Using the rDNA Internal Transcribed Spacer 1 to Identify the Invasive PestRhagoletis cerasi(Diptera: Tephritidae) in North America

Abstract

The cherry-infesting fruit fly Rhagoletis cerasi Loew is a significant commercial pest in Europe that has recently invaded North America. To date, it has been trapped only in Canada and northwestern counties of New York. It has the potential to spread further and threaten production and movement of cherry commodities. Timely diagnosis of the pest will facilitate surveys and quick response to new detections. Adult morphology of the pest is distinct from other flies in North America. However, when flies are significantly damaged on traps or the immature life stages are found in fruits, molecular methods of identification are important to confirm presence and host-use records. Other than DNA sequencing of genes from flies which takes over a day to complete, there are no timely methods of molecular identification for this pest. In this study, we report the first sequence record of the internal transcribed spacer 1 (ITS1) from R. cerasi and develop two diagnostic tests for the pest based on ITS1 differences among species in North America. The tests use loop-mediated isothermal amplification (LAMP) and multiplex, conventional polymerase chain reaction (mcPCR) technologies that target the same region of the R. cerasi ITS1 sequence. Both tests performed well when tested against collections of R. cerasi from North America and Europe, generating Diagnostic Sensitivity estimates of 98.4–99.5%. Likewise, the tests had relatively high estimates of Diagnostic Specificity (97.8–100%) when tested against Rhagoletis Loew species present in North America that also use cherry as a developmental host.

Partial Ribosomal Nontranscribed Spacer Sequences DistinguishRhagoletis zephyria(Diptera: Tephritidae) From the Apple Maggot,R. pomonella

“…The mitochondrial COI barcode primers of Hebert et al (2004) , LEP F1 and LEP R1 ( Table 2 ), were used to amplify the mitochondrial COI region from the same set of flies from which we amplified the NTS and ETS regions. As observed by Barr et al (2021) in European cherry fruit fly Rhagoletis cerasi (L.), the quality of some COI sequence reads from Rhagoletis flies in our sample was low, leading to ‘dirty’ sequences. In two cases, PCR of whole fly Rhagoletis extracts using LEP F1 and LEP R1 resulted in the amplification of COI fragments that were very clean upon sequencing, but yielded a DNA sequence that matched exactly the COI sequence from Wolbachia pipientis (GenBank Accession CP 042445.1 ).…”

“…In two cases, PCR of whole fly Rhagoletis extracts using LEP F1 and LEP R1 resulted in the amplification of COI fragments that were very clean upon sequencing, but yielded a DNA sequence that matched exactly the COI sequence from Wolbachia pipientis (GenBank Accession CP 042445.1 ). This phenomenon has been observed before ( Smith et al 2012 ), and we thus followed the recommendation of Barr et al (2021) and used the primer pair TY-J-1460 and C1-N-2191 ( Simon et al 1994 ) for a number of subsequent COI amplifications, modifying the TY-J-1460 primer to match the known Rhagoletis sequence of the tRNA Tyr gene (TY-J-1460_Rhag; Table 2 ).…”

A multiplex PCR assay for the identification of fruit flies (Diptera: Tephritidae) of economic importance in South Africa

“…Multiplex PCR offers the ability to amplify different DNA targets and different amplicon sizes in a single run. Although the use of multiplex PCR for fruit fly identification has not been well explored, it has shown promising results in differentiating a species of interest, Rhagoletis cerasi Loew, from other tephritid flies in North America as well as fruit fly parasitoid identification 31 , 32 . While the five fruit flies under study can be identified through a variety of existing molecular assays, to date, no assay can identify all five flies simultaneously.…”