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Morphology, Systematics, Evolution
The First Evidence of Nanism in Ixodes (Ixodes) scapularis
(Acari: Ixodidae), Found Parasitizing a Human Host
John Soghigian,
1
Gale E. Ridge,
2
Kirby C. Stafford III ,
3
and Goudarz Molaei
1,4,5
1
Department of Environmental Sciences and Center for Vector Biology & Zoonotic Diseases, The Connecticut Agricultural
Experiment Station, 123 Huntington St., New Haven, CT 06511 (john.soghigian@ct.gov; goudarz.molaei@ct.gov),
2
Department of
Entomology, The Connecticut Agricultural Experiment Station, 123 Huntington St., New Haven, CT 06511 (gale.ridge@ct.gov),
3
Department of Entomology and Center for Vector Biology & Zoonotic Diseases, The Connecticut Agricultural Experiment Station,
123 Huntington St., New Haven, CT 06511 (kirby.stafford@ct.gov),
4
Department of Epidemiology of Microbial Diseases, School of
Public Health, Yale University, 60 College St., P.O. Box 208034, New Haven, CT 06520-8034, and
5
Corresponding author, e-mail:
goudarz.molaei@ct.gov
Subject Editor: Lars Eisen
Received 3 April 2017; Editorial decision 5 May 2017
Abstract
Ixodes scapularis Say 1821, the primary vector of several human pathogens in the northeastern and upper
Midwestern United States, has considerable genetic and morphological variation throughout its range.
Recently, developmental or teratological abnormalities have been observed in this species for the first time, fur-
ther complicating morphological identification. Here, we report the first evidence of nanism (dwarfism) in I.
scapularis, found parasitizing a human host. We used molecular methods and scanning electron microscopy to
identify the specimen. Morphological identification confirmed that the specimen is substantially smaller, ap-
proximately half the size, than a typical I. scapularis female. Here we discuss the recent reports of teratological
abnormalities in I. scapularis, particularly from the Hudson River valley region of the northeastern United
States, and highlight the need for additional studies of teratology in this important species and its potential im-
plications in disease transmission.
Key words: Ixodes scapularis, teratological abnormality, nanism
The blacklegged tick or deer tick, Ixodes scapularis Say 1821, is an
important vector of pathogens responsible for Lyme disease, human
babesiosis (Spielman et al. 1979), granulocytic anaplasmosis
(Pancholi et al. 1995), Powassan encephalitis (Ebel 2010), and
others (Nelder et al. 2016). Lyme disease is the most prevalent
vector-borne disease in North America and is caused by spirochetes
in the Borrelia burgdorferi sensu lato species complex (Pritt et al.
2016). An estimated 300,000 human Lyme disease cases occur
annually in the United States, the majority from the northeast
and upper Midwest (Nelson et al. 2015).
Populations of I. scapularis in the United States can be classified
into two genetic lineages: a diverse “southern clade” found only in
the southeastern United States, and a second lineage, far less geneti-
cally diverse, that is found in both the southern and northern range
of this species (Norris et al. 1996,Van Zee et al. 2013,Sakamoto
et al. 2014). Diversity in morphological (Hutcheson et al. 1995,
Keirans et al. 1996) and behavioral characteristics (Arsnoe et al.
2015,Goddard et al. 2015) historically created difficulties in identi-
fication of I. scapularis. Prior to the widespread establishment of
I. scapularis in northern and central North America, this morpho-
logical variability caused both the misclassification of I. scapularis
nymphs as Ixodes muris (Spielman et al. 1979), and the temporary
elevation of some I. scapularis populations to the separate species,
Ixodes dammini (a name later demoted to junior subjective syno-
nym; Spielman et al. 1979,Oliver et al. 1993). However, the rede-
scription of I. scapularis in 1996 accounted for variation across the
geographic range of this species (Keirans et al. 1996).
Recent studies from Wisconsin (Larson and Paskewitz 2016) and
New York (Prusinski et al. 2015) reported morphological abnormal-
ities in field populations of I. scapularis for the first time.
Abnormalities in tick species have been observed since the late 19th
century (Neumann 1899) and are classified into two categories: lo-
cal, e.g., deformities or absence of specific structures such as legs or
mouth parts in an otherwise normal tick; and general, e.g., nanism
or dwarfism, gigantism, asymmetry or bifurcation of the idiosoma,
and gynandromorphism (Campana-Rouget 1959,Sim~
oes et al.
1992). Both categories are thought to be a result of teratological de-
fects and may be caused by genetic factors and changes in tempera-
ture and humidity during tick development (Buczek 2000,Kar et al.
2015), feeding on exotic hosts (Nowak-Chmura 2012), or exposure
to insecticides or other chemicals (Campana-Rouget 1959,Buczek
et al. 2013). Such defects are rare in ticks, with total incidence of
V
CThe Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America.
All rights reserved. For Permissions, please email: journals.permissions@oup.com 1
Journal of Medical Entomology, 2017, 1–5
doi: 10.1093/jme/tjx111
Research article
general or local abnormalities from 0.028% to 0.2% (Tovornik
1987,Kar et al. 2015,Larson and Paskewitz 2016). These abnor-
malities seem to occur at even lower frequency in Ixodes (Nowak-
Chmura 2012,Kar et al. 2015,Larson and Paskewitz 2016), and of
general abnormalities, only occurrences of gynandromorphism and
asymmetry have been reported in this genus (Prusinski et al. 2015,
Larson and Paskewitz 2016).
Here, we report the first evidence of nanism in I. scapularis, con-
firming the identity of the specimen using molecular markers and
scanning electron microscopy. This specimen was found biting a
child and was submitted for screening of tick-associated pathogens
to the Tick Testing Laboratory at the Connecticut Agricultural
Experiment Station (CAES).
Materials and Methods
Specimen Submission
The specimen was submitted on November 6, 2015 for tick testing
at the CAES. The tick had been removed from a child with no out of
state travel history in Ridgefield, CT, in early November. Following
morphological examination, it was determined the specimen was
not engorged and thus was not tested for tick-associated pathogens.
The specimen was stored in 75% EtOH for further analysis.
Morphological Identification
Initial morphological identification was attempted following
Keirans and Litwak (Keirans and Litwak 1989). Due to the unusu-
ally small size and morphological features of the specimen, we used
an AXIO Scope.A1 (Zeiss, Go¨ ttingen, Germany) with an attached
RT3 camera system (SPOT Imaging, Sterling Heights, MI) to cap-
ture ventral and dorsal images of the specimen, and then proceeded
with scanning electron microscopy (SEM) and molecular
identification.
Scanning electron microscopy was used to visualize the morpho-
logical characters of the specimen. The specimen was dehydrated in
99.8% EtOH and critical point dried (931GL, Tousimis, Rockville,
MD). The dried specimen was mounted on an SEM stub using car-
bon tape, sputter coated with gold/palladium (E5100, Polaron), and
examined with the aid of a Nova NanoSEM 450 (FEI, Hillsboro,
OR) scanning electron microscope.
Following SEM, we used a dichotomous key to the adults of
Ixodes based on scanning electron micrographs (Keirans and
Clifford 1978). Morphological characters of the specimen were
measured in ImageJ2 (Schindelin et al. 2015), using images from
light microscopy or SEM. Morphological structures were defined
following Keirans et al. (1996):breadth for all features were mea-
sured at their broadest point; idiosomal length was based on the
length from the scapular apices to the posterior body margin; and
coxae length from the insertion of the anterior most seta adjacent to
the trochanter diagonally to the posterior tip of the coxa (or the tip
of the internal spur, for coxa I). All measurements are reported in
millimeters.
DNA Extraction and Sequencing
For molecular identification, the tarsi and tibiae of the specimen
were removed for DNA extraction. DNA was extracted from the re-
moved appendages using DNAzol BD (Molecular Research Center,
Cincinnati, OH) according to manufacturer’s recommendations
with minor modifications. Briefly, the sample was homogenized in a
tube containing 400 ml DNAzol BD and two zinc-plated BBs (Daisy
Outdoor Products, Rogers, AK) using a TissueLyser Mixer Mill
(Qiagen, Valencia, CA). The sample was then incubated at 70 C for
10 min, then mixed and centrifuged at 14,000 RPM for 10 min.
Following the addition of 3 ml Poly Acryl Carrier (Molecular
Research Center), DNA was precipitated using 200 ml 100% EtOH.
The DNA pellet was washed twice with 750 ml 75% EtOH, air dried
briefly, reconstituted in 30 mldH
2
0, and stored at 20 C for further
analysis.
The identity of the specimen was confirmed using several molec-
ular markers—the ribosomal internal transcribed spacer 2 (ITS2) re-
gion, the 16S mitochondrial ribosomal region, and the nuclear gene
coding for the serotonin 4 receptor.
ITS2
The full ITS2 region, along with partial sequences of 5.8S and 28S,
was amplified with polymerase chain reaction (PCR). The primers
were TITS2F1, 50-CGA GAC TTG GTG TGA ATT GCA-30, and
TITS2R1, 50-TCC CAT ACA CCA CAT TTC CCG-30(Chitimia
et al. 2009) that yielded fragments of 900 bp. GoTaq G2 Green
Master Mix (Promega, Madison, WI) was used according to the
manufacturer’s protocols in a 50-ml reaction, containing 0.3 mMof
each primer and 2 ml of template DNA. Thermal cycling conditions
included an initial denaturation step of 95 C for 3 min, followed by
35 cycles of 95 C for 45 s, 60 C for 1 min, and 72 C for 75 s.
16S
The mitochondrial 16S region was amplified using primers mt-rrs1,
50-CTG CTC AAT GAT TTT TTA AAT TGC TGT GG-30, and mt-
rrs2, 50-CCG GTC TGA ACT CAG ATC AAG TA-30(Ushijima
et al. 2003) that yielded a fragment size of 456 bp. Reaction condi-
tions for 16S were as described above for the ITS2, except that 1 ml
of DNA template was used. Thermal cycling conditions included an
initial denaturation step of 95 C for 2 min, followed by 35 cycles of
95 C for 30 s, 48 C for 30 s, and 72 C for 45 s, with a final exten-
sion step of 72 C for 5 min.
Serotonin 4
The nuclear region serotonin 4 was amplified with primers S4F, 50-
AAC GAA ACC ACG CTC AAG A-30, and S4R, 50-GTA GCA
GAC AGC GAA CAG CA-30(Van Zee et al. 2013), which yielded a
fragment size of 650 bp. Reaction mixture included 2 ml of DNA
template and 0.5 mM of each primer. Thermal cycling conditions in-
cluded an initial denaturation of 95 C for 2 min, followed by 40 cy-
cles of 95 C for 30 s, 58 C for 30 s, and 74 C for 30 s, with a final
extension step of 74 C for 5 min.
Sequence Analysis
All PCR products were purified using QIAquick PCR Purification
kit (Qiagen), sequenced in both directions at the Keck DNA
Sequencing Center, Yale University (New Haven, CT), and se-
quences were assembled in Geneious version 9 (Kearse et al. 2012).
Geneious was used to query the NCBI’s nr database with the mega-
blast program (Camacho et al. 2009), returning the top 50 most sim-
ilar hits for all sequences. These hits were sorted by “Grade” in
Geneious, a weighted combination of pairwise identity, coverage,
and E score, and each hit was assigned a rank from 1 to 50, relative
to its sorted position. Identity of the specimen was assessed based on
the resulting tables.
2Journal of Medical Entomology, 2017, Vol. 0, No. 0
Results
Specimen Description
Measurements for morphological characters are provided in
Table 1, and descriptors of specific regions are given here.
Body
Scutal color brownish, outline oval, light brown opisthosoma.
Capitula
Auriculae broadly rounded. Palpis with suture between article II and
III distinct, article II 1.7 times longer than article III. Hypostome
dentition 4/4 apically, 3/3 for most of the visible dentitions, and
then 2/2 near base. Cornua small, porose areas semicircular.
Scutum
Carinae absent or not visible, cervical grooves faint, with visible
punctations and setae primarily in marginal areas.
Venter
Spiricular plate oval, genital aperture between coxae IV.
Legs
Tarsi and tibiae removed or damaged and were not used in specimen
description. Coxa I with internal spur. Syncoxae, if present, not visi-
ble. Coxae I–IV with blunt external spur.
Morphological Comparison
Due to the small size of the specimen, identification using morpho-
logical characters alone was difficult. The presence of porose areas
(not shown in figures), a genital aperture between coxae IV, as well
as hypostomal shape and dentition indicated the specimen was an
adult female tick despite a size more typical of a nymph (Fig. 1;
Supp. Fig. 1 [online only]). Comparisons with SEM images nar-
rowed the species identity based on morphological characters to I.
scapularis,Ixodes pacificus Cooley & Kohls, or Ixodes jellisoni
Cooley & Kohls. Due to the small size of the internal spur on coxa I,
further classification of this specimen was not feasible; however,
given the geographic location where the specimen was found, the
tick was preliminarily identified as I. scapularis.
A comparison of the morphological measurements from the
specimen with those reported in the redescription of I. scapularis
(Keirans et al. 1996) revealed that the specimen in question was ap-
proximately half the size of a typical I. scapularis female (Table 1,
Supp. Fig. 1 [online only]). Furthermore, the dentition of the speci-
men, 4/4 at the tip of the hypostome, declining to 3/3 and eventually
to 2/2, was consistent with that of I. scapularis. In addition, the
rounded auriculae (Supp. Fig. 2 [online only]) and elongate internal
spur of coxa I (Supp. Fig. 3 [online only]) were consistent with an
adult female I. scapularis.
Sequence Analysis
Sequence analysis of the three genes used in this study indicated the
specimen in question is most likely I. scapularis. BLAST search re-
sults yielded multiple sequences with >96% pairwise identity be-
tween the ITS2 sequence from the specimen (GenBank KY985362)
and other sequences available on GenBank for I. scapularis. Both
16S (GenBank KY985363) and serotonin 4 (GenBank KY985364),
with exact matches found on GenBank, unequivocally confirmed
the identity of the specimen as I. scapularis. Although our ITS2 se-
quence was not identical to any sequence deposited in GenBank,
only I. scapularis sequences had “Grades” higher than 91%, with
the next nearest taxa sequence belonging to Ixodes persulcatus with
a rank of 28, a “Grade” of 90.5%, and pairwise identity of 81%
(GenBank accession for this I. persulcatus sequence, D88872).
Discussion
Our analysis of this specimen provides the first evidence of nanism
in I. scapularis. The specimen possesses the characters of an adult fe-
male tick, and because the size of this tick falls far outside the range
of previously reported values for female I. scapularis, we conclude
that this tick is a dwarf I. scapularis. Although nanism has been re-
ported in other tick taxa (Campana-Rouget 1959,Sim~
oes et al.
1992), to the best of our knowledge, this is the first report of this
type of abnormality in Ixodes.
General abnormalities such as nanism are extremely rare in
I. scapularis; of all the ticks examined by investigators throughout
Table 1. Measurements of some morphological features of the
dwarf I. scapularis, compared to the reported range in the species
description of I. scapularis
Feature Dwarf
I. scapularis
Reported range
(Keirans et al. 1996)
Length Breadth Length Breadth
Idiosomal length
a
1.47 0.84 2.37–2.7 1.43–1.89
Palpis 0.40 0.13 0.68–0.79 0.17–0.21
Hypostome 0.33 – 0.51–0.59 –
Scutum
a
0.82 0.69 1.23–1.46 1.02–1.30
Spiricular plate 0.19 – 0.33–0.44 –
Coxa I 0.28 – 0.45–0.56 –
Internal spur (Coxa I) 0.07 0.04 0.11–0.17 –
Coxa II 0.25 – 0.37–0.44 –
Coxa III 0.25 – 0.39–0.46 –
Coxa IV 0.22 – 0.30–0.46 –
All measurements are in millimeters and rounded to the nearest hundredth.
a
Measurements taken from light microscopy images (not shown).
Fig. 1. The ventral scanning electron micrograph of a dwarf I. scapularis.
Despite its small size, the specimen has features consistent with an adult
female, such as the genital aperture visible here.
Journal of Medical Entomology, 2017, Vol. 0, No. 0 3
the eastern United States, this is only the seventh reported occur-
rence of any teratological abnormality in this species. Over the past
two decades (1997–2016), the Tick Testing Program at the CAES
has received and examined a total of 88,347 ticks, of which 81,475
(92.5%) have been identified as I. scapularis; however, this marks
the first encounter of an adult I. scapularis of abnormally small size.
Although it is possible that dwarf ticks may be mischaracterized as
nymphs during routine examinations, and thus nanism may occur
more commonly, dwarf I. scapularis have never been reported previ-
ously, suggesting the scarcity of this phenomenon.
It is worth noting that a prior report of general abnormalities in
I. scapularis detailed two gyandromorphic specimens from the
Hudson River Valley region of the State of New York (Prusinski
et al. 2015), and the specimen reported here was submitted from the
same general region. That three of the general abnormalities re-
ported in I. scapularis have been found in the same general geo-
graphical region suggests the possibility that certain environmental
factors might be involved. Research has shown that teratological de-
fects may be caused by variation in temperature or humidity during
tick development (Buczek 2000,Kar et al. 2015). In Europe, the oc-
currence of morphologically abnormal ticks has reportedly increased
(Alekseev and Dubinina 2004,Alekseev et al. 2007) and may be re-
lated to increasing levels of pollution (Alekseev and Dubinina
2008). In another Ixodid, Dermacentor andersoni, extreme size vari-
ation was heritable (de la Fuente et al. 2005), but whether this
applies to the teratological defects found in I. scapularis is
unknown.
Accurate morphological identification of tick specimens is im-
portant for understanding of species abundance and diversity, as
well as determining which specimens require examination for the ev-
idence of infection. As such, teratological defects complicate identi-
fication of tick specimens, but what effect these abnormalities might
have on the ability of I. scapularis to transmit pathogens is un-
known. Studies in I. persulcatus suggest ticks with exoskeletal
anomalies may have greater infections with Borrelia (Alekseev and
Dubinina 2000), and may also have more multiple simultaneous
pathogen infections, than morphologically normal ticks (Alekseev
et al. 2007). Whether these findings can be generalized to include
I. scapularis with teratological abnormalities is unknown.
Here we report the first evidence of nanism, and and an addi-
tional example of a general teratological abnormality, in I. scapula-
ris. Given the difficulties associated with species identification of
tick specimens with morphological abnormalities and potential im-
plications in pathogen transmission (Alekseev and Dubinina 2000,
Alekseev et al. 2007), further investigation of teratologies is needed
in I. scapularis.
Acknowledgments
We are grateful to the Editor and three anonymous reviewers for their con-
structive comments and suggestions on an earlier version of this manuscript.
We would like to thank Saryn Kunajukr (former technician at the CAES Tick
Testing Laboratory) for her technical assistance. SEM was performed with
the assistance of X. Sun at the Biosciences Electron Microscopy Facility of the
University of Connecticut, which is partially funded by NSF grant 1126100.
We are also grateful to Adriana L. Arango-Velez and Katherine Dugas, both
of the CAES, for assistance with light microscopy performed in this study.
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