Long-read RNA-seq delineates temporal transcriptional dynamics in multiplexed and sexed single medfly embryos

Long-read RNA sequencing has great potential to improve genomic characterization of non-model organisms due to its ability to yield full-length genes. Coupled with absolute gene expression quantification, dynamics of development orchestrated at transcript level can be elucidated with high precision. The resolution of this precision can be further improved by studying organisms as close as possible to their basic entities, single cells for example or single embryos. Here, we collected developing embryos of the Mediterranean fruit fly (medfly, Ceratitis capitata ) at hourly time-points for the first 15 hours of development. The medfly is an organism of huge economic importance in agriculture due to its wide host range including apples, pear, citrus, olives, etc. We simultaneously isolated total RNA and genomic DNA from single embryos and sexed the embryos using Y-specific PCR assays. The RNA, spiked with external ERCC standards to aid in absolute quantification, was used to perform Nanopore long-read RNA-seq. We developed a genome-guided transcriptome assembly based on full-length transcripts and identified a total of 22,875 transcripts comprising 3879 novel genes, missed in the current NCBI predicted gene models. We show that, indeed, the absolute quantification of gene expression performs superiorly to relative quantification in highly dynamic systems such as developing embryos. Further, we used unsupervised clustering and lineage tracing algorithms to group and accurately place embryos along a pseudo-temporal development trajectory. We show that medfly embryos undergo successive waves of zygotic genome activation. We discover a dramatic reorganization of maternally deposited mRNA occurring within the first 3 hours of egg laying followed by maternal-to-zygotic transition. We finally identify modules of temporal synexpression and elucidate the biological role of these modules. Together, these results provide the first detailed look at early embryo development in the medfly and should aid in future control efforts of this pest.