Mitochondrial 12S rRNA sequences used to design a molecular ladder assay to identify six commercially available phytoseiids (Acari: Phytoseiidae)

Phytoseiid mites are released to suppress pest mites and prevent crop damage, but they are small (<0.5 mm) and often difficult to identify. Molecular identification could enhance our abilities to monitor the phytoseiid species during rearing and after release into the field. Hence, a portion of the mitochondrial 12S rRNA gene was amplified, cloned, and sequenced from Neoseiulus fallacis (Garman), N. californicus (McGregor), N. cucumeris (Oudemans), Metaseiulus (=Typhlodromus or Galendromus) occidentalis (Nesbitt), Iphiseius degenerans (Berlese), Phytoseiulus persimilis Athias-Henriot, and their prey Tetranychus urticae Koch. The sequence divergences detected among these phytoseiids were much higher at the mitochondrial 12S rRNA gene locus (9.5–45%) than the nuclear rRNA internal transcribed spacer region (ITS-1, 5.8S, and ITS-2) (1.2–23%) (Navajas et al., 1999; Jeyaprakash and Hoy, unpublished). This level of divergence allowed us to design a ‘molecular ladder assayʹ that amplifies a diagnostically different sized DNA band from each of the six phytoseiid species using species-specific primers from the variable regions of the mitochondrial 12S rRNA gene. However, reliable amplification of phytoseiid mitochondrial DNA by Standard PCR was possible only when DNA was prepared by a Puregene method from multiple specimens (>20 females). To amplify mitochondrial 12S rRNA sequences consistently from single females, males, nymphs, larvae, or eggs, DNA was extracted in Chelex and Long PCR (also called High fidelity PCR) was tried, which allowed us to identify individual phytoseiids. Such a sensitive method is needed to confirm pure colonies for research, or to evaluate individuals for contamination during mass rearing, or to confirm identities of field-collected specimens.