Analysing components of the Torso-activation module: Do they pattern the end-terminal regions of two Hymenopteran insect species, Apis mellifera and Nasonia vitripennis?

This project sought to determine if components of the Torso-activation module pattern the embryonic anterior and posterior ends of the honey bee, Apis mellifera, and the parasitoid wasp, Nasonia vitripennis via a patterning process termed terminal patterning. The Torso-activation module is the collective term for the set of proteins Trunk, Torso, prothoracicotrophic hormone (PTTH) and Torso-like (Duncan et al., 2013). Throughout arthropod evolution, the Torso-activation module’s evolutionary history is complex, with different components of it being implemented for a variety of developmental functions among different species (Duncan et al., 2013). The overarching aim of this project was to narrow down when, in the evolutionary history of arthropods, the Torso activation module was adapted to function in end-terminal patterning. An additional aim was to determine the functional role of torso-like in A. mellifera. As this insect species does not express the other components of the Torso-activation module, it is hoped that this research may help to reveal the individual function of torso-like, outside of interacting with the Torso-activation module. The Torso-activation module’s role in terminal patterning appears to have a limited phylogenetic distribution and has only been found in those species that express trunk. To date, it has only been confirmed in the red flour beetle, Tribolium castaneum, and some species of Diptera. It is currently hypothesized that the Torso-activation module has only recently been adapted for the role of terminal patterning, with its older role being that of initiating larval moulting (Duncan et al., 2014). It is worth noting that the genomes of both N. vitripennis and A. mellifera do not contain the full Torso-activation module as the genome N. vitripennis does not contain trunk, and A. mellifera expresses only torso-like. Regardless, if components of the Torso-activation module were found to pattern the end-terminals of N. vitripennis, it would have provided evidence that this adaptation of it evolved more basally in the radiation of the holometabolous insects than what is currently hypothesised (Duncan et al., 2014). Furthermore, it would have been the first known example of the Torso-activation module patterning the end-terminals of an insect that does not express trunk (Duncan et al., 2014). It’s unsurprising then that completion of parental RNAi (pRNAi) experiments in N. vitripennis here suggest that neither PTTH nor torso function to pattern the end-terminal regions in this species. Here, the cuticle structures of larvae collected from N. vitripennis females injected with torso and PTTH dsRNA appeared wildtype. However, these results will have to be confirmed via RT-qPCR. Additionally, these functional pRNAi experiments would benefit from completion of in situ hybridization to visualize where, and at what developmental stage, the transcripts of PTTH and torso localize in N. vitripennis. By determining whether these transcripts localize to the embryo poles, it may be possible to assess if they play a role in patterning the end-terminal regions of this species. Here, several attempts were made to perform in situ hybridization of these two genes. Unfortunately, for a variety of reasons, these attempts were unsuccessful and yielded no informative results. As for A. mellifera, pRNAi experiments performed here suggest that torso-like, functioning independently from the Torso-activation module, may play a role in patterning the anterior terminal end of this species. Here, larvae collected from A. mellifera queens injected with torso-like dsRNA were completely missing the mandible, maxilla, labium and all thoracic segments. This is exciting because it adds yet another example to the ever-growing list of torso-like’s functional roles between different insect species. However, as there are no other known experiments in which pRNAi has been reported on this species, these results will have to be repeated and confirmed via RT-qPCR