Molecular, pharmacological and functional characterization of serotonin and dopamine receptors from Tribolium castaneum and Schistocerca gregaria

Biogenic amines, such as dopamine (DA) and serotonin (5-HT), are major neuroactive substances that influence several important processes in both vertebrates and invertebrates. The aminergic signals are communicated to target cells via binding to receptors in the plasma membrane, mostly G protein-coupled receptors (GPCRs). In insects, three 5-HT receptor families (5-HT1, 5-HT2 and 5-HT7) and four DA receptors (DOP1, DOP2, DOP3 and a dopamine/ecdyson (DopEc) receptor) are identified. However, molecular, pharmacological and functional information about these receptors in insects is poor. The aim of this project was the gain more insight in these receptors in two insect species: the red flour beelte, Tribolium castaneum and the desert locust Schistocerca gregaria. The red flour beetle is a major global pest of stored products for human and animal consumption. It is a well-known model insect, mainly contributing to comparative studies of genetic, developmental and physiological changes during evolution. The genome of the beetle was completely sequences a few years ago, and therefrom we identified several biogenic amine receptors. We cloned two 5-HT receptors, showing sequence similarity to known 5-HT1 or 5-HT7 receptors (called (Trica5-HT1B and Trica5-HT7). Activation of Trica5-HT1B by 5-HT induced a decrease in intracellular cAMP levels, while activation of Trica5-HT7 increased the intracellular cAMP levels. Several known agonists of mammalian 5-HT receptors were tested, but they were much less potent and efficient than 5-HT in activating the receptors. From nine tested antagonists, prazosin, methiothepin and methysergide were the most potent on Trica5-HT1B, all showing competitive inhibition in presence of 5-HT. Ketanserin and methiothepin appeared to be the most potent antagonists of Trica5-HT7. In addition, a DA receptor from T. castaneum was cloned, named TricaDOP3. Receptor activation with DA in mammalian cells increased the intracellular calcium levels and decreased the intracellular cAMP levels in a dose-dependent manner. A pharmacological study identified the mammalian DA receptor agonists (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene and bromocriptine as potent and full agonists. Methysergide was the most potent antagonist and showed competitive inhibition in presence of DA. The assembled data offer important information for the use of pharmacological compounds in functional research on 5-HT and DA receptors in insects and other invertebrates, and contribute to establish a more reliable classification scheme for insect 5-HT and DA receptors. A second pest insect investigated during this project, is the desert locust. Generally these locusts live as innocent, shy individuals in deserts in North-Africa, the Middle-East and the Indian subcontinent. However, certain environmental conditions will lead to an increase in the number of locusts. The resulting increased population densities can eventually induce the formation of large devastating swarms that are a serious threat for the agriculture. Locusts show what is called a density dependent phenotypic plasticity or phase change between a harmless solitarious and a swarm-farming gregarious phase. Solitarious and gregarious locusts differ in several aspects of their physiology and morphology. However, the first characteristic that changes very rapidly is the behavior, from inactive behavior in solitarious locust that try to avoid conspecifics to active behavior in gregarious locusts that are attracted to each other.In the desert locust, as well as in the migratory locust, both 5-HT and DA have been shown to be involved in these behavioral phase changes. In this project, the possible role of several 5-HT and DA receptors to communicate these gregarizing signals in the desert locust were investigated.First, the poor sequence information of 5-HT and DA receptors from the desert locust was supplemented with data from transcriptome databases and amplification studies. In addition, partial sequences of two biosynthetic enzymes of 5-HT and DA were cloned and their transcript levels were examined in different tissues using quantitative real-time PCR. Tryptophan hydroxylase (TRH) was mainly expressed in the CNS, while aromatic amino acid decarboxylase (AADC) was ubiquitously expressed. We also found partial sequences of putative 5-HT1, 5-HT7 and DOP1 receptors and sequences encoding full-length DOP3 and DopEcR receptor. Expression analysis of the receptor genes showed high transcript levels in the CNS, with often higher expression in solitarious compared to gregarious locusts. This is consistent with the neuromodulatory roles of 5-HT and DA in insects, and might indicate roles in differentially regulated processes in both phases. Subsequently, we investigated the effect of a RNA interference (RNAi) induced knockdown of the 5-HT7, DOP1 or DOP3 receptor on the time course of the behavioral phase transition using a behavioral arena assay. Solitarious locusts were shown to display gregarious behavior after only a few hours of crowding. However, knockdown of the receptors did not induce any significant effects on the behavioral gregarization after 2, 4 or 24 hours crowding compared to control-injected animals, neither in terms of activity, nor in attraction/repulsion behavior. Additional studies will have to confirm or disprove these results, together with investigating the role of other biogenic amine receptors in the process of behavioral gregarization of desert locusts.status: publishe