Protein-rich trophallactic contents transferred from reproductives are crucial for termite soldier differentiation in Zootermopsis nevadensis

Polyphenic castes exist for colony success in social animals. A primer pheromone has been suggested as a well-known regulator of caste development, but the basis of chemical communication has remained elusive over the last few decades. In termites, a long-standing hypothesis is that reproductive-secreted juvenile hormone (JH) plays a role as a primer pheromone involved in soldier differentiation. Here, we tested whether exogenous JH is necessary to induce differentiation of soldiers in the dampwood termite Zootermopsis nevadensis. The unique characteristic of this termite is that soldier-destined individuals can be monitored during their ontogeny under natural conditions. Furthermore, oral-anal trophallactic interactions with reproductives are required for soldier differentiation. First, knockdown of JH biosynthetic genes using RNA interference (RNAi) resulted in the reduction of soldier formation but did not affect the frequency of trophallactic interaction from reproductives to RNAi-treated individuals. Next, we compared the effect of parental replacement on soldier differentiation. Old-age reproductives had fewer stimulus effects on the differentiation of soldier-destined individuals compared to young-age reproductives. This difference in stimulus effects was derived from the protein levels within the digestive fluids of the king, but not those of the queen. Consequently, there is little or no possibility that JH itself plays a role as a primer pheromone. Alternatively, we propose that protein-rich nutrients in trophallactic fluids from reproductives are sufficient to induce high JH levels in larvae required for soldier differentiation.