High-throughput sequencing has fundamentally changed how molecular phylogenetic data sets are assembled, and phylogenomic data sets commonly contain 50- to 100-fold more loci than those generated using traditional Sanger sequencing-based approaches. Here, we demonstrate a new approach for building phylogenomic data sets using single-tube, highly multiplexed amplicon sequencing, which we name HiMAP (highly multiplexed amplicon-based phylogenomics) and present bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources and postsequencing consensus calling and alignment. This method is inexpensive and amenable to sequencing a large number (hundreds) of taxa simultaneously and requires minimal hands-on time at the bench (<1/2 day), and data analysis can be accomplished without the need for read mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including some of the most economically important agricultural insect pests. The resulting filtered data set (>150,000-bp concatenated alignment, ~20% missing character sites across all individuals and amplicons) contained >40,000 phylogenetically informative characters, and although some discordance was observed between analyses, it provided unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found high support for the generic status of Zeugodacus and the sister relationship between Dacus and Zeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and the insight the resulting data set provides into relationships of this diverse insect group.

15High-throughput sequencing has fundamentally changed how molecular phylogenetic datasets 16 are assembled, and phylogenomic datasets commonly contain 50-100-fold more loci than those 17 generated using traditional Sanger-based approaches. Here, we demonstrate a new approach for 18 building phylogenomic datasets using single tube, highly multiplexed amplicon sequencing, 19 which we name HiMAP (Highly Multiplexed Amplicon-based Phylogenomics), and present 20 bioinformatic pipelines for locus selection based on genomic and transcriptomic data resources 21 and post-sequencing consensus calling and alignment. This method is inexpensive and amenable 22 to sequencing a large number (hundreds) of taxa simultaneously, requires minimal hands-on time 23at the bench (<1/2 day), and data analysis can be accomplished without the need for read 24 mapping or assembly. We demonstrate this approach by sequencing 878 amplicons in single 25 reactions for 82 species of tephritid fruit flies across seven genera (384 individuals), including 26 some of the most economically-important agricultural insect pests. The resulting dataset 27 (>150,000 bp concatenated alignment) contained >40,000 phylogenetically informative 28 characters, and although some discordance was observed between analyses, it provided 29 unparalleled resolution of many phylogenetic relationships in this group. Most notably, we found 30 high support for the generic status of Zeugodacus and the sister relationship between Dacus and 31 Zeugodacus. We discuss HiMAP, with regard to its molecular and bioinformatic strengths, and 32 the insight the resulting dataset provides into relationships of this diverse insect group. 33

Tephritid fruit flies are destructive agricultural pests that are the targets of expensive population eradication and suppression efforts. Genetic pest management is one of the strategies for reducing or eliminating tephritid populations, relying upon the genetic manipulation of insects to render them sterile or capable of transmitting deleterious traits through gene drive. Currently, radiation, chemical mutagenesis, and transgenic techniques are employed to generate agents for genetic pest management, but new methods must be explored and developed for all tephritid pest species. Targeted mutagenesis induced by nonhomologous end join repair of clustered regularly interspaced short palindromic repeats and the CRISPR associated protein 9 (Cas9) (commonly known as CRISPR/Cas9) has been demonstrated to be an efficient method for creating knock‐out mutants and can be utilized to create germline mutations in Tephritidae. In this paper, we describe detailed methods to knockout the white gene in three tephritid species in the genera Anastrepha, Bactrocera and Ceratitis, including the first demonstration of CRISPR/Cas9 induced mutations in the genus Anastrepha. Lastly, we discuss the variables in tephritid systems that directed method development as well as recommendations for performing injections in remote containment facilities with little molecular biology capabilities. These methods and recommendations combined can serve as a guide for others to use in pursuit of developing CRISPR/Cas9 methods in tephritid systems.

Tephritid fruit flies are among the most invasive and destructive agricultural pests worldwide. Over recent years, many studies have implemented the CRISPR/Cas9 genome-editing technology to dissect gene functions in tephritids and create new strains to facilitate their genetics, management, and control. This growing literature allows us to compare diverse strategies for delivering CRISPR/Cas9 components into tephritid embryos, optimize procedures, and advance the technology to systems outside the most thoroughly studied species within the family. Here, we revisit five years of CRISPR research in Tephritidae and propose a unified protocol for candidate gene knockout in fruit flies using CRISPR/Cas9. We demonstrated the efficiency of our protocol by disrupting the eye pigmentation gene white eye (we) in the melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae). High rates of somatic and germline mutagenesis were induced by microinjecting pre-assembled Cas9-sgRNA complexes through the chorion of embryos at early embryogenesis, leading to the rapid development of new mutant lines. We achieved comparable results when targeting the we orthologue in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), illustrating the reliability of our methods when transferred to other related species. Finally, we functionally validated the recently discovered white pupae (wp) loci in the melon fly, successfully recreating the white puparium phenotype used in suppression programs of this and other major economically important tephritids. This is the first demonstration of CRISPR-based genome-editing in the genus Zeugodacus, and we anticipate that the procedures described here will contribute to advancing genome-editing in other non-model tephritid fruit flies.

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