A novel diagnostic gene region for distinguishing between two pest fruit flies:Bactrocera tryoni(Froggatt)and Bactrocera neohumeralis(Hardy)(Diptera:Tephritidae)

Bactrocera tryoni and Bactrocera neohumeralis are morphologically similar sibling pest fruit fly species that possess different biological attributes,geographic distributions,and host ranges.The need to differentiate between the two species is critical for accurate pest status assessment,management,biosecurity,and maintenance of reference colonies.While morphologically similar,adults may be separated based on subtle characters;however,some characters exhibit intraspecific variability,creating overlap between the two species.Additionally,there is currently no single molecular marker or rapid diagnostic assay that can reliably distinguish between B.neohumeralis and B.tryoni;therefore,ambiguous samples remain undiagnosed.Here we report the first molecular marker that can consistently distinguish between B.tryoni and B.neohumeralis.Our diagnostic region consists of two adjacent single nucleotide polymorphisms(SNPs)within the pangolin(pan)gene region.We confirmed the genotypes of each species are consistent across their distributional range,then developed a tetra-primer amplification refractory mutation system(ARMS)PCR assay for rapid diagnosis of the species.The assay utilizes four primers in multiplex,with two outer universal primers,and two internal primers:one designed to target two adjacent SNPs(AA)present in B.tryoni and the other targeting adjacent SNPs present in B.neohumeralis(GG).The assay accurately discriminates between the two species,but their SNP genotypes are shared with other nontarget tephritid fruit fly species.Therefore,this assay is most suited to adult diagnostics where species confirmation is necessary in determining ambiguous surveillance trap catches;maintaining pure colony lines;and in Sterile Insect Technique management responses.

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

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 tiyoni 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.