Molecular Diagnostics of Enaphalodes rufulus (Coleoptera: Cerambycidae)

M. Brent Kelley, Stephen W. Wingard, Allen L. Szalanski, Fred M. Stephen


Oak-hickory forests in northwestern Arkansas, eastern Oklahoma and southern Missouri have recently experienced an oak decline event with widespread oak mortality. The oak mortality is associated with an outbreak of a native wood-boring cerambycid, Enaphalodes rufulus (Haldeman), the red oak borer. Taxonomic identification, below the family level, of larval Cerambycidae through traditional morphological methods is not usually possible. We employed molecular diagnostics, with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), to distinguish E. rufulus from other closely related species of cerambycids. A portion of the mitochondrial DNA 16S rRNA gene, isolated from legs or thoraxes of adult museum specimens, was amplified and digested with Alu I and Hind III restriction enzymes. Both restriction enzymes independently produced fragments for E. rufulus that were significantly different from any other cerambycid tested. Alu I had one restriction site for E. rufulus and two restriction sites for all other cerambycids tested, while Hind III did not cut for E. rufulus but did cut at one restriction site for all other cerambycids. Eggs, larvae, and pupae of E. rufulus along with an unknown cerambycid larva and pupa were successfully amplified and digested by this method to verify validity of this technique for multiple life stages.

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