Characterization of the complete mitochondrial genome of the New Zealand parasitic blowfly Calliphora vicina (Insecta: Diptera: Calliphoridae).

In this study, the complete mitochondrial (mt) genome of the New Zealand parasitic blowfly Calliphora vicina (blue bottle blowfly) field strain NZ_CalVic_NP was generated using next-generation sequencing technology and annotated. The 16,518 bp mt genome consists of 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and a 1689 bp non-coding region, similar to the two other available C. vicina and most metazoan mt genomes. Phylogenetic analysis showed that C. vicina NZ_CalVic_NP forms a monophyletic cluster with the remaining three Calliphorinae species. The complete mt genome sequence of C. vicina NZ_CalVic_NP is a resource to facilitate future species- and strain-level identification research and investigations into the evolutionary provenance within the Calliphoridae.

The diminished efficacy demonstrated by current members of the Calliphoridae (blowflies) treatments due to the emergence of resistance in blowflies against many classes of insecticides calls for improved DNA-based diagnostics tools. High-level phylogenetic relationships within the Calliphoridae are still largely unresolved primarily due to their large and highly variable mitochondrial (mt) genomes. Calliphora vicina (Robineau-Desvoidy, 1830) NZ_CalVic_NP, was selected for genome sequencing as a representative of an NZ field strain of C. vicina.

The C. vicina specimen was collected from the Palmerston North area (40°21.3′S, 175°36.7′E), and is stored and available upon request from AgResearch Ltd., Grasslands Research Center (accession number: NPY120886). High molecular weight genomic DNA was isolated from entire C. vicina adult males using a modified phenol:chloroform protocol explained in our previous articles (Palevich, Kelly, Ganesh, et al. 2019; Palevich, Kelly, Leahy, et al. 2019; Palevich et al. 2019b). Genomic DNA was prepared for whole-genome sequencing using an Illumina TruSeq Nano library preparation kit (Illumina, Inc., San Diego, CA) according to the manufacturer’s instructions. The Illumina NovaSeq™ 6000 (PE150, Novogene, Beijing, China) platform was used to amplify the entire mt genome sequence. The mt genome was assembled and annotated using the NOVOPlasty pipeline version 3.1 with default parameters (Dierckxsens et al. 2016; Palevich and Maclean 2021), as previously described (Palevich et al. 2019a; Palevich, Maclean, Mitreva, et al. 2019; Palevich et al. 2020).

The length of complete mt genome is 16,518 bp, with the overall 77.8% AT content (BioProject ID: PRJNA667961, GenBank accession number: MW266392). The overall nucleotide distribution for the mt genome is 39.5% A, 13.0% C, 9.2% G, and 38.1% T. The structure of the mt genome is typical of insect mt genomes (Cameron 2014) which consists of 13 protein-coding genes, 22 transfer RNAs, and two ribosomal RNAs. Among these 37 genes, 23 genes encoded on the majority strand while remaining 14 genes encoded on the minority strand. There are eight more complete mt genomes recorded belong to the genus Calliphora (C. vicina, C. vomitoria, C. nigribarbis, and C. chinghaiensis) (Nelson et al. 2012; Chen et al. 2016; Ren et al. 2016; Karagozlu et al. 2019). In comparison, the reported C. vicina NZ_CalVic_NP has the longest complete mt genome and the size difference with the shortest record is 1249 bp (C. chinghaiensis). The main reason for the size difference is the control region. The entire ‘control region’ that is non-coding and AT-rich lies between the 12S rRNA and tRNA-Ile in insect mt genomes, and this area in the C. vicina NZ_CalVic_NP is 1689 bp in length which is the longest among all Calliphora records.

The phylogenetic position of C. vicina NZ_CalVic_NP within the family Calliphorinae was estimated using maximum-likelihood, implemented in RAxML version 8.2.11 (Stamatakis 2014), and the Bayesian inference (BI), implemented in MrBayes version 3.2.6 (Huelsenbeck and Ronquist 2001) approaches using default settings. For analysis, the phylogenetic tree was reconstructed using the complete mitogenome sequences of available blowfly species and isolates retrieved from GenBank with the 13 concatenated mt PCGs genes (Figure 1). Calliphora vomitoria and C. nigribarbis are sister to C. vicina and together with C. chinghaiensis are monophyletic. Overall, the phylogenetic topology is similar to previous studies (Chen et al. 2016), suggesting that the genus Calliphora is monophyletic. This study provides additional complete and curated mitogenome data for the mt genome library of the genus Calliphora. Such a resource will be used in future comparative studies investigating the evolutionary provenance of Calliphora by exploring species- and strain-level diversity.

Figure 1.

A summary of the molecular phylogeny of the Calliphoridae complete mitochondrial genomes. The evolutionary relationship of C. vicina field strain NZ_CalVic_NP (black circle) was compared to the complete mitochondrial genomes of 68 blowfly species or isolates retrieved from GenBank (accession numbers in parentheses) and nucleotide sequences of all protein-coding genes were used for analysis. Phylogenetic analysis was conducted using the Bayesian approach implemented in MrBayes version 3.2.6 (Huelsenbeck and Ronquist 2001) and maximum likelihood (ML) using RAxML version 8.2.11 (Stamatakis 2014). The mtREV with Freqs. (+F) model was used for amino acid substitution and four independent runs were performed for 10 million generations and sampled every 1000 generations. For reconstruction, the first 25% of the sample was discarded as burnin and visualized using Geneious Prime (Kearse et al. 2012). Nodal support is given: Bayes posterior probabilities|RAxML bootstrap percentage. The phylogram provided is presented to scale (scale bar = 0.05 estimated number of substitutions per site) with the species Haematobia irritans from the family Muscidae used as the outgroup.