Background: Lucilia cuprina and L. sericata (family Calliphoridae) are globally significant ectoparasites of sheep. Current literature suggests that only one of these blowfly subspecies, L. cuprina dorsalis, is a primary parasite causing myiasis (flystrike) in sheep in Australia. These species and subspecies are difficult to distinguish using morphological features. Hence, being able to accurately identify blowflies is critical for diagnosis and for understanding their relationships with their hosts and environment. Methods: In this study, adult blowflies (5 pools of 17 flies; n = 85) were collected from five locations in different states [New South Wales (NSW), Queensland (QLD), Tasmania (TAS), Victoria (VIC) and Western Australia (WA)] of Australia and their mitochondrial (mt) genomes were assembled. Results: Each mt genome assembled was ~ 15 kb in size and encoded 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and a control region. The Lucilia species mt genomes were conserved in structure, and the genes retained the same order and direction. The overall nucleotide composition was heavily biased towards As and Ts—77.7% of the whole genomes. Pairwise nucleotide diversity suggested divergence between Lucilia cuprina cuprina, L. c. dorsalis and L. sericata. Comparative analyses of these mt genomes with published data demonstrated that the blowflies collected from sheep farm in TAS clustered within a clade with L. sericata. The flies collected from an urban location in QLD were more closely related to L. sericata and represented the subspecies L. c. cuprina, whereas the flies collected from sheep farms in NSW, VIC and WA represented the subspecies L. c. dorsalis. Conclusions: Phylogenetic analyses of the mt genomes representing Lucilia from the five geographic locations in Australia supported the previously demonstrated paraphyly of L. cuprina with respect to L. sericata and revealed that L. c. cuprina is distinct from L. c. dorsalis and that L. c. cuprina is more closely related to L. sericata than L. c. dorsalis. The mt genomes reported here provide an important molecular resource to develop tools for species- and subspecies-level identification of Lucilia from different geographical regions across Australia. Graphical

The Australian sheep blowfly, Lucilia cuprina dorsalis, is a major sheep ectoparasite causing subcutaneous myiasis (flystrike), which can lead to reduced livestock productivity and, in severe instances, death of the affected animals. It is also a primary colonizer of carrion, an efficient pollinator, and used in maggot debridement therapy and forensic investigations. In this study, we report the complete mitochondrial (mt) genome of L. c. dorsalis from the Northern Territory (NT), Australia, where sheep are prohibited animals, unlike the rest of Australia. The mt genome is 15,943 bp in length, comprising 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and a non-coding control region. The gene order of the current mt genome is consistent with the previously published L. cuprina mt genomes. Nucleotide composition revealed an AT bias, accounting for 77.5% of total mt genome nucleotides. Phylogenetic analyses of 56 species/taxa of dipterans indicated that L. c. dorsalis and L. sericata are the closest among all sibling species of the genus Lucilia, which helps to explain species evolution within the family Luciliinae. This study provides the first complete mt genome sequence for L. c. dorsalis derived from the NT, Australia to facilitate species identification and the examination of the evolutionary history of these blowflies.

Lucilia cuprina, a species of blowfly, consists of two recognized subspecies: L. cuprina cuprina and L. cuprina dorsalis. Although they are morphologically and molecularly similar to each other, they have very different ecological roles. In Australia, L. c. dorsalis is predominantly found in rural areas and is the primary causative agent of sheep myiasis (flystrike), while L. c. cuprina is necrophagous and not a significant pest of livestock in the Americas or elsewhere. Here, we present a chromosome-scale genome assembly for L. c. cuprina and an improved assembly for L. c. dorsalis, enabling comparative genomic analysis between these subspecies. While both genomes share a similar gene content, subspecies-specific genes were identified, which may contribute to their divergent ecological roles -necrophagy in L. c. cuprina and parasitism in L. c. dorsalis. Phylogenetic analyses across target genomic regions reaffirm the close relationship between L. c. cuprina and L. c. dorsalis and position L. sericata as their sister species. Gene mutations linked to diazinon resistance were exclusively observed in L. c. dorsalis, while malathion resistance was detected in both subspecies. Additionally, we identified genes with accelerated evolutionary rates in each subspecies, which may underlie their distinct feeding behaviours. We also conducted a detailed analysis of chemosensory genes, revealing that L. c. dorsalis possesses slightly larger repertoires of all four chemosensory gene families analyzed. In comparison to Drosophila melanogaster, both subspecies exhibit an expanded gustatory receptor clade. Our findings provide valuable insights into the genetic factors underpinning parasitism and insecticide resistance and provide a valuable genetic resource for future research endeavours, including the development of engineered strains aimed at genetic biocontrol strategies. This work enhances our understanding of the evolutionary adaptations for this important blowfly species.

Flystrike (cutaneous myiasis) is caused by blowfly larvae of the genus Lucilia. This disease is a major obstacle to sustainable global sheep and wool production. Flystrike control relies primarily on breech modification surgery (mulesing) and insecticidal treatment; however, control is constantly compromised by the emergence and spread of insecticide-resistance. Preventing the spread of resistance is severely hindered by a limited understanding of genetic variation, structure and gene flow within and among Lucilia populations. Australia is one of the world's largest producers of sheep and wool products, where Lucilia cuprina dorsalis is the major cause of flystrike. Here, we collected 2,034 Lucilia cuprina dorsalis individuals among 86 populations from sheep-grazing regions across the continent. Each fly was genetically characterised at 20,000 loci using DArTseq, a reduced complexity genome sequencing strategy. Three genetically distinct population clusters (i.e., Western Australia, Eastern Australia and Tasmania) were revealed through population structure analyses. This investigation into population structure and gene flow yields significant insights into the genetic composition of diverse L. c. dorsalis populations throughout Australia. These findings will be essential for the sustainable management of flystrike on a global scale and for addressing the ongoing challenge of insecticide resistance.

Blowflies of the genus Lucilia (Insecta: Diptera: Calliphoridae) are important in forensic science, medical research, and agriculture, particularly due to their role as facultative ectoparasites of domesticated sheep. The Australian sheep blowfly, L. cuprina dorsalis , is a primary causative agent of cutaneous myiasis, leading to substantial economic losses to the wool industry. In contrast, the closely related subspecies L. cuprina cuprina does not induce myiasis in sheep, but functions as a necrophagous subspecies in urban regions of Australia. Differences in the physiology, behavior, and host interactions of these subspecies might be attributed to their bacterial community structures as demonstrated in other blowfly species. However, our understanding of the bacterial communities associated with these flies is limited. This study aimed to characterize the microbiomes of two geographically distinct subspecies of L. cuprina — L. c. dorsalis (rural) and L. c. cuprina (urban) using 16S rRNA gene sequencing. The findings indicate that, while both subspecies harbor broadly similar microbial compositions, their respective urban and rural environments influence the prevalence of specific bacterial genera. The bacterial communities of these flies were predominantly composed of members of the phyla Proteobacteria , Firmicutes , Bacteroidetes , and Actinobacteria . The genus Ignatzschineria was highly abundant in L. c. dorsalis , whereas Pseudomonas was more prevalent in L. c. cuprina , highlighting distinct microbial compositions between the two subspecies. These findings provide fundamental insights into the microbial composition of Lucilia species, contributing to a broader understanding of their ecological and biological roles, with potential applications in forensic entomology, medical research, and agricultural pest management. Key points: • The microbial communities of Lucilia cuprina dorsalis and Lucilia cuprina cuprina have a broadly similar composition, but the abundance of particular bacterial genera is notably different. • Proteobacteria is the most abundant phylum in both subspecies. • The genus Ignatzschineria is notably abundant in Lucilia cuprina dorsalis, while Pseudomonas is predominant in Lucilia cuprina cuprina, emphasizing differences between the microbial community members between these subspecies.