Identification of a self-regulatory pheromone system that controls nymph aggregation behavior of rice spittlebug Callitettix versicolor

BACKGROUND: Nymphs of many spittlebug species are known to aggregate in one spittle mass, a behavior which greatly benefits the survival of the developing nymphs. Little is known, however, about the precise mechanisms that induce and regulate aggregation. Here, we investigated the aggregation behavior of nymphs of the rice spittlebug Callitettix versicolor, and analyzed the chemical composition of spittle masses. RESULTS: We identified six n-alkane compounds, namely un-, do-, tri-, tetra-, penta- and hexadecane in the spittle mass. Importantly, we showed that solitary spittle mass (SSM) and aggregation spittle mass (ASM) differed significantly in the amounts and composition of these compounds. While un-, do-, tri-, tetra-and hexadecane were overrepresented in SSM, pentadecane was found at significantly higher levels in ASM. Electrophysiological experiments showed that antennae responses to these six compounds were significantly higher than to both the hexane and the docosane control, which suggests a specific role of the six volatile alkanes as pheromones. In agreement with this hypothesis, behavioral tests revealed that five of the six compounds (e.g. un-, do-, tri-, tetra-, and hexadecane) acted as attractants across a wide concentration range. Thus, these five compounds allow recruitment of additional nymphs to a growing spittle mass. The sixth compound, pentadecane, attracted nymphs at low doses, whereas at higher doses, this effect vanished, suggesting that this alkane functioned as a repellent, thus preventing recruitment of additional individuals to a full aggregation in a spittle mass. CONCLUSIONS: In summary, our study identified a simple, yet fully functional feedback mechanism which allows aggregation at low nymph numbers, while preventing over-crowding beyond a set number of nymphs within one spittle mass. In conclusion, our study provides new insights into C. versicolor development and behavior that should greatly facilitate the identification of new approaches for pheromonal control of this pest.