Molecular differentiation of metastriate tick immatures

Abstract

Hard ticks, family Ixodidae, are divided into two groups, the Metastriata and the Prostriata, based on morphological differences. In the United States, there are four medically important genera of the Ixodidae: Ixodes, Amblyomma, Dermacentor, and Rhipicephalus. Ixodes is the only genus in and representative of the Prostriata, whereas the latter three genera are members of the Metastriata. All developmental stages of the Prostriata can be easily differentiated from the Metastriata using morphology. Similarly, the three Metastriate genera are highly identifiable as adults, yet as immatures, the discriminating characteristics can be difficult to use for differentiation, especially if the specimens are damaged or engorged with blood. All three Metastriate genera represent medically important vectors, thus accurate differentiation is necessary. To this end, we have developed a multiplexed-PCR diagnostic assay that, when combined with RFLP analysis will differentiate between the Metastriate genera--Amblyomma, Dermacentor, Rhipicephalus, and Haemaphysalis based on the length of the PCR amplicon and subsequent restriction digestion profile. The intended use for this diagnostic is to verify morphological identifications, especially of immatures, as well as to identify samples destroyed for molecular analysis, which will lead to more accurate field data as well as implication of vectors in disease transmission.

Fig. 1

Schematic of the multiplex primer combination with predicted amplification product size.

Fig. 2

PCR amplification products from the multiplex reaction. Lanes 1–8 are amplified products from Dermacentor species (lanes 1–3: D. variabilis adult, nymph and larvae; lanes 4–6: D. albipictus adult, nymph and larvae; lane 7: D. andersoni adult; lane 8: D. occidentalis adult). Lanes 9–13 represent amplification from Amblyomma species (lanes 9–11: A. americanum adult, nymph and larvae; lanes 12–13: A. maculatum adult and nymph). Lanes 14–16 are Haemaphysalis leporispalustris adult, nymph and larvae. Lanes 17–18 are Rhipicephalus sanguineus adult and nymph specimens. Lanes 19–21 are Ixodes scapularis adult, nymph, and larvae. The ladder is a 100-bp marker. L, ladder; NC, negative control.

Fig. 3

Ribosomal 16S fragment alignment of the restriction enzyme digestion region (nucleotide 272–276) for TauI restriction enzyme of several species of hard ticks. Species names in bold were tested during this analysis.

Fig. 4

Restriction digestion of genera that were not differentiated with multiplex PCR primer set. Samples were digested with TauI restriction enzyme. D, digested; U, undigested.