Mitochondrial genomic investigation reveals a clear association between species and genotypes of Lucilia and geographic origin in Australia

Abstract

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.

Keywords: Australian sheep blowfly; Lucilia cuprina cuprina; Lucilia cuprina dorsalis; Lucilia sericata; Mt genome; Phylogenetics.

Fig. 1

Linear maps of the circular mitochondrial (mt) genomes of Lucilia species/subspecies reported herein. The name of Lucilia species with their collection region is indicated above plots of gene order. Large bars situated on the mt genome maps indicate the position of protein‐coding genes and ribosomal RNA genes. Dark blue bars with annotated labels demarcate the positions of transfer RNA genes. Features of mt genomes are colour coded as indicated at the bottom of the figure. Protein‐coding genes are colour coded to mt complexes and the other features are coloured by type. Colours of protein‐coding, ribosomal RNA and transfer RNA genes are as follows: pink, cytochrome c oxidase (cox genes); dark green, ATP synthase (atp genes); yellow, NADH dehydrogenase (nad genes); light green, cytochrome b gene (cob); red, ribosomal RNAs and dark blue, transfer RNAs. For protein‐coding and ribosomal RNA genes, those sitting above the black line are on the positive strand, and those below the line are on the negative strand. Standard nomenclature was applied for protein-coding genes and ribosomal RNA genes, whereas for transfer RNA genes, single-letter abbreviations were used. The number of species-specific single nucleotide polymorphisms (SNPs) present in the genes were marked in brown text. Detailed annotations of the mt genomes are provided in Additional file 3: Table S3

Fig. 2

Analyses of protein-coding genes in Lucilia species/subspecies. A Nucleotide diversity (π) of individual protein-coding genes for pairs of Lucilia species/subspecies in this study. B Rates of nonsynonymous substitutions to the rate of synonymous substitutions (Ka/Ks) of individual protein-coding genes for pairs of Lucilia species/subspecies

Fig. 3

A summary of the molecular phylogeny of the Lucilia species/subspecies based on the cytochrome c oxidase subunit I (cox1) gene sequences. The phylogenetic tree was created using Bayesian inference (BI) implemented in MrBayes v.3.2.6. The numbers above the branches represent the posterior probabilities. Each specimen is labelled with the species name, location and GenBank accession number. Mitochondrial (mt) genomes sequenced in this study are colour coded: Lucilia cuprina cuprina (QLD) in pink, Lucilia sericata (TAS) in green, Lucilia cuprina dorsalis (NSW) in brown, Lucilia cuprina dorsalis (VIC) in purple and Lucilia cuprina dorsalis (WA) in blue. The Lucilia cuprina cuprina, Lucilia sericata and Lucilia cuprina dorsalis clades are highlighted in orange, cyan and yellow colour, respectively. The phylogram provided is presented to scale (scale bar = 0.02 estimated number of substitutions per site) with the species Calliphora vicina used as the outgroup

Fig. 4

Molecular phylogeny of dipteran flies. The phylogenetic tree was created using the Bayesian inference (BI) and maximum likelihood (ML) methods. The numbers on the branches indicate bootstrap values from ML (first value) and posterior probabilities from the BI (second value) method. Hyphens "-" indicate node support values are unavailable. Each specimen is labelled with the species name, location and GenBank accession number. Haematobia irritans irritans from the family Muscidae was used as the outgroup. Mitochondrial (mt) genomes sequenced in this study are colour coded: Lucilia cuprina cuprina (QLD) in pink, Lucilia sericata (TAS) in green, Lucilia cuprina dorsalis (NSW) in brown, Lucilia cuprina dorsalis (VIC) in purple and Lucilia cuprina dorsalis (WA) in blue. To the left of the species names, family names (A: Calliphoridae, B: Oestridae, C: Tachinidae, D: Sarcophagidae and E: Muscidae) are reported. The tree branches for subfamilies Luciliinae, Calliphorinae and Chrysominae of the Calliphoridae family are colour coded in red, blue and orange, respectively. The phylogram provided is presented to scale (scale bar = 0.05 estimated number of substitutions per site)