Differentiation of three phenotypically similar Blattella spp.: Analysis with polymerase chain reaction-restriction fragment length polymorphism of mitochondrial DNA

Differentiation of Blattella asahinai Mizukubo, Blattella vaga Hebard, and Blattella germanica (L.) was investigated using polymerase chain reaction-r estriction fragment length polymorphism (PCR-RFLP). DNA fragments correspon ding to mitochondrial CO1,CO1/TL2 (small and large fragments), and ITS2 reg ions were amplified using PCR. The universal primers used for amplifying mi tochondrial CO1 and CO1/TL2 (small and large fragments) were CO1-J-1718/CO1 -N-2191, CO1-J-2441/TL2-N-3014, and CO1-J-1751/TL2-N-3014. Amplification wa s observed with all primer combinations, but diagnostic patterns were found only with AluI digested amplicons of CO1 and CO1/TL2 (large fragments) reg ions. Both inter/intraspecific polymorphism was observed. for the 517 bp CO 1 region between German (A, B) and Asian (A, B, C) cockroach populations. P olymorphic form B of the Asian cockroach (374 and 179 Lp fragments) was nea rly identical to the field cockroach (370 and 178 bp fragments). The CO1 am plicon (517 bp) differentiated German and field cockroaches but not Asian a nd field cockroaches. The CO1/TL2 amplicons (1275 bp) digested with AluI cl early differentiated all three species. The Asian cockroach had a distinct 540 bp fragment, whereas the field cockroach had a 403 bp fragment and the German cockroach lacked both fragments. Intraspecific polymorphs for CO1/TL 2 amplicons (1275 LD) were observed with the German cockroach only. Portion s of the CO1 and TL2 genes were sequenced, revealing 76% identity among Ger man, Asian, and field cockroaches. Sequence data also revealed that German and Asian cockroaches were more closely related to each other than to the f ield cockroach.