Download Temeyer 2016 tick salivary cholinesterase

Journal of Medical Entomology Advance Access published January 21, 2016
Journal of Medical Entomology, 2016, 1–5
doi: 10.1093/jme/tjv252
Research article
Arthropod/Host Interaction, Immunity
Tick Salivary Cholinesterase: A Probable
Immunomodulator of Host–parasite Interactions
Kevin B. Temeyer1 and Alexander P. Tuckow
Knipling-Bushland U.S. Livestock Insects Research Laboratory, U.S. Department of Agriculture-Agricultural Research Service,
2700 Fredericksburg Road, Kerrville, Texas 78028 ([email protected]; [email protected]) and 1Corresponding
author, e-mail: [email protected]
Received 21 September 2015; Accepted 23 December 2015
Abstract
Key words: cholinesterase, acetylcholine, inflammation, parasite–host interaction, parasite immune regulation
The southern cattle tick, Rhipicephalus (Boophilus) microplus
(Canestrini), is the most economically important ectoparasite affecting cattle in the world (Bellgard et al. 2012, Guerrero et al. 2012).
Rhipicephalus annulatus (Say) and R. microplus were eradicated
from the United States through the Cattle Fever Tick Eradication
Program in a protracted effort that began in 1906 and continues
with ongoing inspection and treatment of imported cattle as well as
quarantine and eradication of outbreak infestations within the
United States (Graham and Hourrigan 1977). Rhipicephalus microplus and R. annulatus remain endemic in Mexico and other countries, and reintroduction of the ticks by importation or incursion
constitutes a continuing threat to U.S. cattle producers (Temeyer
et al. 2004b, Giles et al. 2014). Cattle dipping in coumaphos (an organophosphorus acaricide) is the preferred method for cattle treatment at importation inspection stations and for treatment of cattle
before movement within or from quarantined premises (Davey et al.
2003).
Organophosphate (OP) pesticides function by inhibition of
acetylcholinesterase (AChE), an enzyme essential to the function of
central nervous systems for both invertebrate and vertebrate
organisms. OP resistance in insects is often conferred by point mutations in a gene encoding AChE, resulting in production of an altered
AChE enzyme that is insensitive to inhibition (Lee and Bantham
1966, Fournier and Mutero 1994). Initial discovery of genes encoding AChE in ticks and searches for mutations conferring OP resistance failed to identify mutations responsible for production of
AChE insensitive to inhibition by OPs. Subsequent studies revealed
that ticks possess at least three genes encoding AChEs that differ
substantially in amino acid sequence and biochemical properties,
with BmAChE1, BmAChE2, and BmAChE3 exhibiting calculated
Km values of approximately 5–10, 35–40, and 90–100 mM, respectively (Temeyer et al. 2004a, 2010). Quantitative reverse transcription polymerase chain reaction and gene silencing studies indicated
that all three tick AChEs were expressed in the tick synganglion and
that they were capable of functionally complementing one another
(Temeyer et al. 2011, 2012a, 2013a). Further, all three tick AChEs
were present at amplified copy number in genomic DNA and multiple transcripts were simultaneously expressed for each of the
AChEs, providing an explanation for why searches for mutations
conferring resistance had initially been unsuccessful (Temeyer et al.
Published by Oxford University Press on behalf of Entomological Society of America 2016.
This work is written by US Government employees and is in the public domain in the US.
1
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The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is the most economically important
cattle ectoparasite in the world. Rhipicephalus microplus and Rhipicephalus annulatus (Say) continue to
threaten U.S. cattle producers despite eradication and an importation barrier based on inspection, dipping of
imported cattle in organophosphate (OP) acaricide, and quarantine of infested premises. OP acaricides inhibit
acetylcholinesterase (AChE), essential to tick central nervous system function. Unlike vertebrates, ticks possess
at least three genes encoding AChEs, differing in amino acid sequence and biochemical properties. Genomic
analyses of R. microplus and the related tick, Ixodes scapularis, suggest that ticks contain many genes encoding
different AChEs. This work is the first report of a salivary cholinesterase (ChE) activity in R. microplus, and
discusses complexity of the cholinergic system in ticks and significance of tick salivary ChE at the tick–host
interface. It further provides three hypotheses that the salivary ChE plausibly functions 1) to reduce presence of
potentially toxic acetylcholine present in the large bloodmeal imbibed during rapid engorgement, 2) to modulate the immune response (innate and/or acquired) of the host to tick antigens, and 3) to influence transmission
and establishment of pathogens within the host animal. Ticks are vectors for a greater number and variety of
pathogens than any other parasite, and are second only to mosquitoes (owing to malaria) as vectors of serious
human disease. Saliva-assisted transmission (SAT) of pathogens is well-known; however, the salivary
components participating in the SAT process remain to be elucidated.
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Materials and Methods
Tick Strains and Tissues
The R. microplus susceptible strain Deutch was maintained at the
Cattle Fever Tick Research Laboratory (Edinburg, TX) as described
previously (Temeyer et al. 2007). Tick nymphs were collected using
forceps from a patch enclosing the ticks on an infested animal approximately 2 weeks following infestation. Nymphs that molted
into unfed adults were separated by sex. Unfed adult females were
dissected to remove salivary glands and synganglia or microinjected
with acetylthiocholine for toxicity studies and for collection of saliva. Excised salivary glands were placed in isotonic phosphatebuffered saline (PBS), pH 7.5, and stored on ice before salivary
extract preparation. Salivary extract was prepared by grinding 20
pairs of tick salivary glands in 250 ml final volume of PBS and collection of supernatant after centrifugation (3 min, 20,800 g) to remove particulate material.
Microinjection of Unfed Adult Ticks and Collection of
Saliva
Glass needles were pulled from 25-ml glass micropipettes using a
Narishige PC-10 micropipette puller (Narishige Group USA, East
Meadow, NY), according to the manufacturer’s instructions.
Needles were broken from the glass capillary, inspected under a dissecting microscope, and epoxied onto blunt needles of gas-tight
10-ml Hamilton syringes (model 1702SN 26/0.5/PT3, Hamilton
Company, Reno, NV), taking care to avoid any air pockets at the
joint between the glass and metal needle. Unfed adult female ticks
were placed ventral side up onto double-sided sticky pads mounted
on a micromanipulator, and injected with 0.005–0.500 ml acetylthiocholine (various dilutions in isotonic PBS, pH 7.5) by positive displacement (no air in system) using a calibrated timer-activated
screw-driven solenoid (Model M, ISCO Inc., Lincoln NE), as shown
in Fig. 1. Saliva was collected from microinjected ticks by placement
of a glass capillary over the hypostome.
Acetylthiocholine Toxicity
Toxicity of acetylthiocholine to unfed adult female R. microplus
(average weight 2.5 mg) was investigated by microinjection (with
minimal tissue damage) of dilutions of acetylthiocholine (81 mM
stock solution) at the lateral axilla between the first and second or
between the second and third legs. Injections of 30 or 300 nI of 100fold dilution series of acetylthiocholine stock solutions (810 nM to
81 mM) were performed to give final concentrations (based on
2.5 mg wt of uninjected ticks) of from 1 nM to 10 mM. Injected ticks
were placed in packets and incubated at 28 C and 92% humidity, as
described for the larval packet assay (Miller et al. 2002), and packets were monitored at 1, 2, 3, 4, and 8 h. Mortality was recorded
after 24 h. Control injections with water alone resulted in no
mortality.
AChE Microplate Assay
AChE activity and biochemical characterization in tick saliva or extracts of salivary glands or synganglia were determined in microplate assays using triplicate samples as described previously
(Temeyer et al. 2010), except that data for calculation of Km and
Vmax were analyzed and plotted by nonlinear regression using
GraphPad Prism ver. 5.0 (GraphPad Prism, Inc., La Jolla, CA).
Comparison of salivary gland extract with recombinant BmAChE1
utilized 5 ll per well of either salivary gland extract or 500-fold dilution (in PBS) of BmAChE1 baculoviral supernatant (Temeyer et al.
2010). Substrate preference for acetylthiocholine versus butyrylthiocholine in tick salivary gland extracts was tested at a final substrate
concentration of 100 lM.
Results
Acetylthiocholine delivered to unfed female ticks by microinjection
resulted in rapid tick paralysis at final equivalent concentrations of
1–10 mM, while lesser concentrations resulted in slowed movement
and rapid onset of copious salivation. After 3 h, approximately 50%
of ticks exposed at calculated internal concentrations of 1 mM acetylthiocholine exhibited normal movement and 50% moved very
slowly with progression to 20% mortality at 4 h, 30% mortality by
8 h, and 60% mortality at 24 h. Acetylthiocholine at final equivalent
concentrations of 100 mM or less resulted in minimal (5%) mortality at 24 h post-injection, while 100% mortality occurred at 10 mM.
At 24 h post-injection, dead ticks appeared desiccated and were frequently “cemented” to the packet by their mouthparts in similar
fashion as ticks killed by exposure to coumaphos at the IC50 concentration (data not shown).
AChE assay of tick saliva, or extracts prepared from excised synganglia or salivary glands all exhibited cholinesterase activity using
acetylthiocholine as substrate. The available sample of tick saliva
was very limited, but yielded an apparent Km for acetylthiocholine
of 10-12 mM. AChE activity was verified in salivary gland extracts
by substrate preference for acetylthiocholine (AcSCh) over butyrylthiocholine, exhibiting 44-fold preference for AcSCh. Total AChE
activity was approximately 2.4-fold higher in synganglial extract
compared with extract from an equivalent number of paired salivary
glands. AChE activity in tick salivary gland extract was compared
with that of recombinant BmAChE1 expressed in baculoviral supernatant. As shown in Fig. 2, the relative response to increasing substrate concentrations was very similar for recombinant BmAChE1
and salivary gland extract. Calculated Km values for acetylthiocholine were 8.755 mM and 13.62 mM for recombinant BmAChE1 and
salivary gland extract, respectively; however, the 95% confidence
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2011). Genotyping surveys identified multiple mutations associated
with resistance for each of the three tick AChEs, and baculoviral expression and biochemical characterization of recombinant AChEs
have to date confirmed OP-resistance mutations in at least two of
the three tick AChEs (Temeyer et al. 2007, 2009, 2012b, 2013b).
The genome of the related hard tick, Ixodes scapularis, is available,
and appears to contain many genes that putatively encode AChEs
(Temeyer et al. 2013a). In addition, the R. microplus genome also
appears to contain many genes that putatively encode AChEs
(Bellgard et al. 2012). The present work reports a salivary cholinesterase (ChE) activity in R. microplus, and discusses the complexity
of the cholinergic system in ticks and the functional significance of
tick salivary ChE at the tick–host interface. It is the first report of
tick salivary ChE and provides three hypotheses that tick salivary
ChE may plausibly function 1) to reduce presence of potentially
toxic acetylcholine present in the extremely large bloodmeal
imbibed during rapid engorgement, 2) to potentially modulate the
immune response (innate and/or acquired) of the host to tick antigens and subsequent exposures, and 3) to influence tick transmission
and establishment of pathogens to the host. The last hypothesis is
significant because ticks are vectors for a greater number and variety
of pathogens than any other parasite, and are second only to mosquitoes (owing to malaria) as vectors of serious human disease.
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Fig. 2. Comparison of R. microplus acetylcholinesterase activity in salivary
gland extract with recombinant BmAChE1. The plot shows increasing substrate (acetylthiocholine) concentration on the X-axis versus initial velocity
(DOD/min) on the Y-axis. Solid lines are best fit curves.
intervals were 7.1–17.1 and 12–20.1, indicating that the Km values
were not significantly different from one another. The previously reported Km values for BmAChE2 and BmAChE3 are quite different
from that of BmAChE1 (Temeyer et al. 2011). These results suggest
that although of unknown identity, the ChE activity present in tick
saliva exhibits kinetic properties similar to BmAChE1.
Discussion
Acaricidal activity of organophosphate is attributed to inhibition of
AChE and subsequent accumulation of the neurotransmitter acetylcholine causing overstimulation and failure of the tick nervous system resulting in death. This paradigm would predict that parenteral
administration of acetylcholine in sufficient concentration to overwhelm the available AChE should exhibit toxic effects on ticks.
Microinjection of acetylthiocholine appeared to produce an equivalent toxicological profile as that observed to result from organophosphate intoxication (i.e., dose-dependent response, with dead
ticks exhibiting apparent desiccation and attachment to packets resulting from excessive salivation). The reported Km values for bovine
erythrocyte AChE (140 mM acetylthiocholine, Mendelson et al.
1998; 220 mM acetylthiocholine, Montenegro et al. 2009) are 1020-fold higher than that of tick ChE present in saliva or salivary extract of R. microplus (10–14 mM acetylthiocholine). During rapid
engorgement, ticks ingest several hundred times their unengorged
body weight in host blood in a 3-5-d period, with most of the blood
intake occurring in the final day of rapid engorgement, suggesting
that one function of tick salivary AChE may be to detoxify the host
bloodmeal by hydrolyzing potentially toxic levels of acetylcholine
(Fujii et al. 1995) imbibed during rapid engorgement. Interestingly,
bedbugs have also been reported to contain AChE as a significant
component of their saliva (Francischetti et al. 2010, Seong et al.
2011).
In addition to a plausible role of tick salivary ChE for detoxification of the bloodmeal, the prolonged attachment of ticks to the host
may suggest another potentially significant role for tick salivary
ChE. Tissue damage and introduction of “foreign” material into the
host during and subsequent to tick attachment would be expected to
activate an innate immune and inflammatory response by the host
(Francischetti et al. 2009). Phospholipids and other materials
released from damaged tissues would be converted to proinflammatory eicosanoids, arachidonic acid, and prostaglandins.
Dendritic cells and other immune sentinel cells would initiate an inflammatory response, including release of cytokines and other
immunostimulatory molecules to increase blood flow (vasodilation)
and recruit various cellular components of the immune system to the
wound site and to guide appropriate generation of subsequent innate
and acquired components of the immune response (Saeed et al.
2005). Tick saliva contains a myriad of immunomodulatory components (Mathias et al. 2011, Kot
al et al. 2015) that work together to
modify innate and adaptive host immune response to allow
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Fig. 1. Photographic depiction of microinjection setup for R. microplus. Upper left, complete setup with microinjector module, Hamilton syringe, dissecting microscope, ticks on micromanipulator. Upper right, ticks on micromanipulator. Lower left, size of unfed adult ticks. Lower right, microscopic view of microinjection.
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In addition, chronic inflammation is a key component of many
serious diseases, including Alzheimer’s disease, amyotrophic lateral
sclerosis, multiple sclerosis, Parkinson’s disease, myasthenia gravis,
arthritis, asthma, as well as cardiovascular disease (Shytle et al.
2004, Nizri and Brenner 2013). Parasitic cholinergic capitalization
on complexities of vertebrate neural-immune integration may provide a “Rosetta Stone” for further deciphering regulatory pathways
and mechanisms controlling vertebrate immune response and regulation of inflammation.
In summary, this is the first report of tick salivary ChE and provides three hypotheses that the salivary ChE plausibly functions 1)
to reduce presence of potentially toxic acetylcholine present in the
extremely large bloodmeal imbibed during rapid engorgement, 2) to
potentially modulate the immune response (innate and/or acquired)
of the host to tick antigens and subsequent exposures, and 3) to influence tick transmission and establishment of pathogens to the
host. The last hypothesis is significant because ticks are vectors for a
greater number and variety of pathogens than any other parasite,
and are second only to mosquitoes (owing to malaria) as vectors of
serious human disease. Elucidation of the effects of tick cholinesterase in altering the responses of specific immune cell populations to
various activating factors is suggested as a promising area for further
study. We would welcome immunological collaboration to further
investigate the hypotheses presented in this study.
Acknowledgments
We thank Kristie Schlechte, Dave Krska, and Jason Tidwell for technical support and help with tick dissection. USDA is an equal opportunity provider
and employer. This work was funded by USDA appropriated funds (CRIS #
6205-32000-031-00D and 3094-32000-036-00). A.P. Tuckow was supported
by a USDA, ARS, Postdoctoral Research Program award to K.B. Temeyer.
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maintenance of blood availability (maintain vasodilation), and inhibition of wound closure and healing. The anti-inflammatory nature of systemic release of acetylcholine resulting from vagal nerve
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2006, 2008). The injection of tick salivary cholinesterase at the tick
attachment site would be expected to substantially reduce local concentrations of acetylcholine, thereby altering the relative activity of
nicotinic and muscarinic acetylcholine receptors, hypothetically affecting production and release of various cytokines, interferons,
interleukins, TNF, etc., from activated immune cells. There is substantial evidence supporting the integration of cellular cholinergic
signaling systems with other cellular pathways controlling immune
system function (Razani-Boroujerdi et al. 2007, Rosas-Ballina et al.
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competence of many immune component cell populations suggests
that these cells have the capacity to maintain homeostatic local concentration of acetylcholine, thereby providing a limitation on response to changes in acetylcholine receptor activation and
preventing runaway immune responses to temporal immune stimulation. Localized presence of tick salivary ChE possessing a significantly lower Km than mammalian AChE would therefore be
expected to prevent return to homeostatic balance as long as the tick
ChE remained present, suggesting an experimental protocol for future experiments, although choice and separation of immune cell
subsets may be critical to identify regulatory changes in production
of cytokines, interferons, TNFs, or other immunoregulatory products. It is therefore reasonable to conclude that tick salivary cholinesterase likely works together with other immunomodulatory
components of tick saliva to modify the resulting immune response
of the host. In particular, the reduced local concentration of acetylcholine might be expected to support the pro-inflammatory vasodilation response, helping to maintain availability of host blood to
the feeding tick.
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components have not yet been identified. Therefore, a possible additional consequence of the proposed immunomodulation by tick salivary ChE may be the enhancement of pathogen transmission and
establishment within the host.
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