotopic evidence that the Si cycle has been significantly altered in the Baltic Sea catchment by human activities. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Lake Malawi is the second largest lake in Africa, supporting diverse populations of endemic cichlids and supplying essential water resources to Malawi, Mozambique and Tanzania. However, population growth, rapid deforestation and intensive agriculture, especially in the southern catchments, have accelerated soil erosion during the last half century. These anthropogenically-disturbed catchments have caused rivers to transport greater sediment loads into Lake Malawi than rivers within forested catchments. Lake Malawi?s immense size and oligotrophic nature may retard detection of inputs of external contaminants. Reversing the effects of increased nutrient loading to Lake Malawi once observed would likely take generations, as the residence time of water is over 140 years. Therefore, sensitive metrics are required to assess the effects of land use change and climate variability in Lake Malawi in advance of deleterious effects. In this study, paleolimnological analyses of four sediment cores collected in 1997 and 1998 along a longitudinal transect of Lake Malawi, dated with ²¹⁰Pb analyses and analyzed for biogenic silica and sedimentary diatom assemblages, were used to create a long-term water quality dataset. These four sites span gradients of land use and latitude in order to reconstruct limnological conditions over the whole lake during the last 300 years. Paleoecological results indicate that patterns of diatom assemblage change are not uniform lake wide. Southern cores contain evidence of nutrient enrichment starting as early as ca. 1940, indicated by increased silica, carbon and nitrogen burial. By ca. 1970, increased rates of sedimentation, diatom influx and changes in diatom community composition, characterized by increased percent abundance of eutrophic diatom taxa, are attributable to accelerated enrichment by terrestrial soil erosion. The succession of diatoms in southern Lake Malawi begins with high percent abundance of <i>Aulacoseira nyassensis</i> and <i>Fragilaria africana</i>, which thrive in nutrient-rich waters, followed by a shift towards diatom taxa with reduced silica requirements by ca. 1980 (e. g. <i>Stephanodiscus nyassae, S. minutulus, S. muelleri, Cyclostephanos</i> and small <i>Nitzschia</i> species. ), a pattern comparable to the eutrophication-induced decline in silica to phosphorus ratios in Lake Victoria. In Lake Malawi, evidence of eutrophication extends to the mid lake as indicated by similar diatom assemblage changes in the sediment core from the central region. Diatom stratigraphies from the north end of the lake indicate no observable impacts of land use change on the northern basin of Lake Malawi during the past 350 years. However, a nine-meter rise in water level ca. 1860 AD appears to have resulted in elevated diatom influxes at that time comparable to the recent eutrophication-induced diatom influxes of the southern cores. The effects of this rise in water level was recorded in all three measured sites, southern, central and northern Lake Malawi, indicating lake-wide increased productivity, yet changes to the diatom community composition were imperceptible. This study shows evidence of recent cultural eutrophication altering limnological conditions with impacts to the biogeochemical cycling of silica, the available silica to phosphorus ratios and the biotic communities of a large portion of Lake Malawi. Thus, providing an early warning that proper stewardship of Lake Malawi requires effective management of land-use practices within the catchment to reduce soil erosion and avoid widespread water quality deterioration of this great lake.