Elisabeth Stafflinger:
The characterization of new hormonal systems in arthropods with a focus on neuropeptide GPCRs

Date: 15-12-2009    Supervisor: Cornelis J. P. Grimmelikhuijzen

Neuropeptides and their G-protein coupled receptors (GPCRs) occupy a high hierarchical position in the physiology of animals, because they steer important processes such as reproduction, development, and behaviour. Within the last nine years, several genomes have been sequenced providing the basis of genome comparisons and analysis. This yields new insights in GPCR evolution and ligand co-evolution, but also uncovers many new hormonal systems. In this thesis, I characterized a group of neuropeptide GPCRs and their ligands in arthropods and investigated GPCR evolution and co-evolution of their ligands.

Previously, it has been found that the locust Locusta migratoria has a peptide related to the oxytocin and vasopressin peptides from mammals, but in the sequenced genomes of the fruit flies, mosquitoes, the honey bee, and the silkworm, no oxytocin/vasopressin-like peptide or receptor could be found. When the genome of the red flour beetle Tribolium castaneum was sequenced and released, I found a peptide closely related to the oxytocin and vasopressin peptide that I called inotocin (for insect oxytocin/vasopressin-like peptide). Additionally, I could also find a receptor structurally highly related to the receptors from the oxytocin/vasopressin family, that I therefore called inotocin receptor. Oxytocin, vasopressin, and related peptides are often correlated with reproduction. Using RNAi, it could be excluded that this system is responsible for any function involved in oviposition. I could also annotate the inotocin system (receptor and peptide) in the parasitic wasp Nasonia vitripennis. Furthermore, I found an oxytocin/vasopressin-like peptide and cloned an oxytocin/vasopressin-like receptor from Daphnia pulex, a crustacean belonging to Branchiopoda. Branchiopods are regarded to be the direct ancestors of insects; finding this system in D. pulex therefore explains the occurrence of the inotocin system in insects in an evolutionary perspective.

Additionally, a new hormonal system, structurally intermediate between the AKH and the corazonin system was found. We therefore called the peptide ACP (for AKH/corazoninrelated peptide). Since both the ACP and its receptor are intermediate between the AKH/AKHR and corazonin/corazonin receptor, this is a prominent example of receptor/ligand co-evolution, probably originating from receptor and ligand gene duplications. So far, the ACP system could be found in the mosquitoes Anopheles gambiae, Aedes aegypti, and Culex pipiens, the silkworm Bombyx mori, T. castaneum, N. vitripennis, and the bug Rhodnius prolixus but not in all the other sequenced insect genomes. Bioassays showed that only ACP can activate the ACPR, but not AKH or corazonin, showing that this system is an independent signalling system.

The work in this thesis describes unknown hormonal systems in arthropods and gives insights into their evolution. Furthermore, it shows that neuropeptide/receptor couples can easily duplicate or disappear during insect evolution.