Molecular Differentiation of Metastriate Tick Immatures

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Molecular Differentiation of Metastriate Tick Immatures

Postby Yvonne » Mon 4 Jan 2010 9:15


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.


Molecular evaluation of ticks for disease
transmission and phylogenetic studies has increased
tremendously over the past ten years
(Sparagano et al. 1999). A considerable amount
of this research is being carried out by personnel
with little or no training in acarine taxonomy
and morphology. Mis-identification can
easily lead to erroneous results and, in the case
of pathogen transmission, could implicate the
incorrect vector species. Additionally, once the
specimen is destroyed during DNA extraction,
anomalous results are difficult to verify. Although
voucher specimens should always be
retained and identification verified by qualified
taxonomists, the need to validate the identification
of extracted tick DNA is crucial. Using
established molecular markers for PCR amplification
and sequencing can be extremely cost
prohibitive, especially for population studies or
surveys where possibly hundreds of samples
are processed.

In the mid-Atlantic and northeastern regions
of the United States, the main tick genera commonly
encountered—Ixodes, Dermacentor, Amblyomma,
Haemaphysalis, and Rhipicephalus—have
overlapping seasonal activity and distributions,
and most importantly have the potential
to pose as threats to public health. Subsequently,
tick collections often consist of more
than one tick species at any given time. Misidentification
of tick samples could lead to inaccurate
disease transmission rates, incorrect
host association as well as mis-interpretation of
geographic and habitat range.

Three of the tick species used in this study—
D. variabilis, A. americanum, and I. scapularis—
are predominant ticks commonly encountered
in Maryland and are all medically important.
Several other tick species were included in this
study even though they are either not considered
medically important or are not commonly
found in Maryland. We felt it was critical to include
these species to create a tool that would
be useful to investigators in other regions of
North America. Although the list of species
used in this investigation is not exhaustive, we
could speculate on the ability of the speciesspecific
internal primers to anneal to template
DNAs from species that were not tested, based
on the alignments provided in Table 1.

Internal Dermacentor- and Amblyomma-specific
primers were designed to amplify all
species of these respective genera. One exception
was A. maculatum. The expected Amblyomma-
specific fragment could not be amplified
from representatives of this species. The 16S
gene alignment based on the Amlyomma sequences
from genebank illustrates that the internal
primer region is highly polymorphic
with multiple base changes in A. maculatum
compared to A. amblyomma. Attempts at creating
a primer with degenerate bases at the predicted
mutation sites was not successful (data
not shown). The distribution of A. maculatum is
limited to the coastal regions of the southeastern
United States. Specimens have been found
at remote inland locations but populations do
not generally become established. In addition,
this tick is not common throughout much of the
eastern United States

Due to limited variability in the small 16S
rRNA target sequence on which this diagnostic
is based, a conserved primer set could not
be created to differentiate between Rhipicephalus,
Haemaphysalis, and Ixodes. This was
partially overcome by using a restriction en-
zyme, TauI, which specifically cleaves Rhipicephalus,
leaving both Haemaphysalis and Ixodes
template DNAs uncut (Fig. 4). Further differetiation
of Ixodes ticks should not be problematic
as the Prostriata can be easily differentiated
from the Metastriata using traditional morphological
characteristics on all life stages, both
engorged and un-engorged. Therefore, the
derived PCR-RFLP-based diagnostic is only
intended for differentiation of the four Metastriate
genera; Dermacentor, Amblyomma, Rhipicephalus,
and Haemaphysalis (Fig. 2). The molecular
diagnostic described here will allow
investigators to confidently discriminate between
these Metastriate genera, quickly, easily
and inexpensively, either as a confirmation of
morphological identifications or verification
for material processed in the laboratory for vector
genetics or pathogen surveillance
Listen to all,
plucking a feather from every passing goose,
but follow no one absolutely

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