Pheromone and population genetics analyses of Clavigralla species in Africa

Cowpea (Vigna unguiculata (L.) Walp) and common bean (Phaseolus vulgaris (L.)) are major sources of protein for human and animal consumption. Production of these crops is hampered by insect pests, especially the complex of brown spiny bugs of the genus Clavigralla (Hemiptera: Coreidae) which causes yield loss of up to 100% in various parts of Africa. The current practice of pesticide application to control these species is not efficient and has negative impacts on human health and the environment. These species are widely distributed in Africa and has a wide range of host plants, suggesting variability in genetics and chemical profiles of this pest. Aggregation behavior is observed in Clavigralla spp. from the nymph to adult stages, indicating the involvement of semiochemicals. Olfactometer assays showed that the egg parasitoid, Gryon species (Hymenoptera: Scelionidae) could potentially be biocontrol agent for Clavigralla spp. Gryon fulviventris Crawford (Hymenoptera: Scelionidae) was attracted to the volatiles released by C. tomentosicollis males, suggesting involvement of semiochemicals which have not been identified yet. Additionally, this attractive compound appears to be a male pheromone of which the bio-chemical composition, and its effect on the behaviour of Gryon sp. have not been elucidated.The aim of this study was to investigate the diversity of the Clavigralla species complex on crops in Bénin and Kenya, to elucidate aspects regarding the pheromone responsible for aggregation behavior of Clavigralla spp., to do a population genetics analyses of the Clavigralla species group. To achieve these objectives, detailed knowledge on the levels of parasitism of Clavigralla spp., cuticular chemistry that may influence parasitoid – pest interactions, the chemical profiles, the identity and genetic variability, and semiochemical cues mediating aggregation behavior and attraction in Clavigralla species and Gryon sp. respectively are required.Both live and dead ethanol preserved samples of the pests as well as their eggs were collected in West Africa (Bénin) and East Africa (Kenya). Colonies were established in an insectary and egg parasitoids were recorded. Additionally, parasitism and egg cuticular chemistry were investigated. A Y-tube olfactometer was used to investigate the effect of male and female headspace volatiles of Clavigralla spp. on their conspecifics. Headspace volatiles of both sexes of C. tomentosicollis, C. shadabi and C. elongata adults were collected and analyzed. Active-components to both pest and parasitoid antennae were identified by coupled GC/electroantennographic detection (GC/EAD) and GC/MS respectively. Olfactometer assays were performed to determine the effect of male-specific compound(s) on behavior of both the pest and egg parasitoid, Gryon sp. The genetic diversity of the three Clavigralla species collected in Kenya and Bénin and their identity were established using DNA barcoding and Cytb primers and different molecular tools (MEGA 7, NJ, K2P, BLAST).The parasitism assays conducted with Gryon sp. showed a higher incidence of parasitism of C. tomentosicollis eggs than that of C. elongata. The GC/MS analysis of cuticular extracts obtained from C. tomentosicollis and C. elongata parasitized and unparasitized eggs identified 15 compounds of which the amount varied between the two species. Furthermore, the Y-tube olfactometer bioassays conducted with group of males and females of C. tomentosicollis showed that volatiles released by groups of males were strongly attractive to both sexes. Antennae of both sexes of C. tomentosicollis detected identical components, including a male-specific component (isopentyl butanoate) which was also detected by antennae of the egg parasitoid. Likewise, in olfactometer bioassays with the synthetic of this male-specific compound, both the pest and the egg parasitoid were significantly attracted. GC/MS analyses of headspace volatiles of the three Clavigralla species identified 31 components. A heat map generated from the chemistry of Clavigralla spp. volatiles showed separation of the three species with a higher concentration of the components in C. tomentosicollis volatiles compared to the other two species. A close similarity between C. tomentosicollis and C. elongata was also observed. Genetic analyses showed very low variability within the different Clavigralla species and populations. Great variability was observed between C. tomentosicollis and the other two species. These results suggest that the alkanes present in the egg cuticula as well as isopentyl butanoate could serve as semiochemicals for Gryon sp., facilitating host finding and parasitism and that isopentyl butanoate is the aggregation pheromone for both sexes of C. tomentosicollis. These compounds are, therefore, potential candidates for future use as tools in management of these pests. Results on the genetic characteristics and distribution ranges of Clavigralla spp. will contribute to development of management strategies of these pests in Africa.