First evidence of wasp brood development inside active nests of a termite with the description of a previously unknown potter wasp species

Potter wasps (Vespidae: Eumeninae) are known to exhibit not only sophisticated preying strategies but also a remarkable ability to manipulate clay during nest building. Due to a mixture of plasticity in building behaviour and flexibility in substrate preferences during nest-building, the group has been reported nesting in a variety of places, including decaying nests abandoned by termite species. Yet, evidence of wasps nesting inside senescent termite mounds is poorly reported and, to date, accounts confirming their presence inside active colonies of termites are absent. Here, we address a novel intriguing association between two species from the Brazilian Cerrado: a previously unknown potter wasp (nest invader) and a termite species (nest builder). Besides scientifically describing Montezumia termitophila sp. nov. (Vespidae: Eumeninae), named after its association with the termite Constrictotermes cyphergaster (Silvestri, 1901) (Termitidae: Nasutitermitinae), we provide preliminary information about the new species' bionomics by including (i) a hypothetical life cycle based on the evidence we collected and (ii) a footage showing the first interaction between a recently ecloded wasp and a group of termites. In doing so, we attempt to provoke relevant discussions in the field and, perhaps, motivate further studies with the group. Finally, we describe a solution to efficiently detect and sample termitophilous species from termite nests, an intrinsic yet challenging task of any studies dealing with such a cryptic biological system.Potter wasps (Vespidae: Eumeninae) are known to exhibit not only sophisticated preying strategies but also a remarkable ability to manipulate clay during nest building. Due to a mixture of plasticity in building behaviour and flexibility in substrate preferences during nest-building, the group has been reported nesting in a variety of places, including decaying nests abandoned by termite species. Yet, evidence of wasps nesting inside senescent termite mounds is poorly reported and, to date, accounts confirming their presence inside active colonies of termites are absent. Here, we address a novel intriguing association between two species from the Brazilian Cerrado: a previously unknown potter wasp (nest invader) and a termite species (nest builder). Besides scientifically describing Montezumia termitophila sp. nov. (Vespidae: Eumeninae), named after its association with the termite Constrictotermes cyphergaster (Silvestri, 1901) (Termitidae: Nasutitermitinae), we provide preliminary information about the new species' bionomics by including (i) a hypothetical life cycle based on the evidence we collected and (ii) a footage showing the first interaction between a recently ecloded wasp and a group of termites. In doing so, we attempt to provoke relevant discussions in the field and, perhaps, motivate further studies with the group. Finally, we describe a solution to efficiently detect and sample termitophilous species from termite nests, an intrinsic yet challenging task of any studies dealing with such a cryptic biological system.

Methods
We collected 13 C. cyphergaster nests from one site in the Brazilian Cerrado (Ratter et al., 1997) near the municipality of Divinópolis (20º08'20"S, 44º53'02"W), State of Minas Gerais. This area is classified as ‘equatorial savanna with dry winters’ (Kottek et al., 2006). The 13 termite nests, distributed sparsely in an area of 20 hectares and never neighboring one another, were collected on March 2018 (between 08:00 and 12:00) and immediately transported to the headquarters of property. Then, they were kept in individual cages for 5 days until nest inspection (Fig. S2: 5). These cages, made of cubic wire frames surrounded by a thin tulle fabric, were designed to contain adult wasps after eclosion. As for verifying whether M. termitophila’s nesting takes place entirely inside C. cyphergaster nests, it would be necessary to (i) obtain empirical evidence of adult wasps ecloding from the inside of termite nests and (ii) document the presence of wasp’s offspring in termite nests, by detecting wasp brood cells integrated between termite galleries containing wasp immature stages. Thus, to search for immature stages of wasps (eggs, larvae and pupae) we thoroughly inspected the content of termite nests by peeling off its surface in order to expose subsequent layers of nest galleries (Fig S2: 5). To prevent damaging the biological material we used scoopulas (stainless-steel spatulas of application in chemistry) and collected individuals using entomological tweezers. As opposed to the method of merely breaking C. cyphergaster nests into smaller pieces and superficially checking for termitophiles (as conducted in previous studies with the species), the protocol mentioned above allows a more refined inspection with preservation of nest structures. Only by preserving such structures, we were able to later document and elucidate the positioning and architectural features of wasp brood chambers inside the termite nest.