Evolutionary Developmental Biology in the Mayfly Hexagenia limbata, with a focus on Hox Genes

Evolutionary developmental biology has become a prominent field for investigating how developmental processes operate and change over time. An integrative approach encompassing morphological and genetic data is fundamental to evo devo; such integration has proven key to understanding how changes in gene expression and function can create novel phenotypes. While this approach has been successfully applied to well known model organisms, it has become apparent that truly understanding the breadth and evolution of developmental processes requires comparisons between many different organisms. Comparative studies thus require research to continually expand beyond traditional model organisms, especially in regard to species that feature novel traits. This has been particularly true for understanding the unique development of body plans in arthropoda, which are based primarily upon repeating segments that form phenotypically distinct units called tagmata. Tagmata often bear unique appendages, and generally define each of the major arthropod classes. However, while the body plan and developmental genetics of arthropods like the fruit fly Drosophila melanogaster have been extensively studied, much remains unknown about how developmental genes changed over time to produce aspects of the insect body plan that are unique, such as wings, or that notably diverge from the typical body plan of most insects. In this regard, mayflies (Ephemeroptera) are particularly appealing candidates for evo devo research. Mayflies are one of the earliest diverging group of winged insects, placing them at a phylogenetically important position amongst the pterygotes. The body plan of their nymphal stages is also different from most insects, as it bears functionally diverse abdominal appendages called gills. These gills are a key part of environmental adaptation in mayflies, and may share structural homology with the appendicular appendages seen throughout arthropoda. While mayflies have been studied extensively in regard to their ecological importance and phylogenetic position, little is known about their developmental genetics, especially in regard to appendage development. Thus, the purpose of this work is to bring an evo devo framework to mayflies by 1) describing their embryonic and nymphal ontogeny, especially in regard to their thoracic and abdominal appendages, 2) describing the expression of the Hox genes Antennapedia (Antp), Ultrabithorax (Ubx) and Abdominal-A (AbdA) during embryogenesis, and 3) identifying sequences for a suite of key developmental and cell signaling genes in a first instar nymphal transcriptome. In order to do accomplish these objectives, we have focused upon the widely dispersed and large bodied mayfly, Hexagenia limbata (Ephemeridae). Morphological descriptions of H. limbata ontogeny revealed that embryogenesis proceeds in a manner consistent with that seen in other studied mayfly species, with posterior body segments gradually appearing from the extending germ band. Likewise, nymphal appendage development in the first three instars was consistent with published literature on Hexagenia mayflies, namely in regard to the simultaneous development of five gill pairs on the abdomen and the similar size and shape of all three thoracic legs. We found that appendage development from post-third instars follows two main themes: the thoracic legs remain similar in size and shape, while the gills gradually increase in complexity by adding gill branches in a distal to proximal manner, as seen in other burrowing mayflies. While we were unable to assess Hox expression in nymphs, Antp and Ubx/AbdA expression during embryogenesis was highly conserved with that seen in non-holometabolous insects, especially Thermobia domestica (Zygentoma) and Gryllus bimaculatus (Orthoptera). Antp was strongly expressed in the posterior labial segment through the thorax, with a narrow stripe of expression down the central abdomen that is commonly associated with nervous system development. The combined expression profile for Ubx/AbdA ranged from the posterior T2 to the tenth abdominal segment, with strong staining seen at the T3/A1 border. These findings suggest that if Antp and Ubx/AbdA expression play a role in gill development, it is likely due to changes in gene function, expression during the first instar when gills develop, or changes to downstream appendage patterning gene regulation. Transcriptomic sequencing was done using whole body, first instar mRNA, from which 70,507,832 reads were generated. De novo assembly resulted in a 93,561 contig database, with 60,861 of these having ≥ 10X average coverage. Two workflows were designed in order to assess and identify developmental and cell signaling genes of interest from this database. Our gene-specific workflow confirmed homologous sequence identity for 78 putative sequences belonging to 53 developmental and cell signaling genes of interest, with most top BLASTx hits matching insect homologs. Our second, Gene Ontology (GO) workflow filtered our contig database via a > 99 reads per contig criteria and provided a high quality contig database of 37,023 contigs, from which 9,813 contigs were successfully mapped and annotated with GO terms. Most top BLASTx hits for these contigs were also from insect taxa, and the level two GO term profile for these contigs largely resembles those reported for other insect transcriptomes. This suggests that our transcriptome successfully represents a broad sampling of mRNA transcripts commonly found in whole body insect transcriptomes. Furthermore, GO term annotation tagged 841 and 996 of these contigs as relevant to developmental processes and signaling, respectively, indicating that additional candidate developmental genes or possible orthologs could be identified. Future evo devo research on mayfly body patterning would greatly benefit from two distinct initiatives. First, sequences identified in the transcriptomic dataset can be used to synthesize riboprobes for in situ hybridization, enabling expression studies for many of the putative developmental genes identified. Such sequences can also be used to synthesize either dsRNA for RNAi or specific DNA sequences for CRISPR/cas9, which would allow functional experiments during mayfly development and potentially reveal both the genetics of gill development and how it compares to limb development in other insects. Second, developing molecular techniques in first instar nymphs would directly address how the mayfly body plan changes, since the sudden development of abdominal gills occurs in the second nymphal instar. With these initiatives, there remain abundant opportunities to investigate the development and evolution of mayflies' novel body plan.