1. Leptocybe invasa is native to Australia and induces galls on various species of Eucalyptus. Two genetically distinct lineages of this wasp have been detected outside its native range, namely, Leptocybe Lineage A and Leptocybe Lineage B.
2. The parasitoid Selitrichodes neseri was released in South Africa as a biological control agent against L. invasa. Another parasitoid of L. invasa, Quadrastichus mendeli, as well as Megastigmus zebrinus (parasitoid) and Megastigmus pretorianensis (role unknown), have also been recorded emerging from L. invasa galls. The objective of this study was to investigate the interactions between the different hymenopterans associated with L. invasa galls in South Africa.
3. L. invasa galls were dissected and species‐specific primers and restriction enzymes were used to identify the larvae where interactions were noted.
4. S. neseri, Q. mendeli and M. zebrinus were confirmed to parasitize Leptocybe Lineage A, and S. neseri was confirmed to parasitize Leptocybe Lineage B. Furthermore, there were direct interactions between these parasitoids, where parasitoids were found parasitising each other. The gall forming experiment confirmed that M. pretorianensis is not a gall former, but other potential roles remain uncertain.
Supporting Information: Table S1. Species specific primers and their annealing temperatures for the hymenopteran species associated with Leptocybe invasa galls and the expected fragment sizes amplified by each species‐specific primer for the region Cyt b.
Table S2. The fragment sizes of digested Cyt b amplicons using the AseI restriction enzyme for Leptocybe invasa (A and B), Selitrichodes neseri, Megastigmus zebrinus, Megastigmus pretorianensis and Quadrastichus mendeli.