Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Abstract

Accurate species-level identifications underpin many aspects of basic and applied biology; however, identifications can be hampered by a lack of discriminating morphological characters, taxonomic expertise or time. Molecular approaches, such as DNA "barcoding" of the cytochrome c oxidase (COI) gene, are argued to overcome these issues. However, nuclear encoding of mitochondrial genes (numts) and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications. One insect group for which these molecular and morphological problems are significant are the dacine fruit flies (Diptera: Tephritidae: Dacini). We addressed these issues associated with COI barcoding in the dacines by first assessing several "universal" COI primers against public mitochondrial genome and numt sequences for dacine taxa. We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region. Our new primers were tested alongside universal primers on a selection of dacine species, including both fresh preserved and decades-old dry specimens. Additionally, Bactrocera tryoni mitochondrial and nuclear genomes were compared to identify putative numts. Four numt clades were identified, three of which were amplified using existing universal primers. In contrast, our new primers preferentially amplified the "true" mitochondrial COI barcode in all dacine species tested. The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable. Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.

Molecular identification of Bactrocera passiflorae (Diptera: Tephritidae): Challenge and solution for DNA barcoding

“…The analysis also revealed that the Mt-COI obtained in this study are closely related to the B. passiflorae COI in the database, with a mean p-distance of 0.0075, but are more distant from the NUMT sequences (Table 3). Similarly, the individual sequences of NUMT type 1 obtained in this study are also closely related to previously reported NUMT sequences of B. passiflorae (Krosch et al, 2020), with a mean p-distance of 0.0047 between the two groups (…”

“…We therefore developed novel primers to specifically amplify the true Mt-COI of B. passiflorae. By cloning the PCR products of NUMT pseudogenes, we were able to determine that the NUMT copies of B. passiflorae, NUMT type 1 share 98-100% identity to the NUMT COI identified previously (Krosch et al, 2020) where the NUMT type 2 is novel sequence identified by this study.…”

In the footsteps of Wallace: population structure in the breadfruit fruit fly, Bactrocera umbrosa (F.) (Diptera: Tephritidae), suggests disjunction across the Indo‐Australian Archipelago

“…Sequences were checked and aligned by eye in BioEdit (Hall ), any ambiguous bases were recoded as missing data and presence of pseudogenes or internal stop codons (Krosch et al . ) assessed by alignment of protein sequences in MEGA4 (Tamura et al . ).…”

The Potency of Angle Measurement and Comparison of Vein Lengths in Distinguishing Bactrocera Species Complexes

“…Perrard et al (2014) confirmed that the genetic difference between wasp species had a stronger influence on the wing shape than other biotic or abiotic factors. Genetically, species complexes in genus Bactrocera are paraphyletic each other due to descent from different ancestral lines (Boykin et al, 2014;Krosch et al, 2020). However, the application of angle measure of cell br and ratio vein r-m : dm-cu could demonstrate had a lack in showing the relationship between subgenus Bactrocera and Zeugodacus as phylogenetic tree showed.…”