Mitochondrial genome of Bactrocera fruit flies (Tephritidae: Dacini): features, structure, and significance for diagnosis

Abstract

Background: True fruit flies (Diptera: Tephritidae) are among the most destructive pests of fruit and vegetables worldwide and are on the top of quarantine pest lists. To respond effectively to a fruit fly invasion, we need to identify the species rapidly and reliably to understand its biological features and guide response decisions. Molecular techniques have been used to improve the diagnostic ability circumventing many difficulties of morphological identification. However, the commonly used Cytochrome Oxidase I (COI) gene lacks sufficient variation to distinguish species within Bactrocera species complexes. Here we conducted mitochondrial genome sequencing to identify additional genetic markers that could aid diagnosis of Bactrocera fruit fly species.

Results: We assembled 82 complete mitochondrial genomes from 16 Bactrocera species, including 13 species for which no mitochondrial genome data were previously available, as well as one species each from Dacus aneuvittatus, Dirioxa pornia and Zeugodacus gracilis. Phylogenetic analysis of the Tephritidae family confirmed the monophyly of the Bactrocera genus but could not properly resolve species within species complexes. Comparative mitochondrial genome analysis revealed that intergenic spacer and NADH dehydrogenase genes, specifically ND2 and ND6, harbour enough variations for new specific real-time PCR assays. Based on these findings, six TaqMan-based real-time PCR assays targeting ND2, COI, and CO3 genes were successfully designed and assessed for their specificity and sensitivity in detecting Bactrocera curvipennis, a member of the B. tryoni complex. Of these, one real-time PCR assay targeting the ND2 gene proved to be the most specific and sensitive. It detects B. curvipennis specifically at the level of 1 copy/µL of target DNA.

Conclusions: Mitochondrial sequence analysis and comparative studies indicate that mitochondrial genomes offer valuable genetic markers for accurate diagnosis of Bactrocera fruit flies. The successful development of the B. curvipennis real-time PCR assay highlights the importance of having additional genetic markers to advance the molecular diagnostics in economically important Bactrocera species.

Keywords: Bactrocera curvipennis; Biosecurity; DNA barcoding; Intergenic spacer; Molecular identification; Phylogeny; Species-specific real-time PCR.

Fig. 1

Reconstructed phylogenetic tree of Tephritidae fruit flies inferred from the PCGs dataset with partitions using RAxML. Maximum likelihood (ML) bootstrap support values are shown at each node. Species and genera clades have been collapsed to improve visualization of the tree’s topology. The Bactrocera tryoni complex includes B. tryoni and B. neohumeralis, while the B. dorsalis complex comprises primarily B. dorsalis and three B. carambolae specimens. The number of specimens used in each collapsed clade is specified. Boxes highlight subfamilies and brackets denote well-supported Bactrocera clades selected for further analyses

Fig. 2

Mitochondrial genome comparative analysis of Bactrocera fruit flies. A Schematic representation of mitochondrial gene arrangement. Protein-coding genes (PCGs), rRNAs, tRNAs, and the control region (CR) are shown in green, cyan, pink, and orange, respectively. Box plots of the genome length, GC content, and control region length are provided for selected Bactrocera clades. B Detailed schematic of major intergenic spaces (IGS) within Bactrocera, showing the boxplots of the lengths of the trnQ-trnM, trnC-trnY, and trnR-trnN IGS for selected clades. Each data point represents individual Bactrocera specimens, with point colours indicating different Bactrocera clades shown on Fig. 1. The number of specimens used for each clade is indicated in the pruned phylogenetic tree

Fig. 3

Evolutionary analysis of mitochondrial protein-coding genes (PCGs) in Bactrocera fruit flies. A Sliding window analysis of nucleotide diversity (Pi) across the PCGs, using a 100 bp window with a 20 bp step. Gene names and Pi values are indicated above or below the schematic representation of the genes. B Boxplots showing synonymous (Ks) and nonsynonymous (Ka) substitution rates for each PCG, with each data point represents an individual Bactrocera specimen. Point colours correspond to different Bactrocera clades shown on Fig. 1. C Boxplots of intra-specific and inter-specific pairwise genetic distances across PCGs to assess the barcoding gap. The median values for nucleotide diversity, Ka/Ks ratio, and interspecific genetic distances for the COI gene are indicated by the blue dotted line

Fig. 4

Performance comparison of the specificity of real-time PCR assays designed for Bactrocera curvipennis detection. A Primer and probe sequences for each of the three assays targeting the ND2, COI, and CO3 genes, represented by color-coded boxes. B Schematic overview of amplicon positions of each assay on the B. curvipennis mitochondrial genome (BCNC1). C–E Boxplot of Cq values for specific and non-specific amplifications across different samples, cut-off set at 36 cycles (grey dotted line). Neg: no amplification within 40 cycles. F-H Boxplot of relative fluorescence unit (RFU) for specific and non-specific amplifications, fluorescence threshold set at 100 RFU (grey dotted line). Primers (F: forward; R: reverse) and probes (P) sequences of each assay are shown in the same colour of boxes. Each data point represents samples from different Bactrocera clades shown on Fig. 1

Fig. 5

Sensitivity of the ND2 real-time PCR assay for Bactrocera curvipennis detection. The gBlock synthetic template (Bac_TC_allin_control), containing concatenated primer and probe binding sites, was serially diluted, and tested with the singleplex real-time PCR assay. Linear regression of gBlock concentrations against Cq values generated a standard curve, plotting Cq values against the log copy number (range = 10⁷ copies–1 copy) of the gBlock

Fig. 6

Specificity of the ND2 real-time PCR assay for Bactrocera curvipennis detection. Reconstructed phylogenetic tree of the samples used in the development of the assay, inferred from the COI alignment using IQ-TREE. The boxplot of Cq values and bars representing the number of specimens displayed on the tree. Positive results with early amplification (Cq < 30) are indicated between the green dotted lines, while the grey dotted line represents the assay cut-off at 36 cycles