Biological and Physiological Characterization of in Vitro

Journal of Insect Physiology 75 (2015) 73–79

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Journal of Insect Physiology

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Biological and physiological characterization of in vitro blood feeding in nymph and adult stages of Ornithodoros turicata (Acari: Argasidae) q ⇑ Hongyuan Zheng a,b, Andrew Y. Li a, , Pete D. Teel c, Adalberto A. Pérez de León a, Janakiram Seshu d, Jingze Liu b a USDA-ARS Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX 78028, USA b Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology of Hebei Province, College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, China c Texas A&M AgriLife Research, Department of Entomology, Texas A&M University, College Station, TX 77843, USA d South Texas Center for Emerging Infectious Diseases and Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249, USA article info abstract

Article history: Biological and physiological aspects of blood feeding in nymph and adult Ornithodoros turicata were Received 5 September 2014 investigated using an in vitro technique combined with electrophysiological recordings and respirometry. Received in revised form 27 February 2015 The duration of blood feeding through a ParaﬁlmÒ membrane was similar (19.2–22.6 min) in both Accepted 4 March 2015 developmental stages. The mean (±SD) size of blood meal ingested by nymphs, females, and males Available online 14 March 2015 was 44.2 ± 17.9, 150.6 ± 48.7, and 74.2 ± 36.9 mg, respectively, representing a 2.5-, 2.8- and 3.0-fold increase from their respective unfed weights. Electrophysiological recordings of the pharyngeal pump Keywords: during blood feeding revealed that ticks ingested blood at a rate of 6.1–6.4 suctions per second. Mean Relapsing fever tick blood volume ingested per suction was 0.013 l in females and 0.007 l in both males and nymphs. Ornithodoros turicata l l 2 Blood feeding Blood meal size (mg) correlated with unfed body weight (mg) (r = 0.50, p < 0.05) and with blood volume 2 Pharyngeal pump ingested per suction (r = 0.71, p < 0.05). Unfed ticks exhibited a circadian ventilation rhythm with dis- Gas exchange continuous gas exchange pattern during the daytime and continuous pattern during nighttime. Mean Water loss _ À1 standard metabolic rates (SMR, VCO2 ) in unfed nymphs, females and males of 1.4, 3.0 and 0.9 llh increased to 2.0, 5.7 and 2.4 llhÀ1, respectively, after a blood meal. SMR correlated positively with blood meal size (r2 = 0.89, p < 0.05). Mean coxal ﬂuid weight excreted after a blood meal in nymphs, females, and males was 8.7, 20.0, and 7.7 mg, respectively, which represents 27.0%, 23.4% and 26.7% of their blood meal size. This study revealed biological and physiological characteristics of soft tick blood feeding and metabolism important to tick survival. Published by Elsevier Ltd.

1. Introduction Oklahoma, Texas and Florida; it is also found in central Mexico (Cooley and Kohls, 1944). O. turicata is a vector of Borrelia turicatae, Ornithodoros ticks (family Argasidae, also known as ‘soft ticks’) a spirochete responsible for tick-borne relapsing fever in humans are vectors of Borrelia spirochetes that cause tick-borne relapsing and dogs (Roscoe and Epperly, 2005; Whitney et al., 2007). This soft fever in the western United States (Schwan et al., 2009), and the only tick species is able to transmit ASFV in the laboratory (Butler and recognized biological vectors of African swine fever virus (ASFV) Gibbs, 1984; Hess et al., 1987). Life cycle characteristics of O. turi- (Hess, 1981; Vial, 2009; Ravaomanana et al., 2010; Boinas et al., cata, including the developmental stages as well as ovipositional 2011; de Carvalho Ferreira et al., 2014). The relapsing fever tick, behavior of adult females, have been previously described by Beck Ornithodoros turicata, is distributed in the United States, including et al. (1986). Although the basic biology and certain aspects of feed- California, Utah, Arizona, New Mexico, Colorado, Kansas, ing physiology have been reported in Ornithodoros tick species, including O. turicata (Kaufman et al., 1981; Kaufman and Sauer, 1982), the physiological mechanism of blood feeding and changes q This article reports the results of research only. Mention of a proprietary in metabolism associated with blood feeding remain to be studied. product does not constitute an endorsement or a recommendation by the USDA for Ticks have evolved efficient physiological mechanisms to regu- its use. The USDA is an equal opportunity provider and employer. late water balance and gas exchange, critical for survival in adverse ⇑ Corresponding author at: USDA, ARS, Invasive Insect Biocontrol and Behavior environmental conditions (Kestler, 1985; Hadley, 1994). Previous Laboratory, Beltsville Agricultural Research Center, 10300 Baltimore Avenue, Beltsville, MD 20705, USA. Tel.: +1 (301) 504 5401; fax: +1 (301) 504 5104. studies in Ixodid ticks demonstrated two distinct gas exchange E-mail address: [email protected] (A.Y. Li). strategies, described as discontinuous CO2 release in fasting and http://dx.doi.org/10.1016/j.jinsphys.2015.03.005 0022-1910/Published by Elsevier Ltd. 74 H. Zheng et al. / Journal of Insect Physiology 75 (2015) 73–79 continuous in engorged ticks (Rechav and Fielden, 1995; Fielden (diameter = 32 mm, length = 40 mm), one end of which was sealed et al., 1999). In Argasid ticks, water balance is maintained by water with a piece of stretched ParafilmÒ (American National Can, excretion through coxal organs, which functionally is homologous Neenah, WI), and a 6-well cell culture plate (Corning to a vertebrate filtration-resorption renal system (Kaufman, 2010). Incorporated, Corning, NY). Cattle blood collected from healthy Excess water is excreted from coxal glands during and/or shortly one-year old calves was mechanically defibrinated immediately after feeding depending on the species. Coxal fluid may contain before being stored at 4 °C. Culture plate wells were filled with tick-borne pathogens and provide a pathway for transmission to 3 ml of cattle blood and placed in a water-filled large glass Petri susceptible hosts (Gaber et al., 1984; Kleiboeker et al., 1998; dish maintained at 38 ± 1 °C over a heating plate. One feeding tube Lopez et al., 2011). The objective of this study was to characterize was placed into each culture plate well such that the membrane biological parameters and physiological mechanisms associated was slightly submerged in blood (1–2 mm below the blood level) with blood-feeding in nymph and adult stages of O. turicata. and ticks were individually placed to allow feeding through the membrane. 2. Material and methods 2.3. Measurements of volumes of blood meal intake and coxal fluid 2.1. Ticks secretion

Fifth instar nymphs and unfed adult O. turicata used in this Each tick was weighed before, and immediately after feeding to study were from a colony maintained at the Tick Research obtain weight of blood ingested. Blood feeding duration was deter- Laboratory, Texas A&M AgriLife Research, College Station, TX. The mined by recording the time from tick attachment to withdrawal colony was established from specimens collected from a natural from the membrane. Fed ticks were placed individually in glass cavern in Travis County, TX in 1992 and were maintained under vials, maintained under conditions described above, and weighed 14L:10D photoperiod, 25 ± 3 °C and 80–85% relative humidity. again at 24 h to measure weight loss as an estimate of coxal fluid Ticks were brought to the USDA, ARS, Knipling-Bushland U.S. secretion (McCoy et al., 2010). Livestock Insects Research Laboratory in Kerrville TX, where they were maintained at 25 ± 2 °C, 80 ± 5% relative humidity, and 2.4. Electrophysiological recording of feeding activity 12L:12D photoperiod prior to use. A fine silver wire electrode (0.076 mm diameter; A-M systems 2.2. In-vitro blood feeding Inc., Carlsborg, WA) was attached to the anterior dorsum of the tick using a small drop of nail enamel. A second silver wire electrode A previously reported ParafilmÒ based artificial blood feeding was placed in the blood well. The electrodes were connected to technique (Schwan et al., 1991) was modified to feed O. turicata an AC amplifier (A-M systems Inc. Carlsborg, WA). Upon tick (Fig. 1). Briefly, the feeding unit consisted of a clear plastic tube attachment to the membrane, an electric circuit was formed

Fig. 1. (A) The in vitro membrane feeding system, including a heating plate, a large glass Petri dish with water, a cell culture plate, and a feeding unit. Two thermometers were used to measure temperature in the water bath (yellow) and blood in the well (white). The insert shows the recording electrode glued to the back of the tick, and reference electrode placed in the blood. (B) An empty feeding unit (right) and a second one fitted with membrane (left). (C) Top view of the feeding unit placed in blood in a well on the cell culture plate soaked in warm water bath. (D) A nymph engorging on the membrane in the feeding unit. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) H. Zheng et al. / Journal of Insect Physiology 75 (2015) 73–79 75 permitting pharyngeal muscle contractions to be detected by the volume of blood ingested per suction, feeding duration as well as AC amplifier. Contraction signals were amplified 1000x and fed _ standard metabolic rate (SMR, VCO2 ), were analyzed using cor- to a CED digital data acquisition system with Spike 2 software _ relation matrices (95% confidence). Statistical comparisons of VCO (Cambridge Electronic Design Limited, Cambridge, England). The 2 between unfed and fed ticks of nymphs, adult females and males digitizer sampling frequency was 2 kHz, with low and high signal were also performed using t-test (p < 0.05). All statistical analyses filter setting at 10 Hz and 5 kHz, respectively. Contraction data were carried out using STATISTICA Version 6.0 (StatSoft, Tulsa, OK). were displayed in real time on a computer monitor and saved at the end of the experiment. Blood ingestion during membrane feeding was recognized as rhythmic waveforms with each peak 3. Results representing one pharyngeal suction (Li – unpublished). To calculate frequency of pharyngeal muscle contraction or suction 3.1. Characteristics of in vitro blood feeding and function of pharyngeal frequency during feeding, the number of suctions in 5 s was mea- pump sured for every 3 min for each individual tick in the group. The total number of suctions during the entire course of blood feeding All ticks tested in this study blood fed successfully. Results was estimated by multiplying suction frequency with feeding obtained with the modified in vitro feeding system are summarized duration. Blood volume ingested per suction was calculated by in Table 1. The mean weight of unfed adult females (39.9 ± 9.4 mg) dividing the total blood ingested (mg) by the total number of suc- was significantly higher than those of unfed nymphs tions. To calculate blood volume ingested by ticks that were ini- (12.6 ± 2.7 mg) and adult males (18.6 ± 5.9 mg) (p < 0.05). The tially measured by weight in the experiments, a mean blood mean body weights of fed nymphs, females, and males were weight/volume conversion rate (1.07 llmgÀ1) was estimated by 44.2 ± 17.9, 150.6 ± 48.7, and 74.2 ± 36.9 mg, respectively. measuring the weight of 0.1, 0.2, and 1 ml of cattle blood. Whereas mean blood feeding duration was similar (19.2– 22.6 min), unfed females ingested significantly more blood than unfed males and nymphs (p < 0.05), resulting in a 3.7-, 3.9-, and 2.5. Respirometry 3.5-fold increase in bodyweight, respectively. There was a significant correlation between amount of blood ingested and unfed body Unfed and newly blood-fed nymphs and adults were subjected weight (r2 = 0.50, p < 0.05) (Fig. 2). No correlation was found to respirometry analysis to estimate changes in metabolic rate and between mean blood meal size and feeding duration (p > 0.05). water loss associated with blood feeding. Engorged ticks were weighed immediately upon withdrawal from the membrane before being placed into the glass tube chamber. Simultaneous recordings Table 1 of CO emission and H O loss of an individual tick were achieved 2 2 Measurements of in vitro blood feeding through a ParafilmÒ based artificial blood by using a flow-through CO2/H2O analyzer (Model Li-7000, feeding system by 5th instar nymphs and adults of Ornithodoros turicata (mean ± SD). Li-COR, Inc., Lincoln, NE). Air flow was generated by an air pump Biological Nymphs (n) Females (n) Males (n) (Dyna-Pump Model #2; Neptune Products Inc., Dover, NJ) regu- parameter lated with an air flow controller (Matheson Tri-Gas, Inc., Basking Ridge, NJ) to sustain 35 ml minÀ1. Room air flowing into the pump Weight (mg) Unfed 12.6 ± 2.7 (10) 39.9 ± 9.4 (10) 18.6 ± 5.9 (12) was scrubbed of CO2 and H2O vapor by passing air through two Fed 44.2 ± 17.9 (10) 150.6 ± 48.7 (10) 74.2 ± 36.9 (12) Ò connecting columns of Drierite desiccant (W.A. Hammond Feeding duration 19.2 ± 10.2 (10) 19.9 ± 4.9 (9) 22.6 ± 8.6 (11)

Drierite Co. Ltd., Xenia, OH) and Ascarite II CO2 absorbent (min) (Thomas Scientiﬁc, Swedesboro, NJ) before clean air reached the Blood meal (mg) 31.5 ± 16.8 (10) 110.7 ± 46.3 (10) 55.7 ± 32.8 (12) Suction frequency 6.1 ± 0.5 (5) 6.4 ± 0.9 (6) 6.3 ± 0.6 (6) tick chamber. Clean air carried CO2 and water vapor released by (# sÀ1) the tick to downstream sensor detection and recording. Only one Volume per 0.007 ± 0.003 (5) 0.013 ± 0.003 (6) 0.007 ± 0.003 (6) tick was monitored for each 24 h respirometry run. suction (ll) Respirometry experiments were carried out at room temperature (27 ± 2 °C). The glass tube chamber containing the tick was placed inside a light-proof locomotion monitor with an infrared light source and a detector. Li-COR Li-7000 data acquisition software (Version 2.0.0) was programmed to collect CO2 and H2O measurements at 5 s intervals for 24 h. Data analysis was performed using ExpeData software (Sable Systems, Inc., Las Vegas, NV). The procedure for analyzing metabolic rate in ticks was described previously (Zheng et al., 2013). The parameters calculated and analyzed from the respiratory recordings include (1) standard _ _ metabolic rate VCO2 , (2) burst VCO2 , (3) burst duration and interburst interval for CO2 emission. Water vapor recordings were baseline-corrected and converted to mg hÀ1. Coxal ﬂuid volume was obtained by calculating the area of excretion peak. Each tick was also weighed before and after respiratory measurements to obtain amount of body mass loss during the 24 h recording.

2.6. Data analysis

Graphs were prepared using SigmaPlot software (Systat Software, Inc., San Jose, CA). Relationships between blood meal size Fig. 2. Relationship between the unfed body weight (mg) of Ornithodoros turicata (in mg) and various parameters related to blood feeding and coxal ticks (nymphs and adults) and the size (mg) of blood meal obtained through a fluid secretion, including unfed body weight, suction frequency, ParafilmÒ based artificial feeding system. 76 H. Zheng et al. / Journal of Insect Physiology 75 (2015) 73–79

Fig. 3. Electrophysiological recording of in vitro blood feeding of Ornithodoros turicata through artiﬁcial membrane (ParaﬁlmÒ), showing membrane penetration by mouthparts salivation-like activity, rhythmic pumping or suction of blood, and withdrawal of mouthparts after repletion.

nymphs and adults showed a circadian rhythm characterized by

a continuous gas (CO2) exchange pattern (CGEP) during the nighttime hours of the day alternating with a discontinuous exchange pattern (DGEP) during the daytime hours (Fig. 5A). In contrast, ele- ven out of ﬁfteen blood fed nymphs and adults demonstrated a more or less regular DGEP during the 24-h post blood-feeding period (Fig. 5B), while the rest showed a continuous ventilation pattern that lasted for over 18 h (data not shown). Measurements of

metabolic parameters associated with CO2 release are summarized in Table 2. The standard metabolic rate (SMR) was estimated as the _ À1 mean CO2 emission rate (VCO2 llh ) during the 24-h period. No signiﬁcant increase in SMR was observed after feeding in nymphs and females, while males demonstrated a signiﬁcantly increased _ VCO2 after the blood meal (p < 0.05; Table 2). All blood fed ticks showed a higher gas exchange than unfed ticks. The higher CO2 release frequency in blood fed ticks was accompanied by a shorter

CO2 burst duration and a shorter inter-burst interval (Table 2). The _ burst VCO2 remained unchanged in fed nymphs while fed adults Fig. 4. Relationship between volume (ll) of blood ingested per suction and blood showed only a slight increase when compared to those of the unfed meal size (mg) of nymph and adult Ornithodoros turicata fed using a ParaﬁlmÒ based membrane feeding system. ticks (Table 2). A signiﬁcant positive correlation was found between SMR and the blood meal size (r2 = 0.89; p < 0.05) (Fig. 6A).

Results of electrophysiological recording experiments showed 3.3. Body mass loss and coxal fluid excretion four distinct phases of in vitro blood feeding activity in O. turicata: the initial penetration of the paraffin membrane, salivation-like Mean weight loss recorded from unfed nymphs, males, and activities, rhythmic suctions of blood, and the withdrawal of females during 24 h of respirometry was 0.3, 0.2, and 0.4 mg, mouthparts upon repletion (Fig. 3). Each peak during rhythmic respectively (Table 3). Mean weight loss recorded from blood fed suctions represented a cycle of pharyngeal pump action. The mean nymphs, males, and females during 24 h of respirometry was rate of blood suction or pharyngeal pumping was similar, ranging 10.8, 9.7, and 29.7 mg, respectively (Table 3). The amount of coxal from 6.1 to 6.3 per second, among nymphs and adults tested fluid excretion after a blood meal during 24 h of respirometry,

(Table 1). The mean volume (0.013 ll) of blood ingested per suc- measured using the CO2/H2O analyzer, was 8.7, 7.7, and 27.0 mg tion in females was significantly higher than those (0.007 ll) of respectively for fed nymphs, males, and females (Table 3). nymphs and males (p < 0.05). Statistical analysis revealed a signifi- Elimination of excess water started soon after completion of blood cant correlation between mean blood meal size and blood volume feeding and lasted for hours (Fig. 5B). Coxal fluid excretion was ingested per suction (r2 = 0.71; p < 0.05) (Fig. 4). occasionally observed to occur after the 24 h period of recording in this study. Defecation was not observed from any of the fed ticks

3.2. Carbon dioxide (CO2) release during the 24 h recording period. Therefore, reduction of tick body weight during the 24 h post feeding period was due mainly to Unfed and fed nymphs and adults of O. turicata demonstrated coxal ﬂuid excretion. It is speculated that water loss through different gas (CO2) exchange patterns. Thirteen out of 15 unfed respiration (not measured in these experiments) also contributed H. Zheng et al. / Journal of Insect Physiology 75 (2015) 73–79 77

Fig. 5. Comparative gas exchange (CO2 release, red) and respiratory water loss (green) between (A) an unfed female and (B) a fed female of Ornithodoros turicata. The upper plates show 24 h recordings. Middle and lower plates in A show details of CO2 and H2O recordings over 1.5-h periods of the unfed female during the daytime (1.5–3.0) and nighttime (5.0–6.5) h, respectively. Middle and lower plates in B show details of the CO2 and H2O recordings over 1.5-h periods of the fed female during the daytime (0.0–1.5) and nighttime (10.0–11.5) hours, respectively. Note the large water peak in the middle plate (B) is coxal ﬂuid excretion. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Table 2 Ò Measurements (mean ± SD) of CO2 release before and after a blood meal by Ornithodoros turicata acquired through a Paraﬁlm based artiﬁcial blood feeding system.

Measurement Nymphs Females Males Unfed Fed Unfed Fed Unfed Fed

_ À1 1.4 ± 0.7 2.0 ± 0.9 3.0 ± 2.5 5.7 ± 3.5 0.9 ± 0.1a 2.4 ± 0.8b VCO2 (llh ) Animals showing DGEP/sample size 5/5 4/5 5/7 3/5 5/5 4/5 Cycle frequency (# hÀ1) 3.3 ± 1.4 5.2 ± 1.6 2.6 ± 1.1 4.5 ± 1.7 2.1 ± 0.9a 5.5 ± 0.7b _ À1 0.2 ± 0.0 0.2 ± 0.1 0.8 ± 0.8 1.3 ± 1.4 0.2 ± 0.1 0.4 ± 0.2 Burst VCO2 (llh ) Burst duration (min) 7.2 ± 1.5a 4.2 ± 0.8b 13.3 ± 9.0 8.0 ± 3.8 7.1 ± 1.3a 4.9 ± 0.6b Burst interval (min) 15.5 ± 9.6 10.7 ± 8.7 14.6 ± 9.4 8.3 ± 3.5 28.6 ± 10.1a 6.7 ± 0.9b

Means of unfed and fed in the same developmental stage followed by different letters differ significantly (p < 0.05). DGEP = discontinuous gas exchange pattern. to the overall body weight loss observed. Blood fed nymphs, 4. Discussion females, and males lost 21.9%, 20.9% and 18.5% their body weight, respectively during the 24 h immediately after feeding. The Ticks are extraordinary animals that attract much attention amount of coxal fluid excretion accounted for 27.0%, 23.4%, and because of their considerable medical and veterinary importance 26.7% of the blood meal for nymphs, females and males, respec- as well as successful biological and physiological adaptations tively (Table 3). Results of correlation analysis revealed a signifi- (Jongejan and Uilenberg, 2004; Sonenshine and Roe, 2014). Here cant linear relationship between the amount of coxal fluid we revealed new aspects of blood feeding in O. turicata that con- excretion (mg) and the blood meal size (r2 = 0.87; p < 0.05) tribute to our understanding of the physiological basis of blood (Fig. 6B). feeding and water balance in soft ticks. 78 H. Zheng et al. / Journal of Insect Physiology 75 (2015) 73–79

It is equally possible that ticks may have ingested more blood and therefore took longer to complete feeding in vivo than on an artificial feeding system. We are unable to test such hypothesis because blood meal size was not provided in the previous study (Beck et al., 1986). The rhythmic signal patterns recorded during tick feeding were produced from electric conductance of fluid/blood to open and close the pharyngeal pump. Each peak of blood ingestion waveforms (Fig. 3) represents one blood sucking cycle – the opening and closing of the pharyngeal pump. This observation has been previously verified in a similar work by these authors in the ixodid tick, Amblyomma americanum, in which each pharyngeal pump action was coupled with blood ingestion through a capillary tube (Li, unpublished data). The rate of pharyngeal pump activity (6/s) observed in O. turicata from the current study was also similar those observed in A. americanum. The other electric pattern lasting from 3 to 17 s observed prior to the start of rhythmic blood pumping activities occurred in 14 out of 20 tick preparations; we inter- preted such activities as ‘‘salivation’’ without attempting to observe actual salivation. Therefore, the designation of this pattern prior to the rhythmic ingestion pattern as ‘‘salivation’’ needs to be validated through further observation. The significant correlation between unfed weight and blood meal size in the current study concurs with observations reported for Ornithodoros hermsi (McCoy et al., 2010). Larger ticks consumed more blood. The pharyngeal pump was found to be highly efficient allowing ticks to make over 6 suctions per second during feeding. Blood feeding duration (19.2–22.6 min) and rate of suction (6.1– 6.3 per second) were similar between nymph and adult ticks, permitting the final blood meal size a tick can achieve to be estimated by pharyngeal pump efficiency (the volume of blood per pumping cycle or suction). The significant positive linear correlation between the blood meal size and blood volume pumped per suction provided direct evidence supporting such a hypothesis. Adult females were most efficient, with a pumping rate (0.013 ll/suction) twice as high Fig. 6. Relationship between blood meal size (mg) and standard metabolic rate as that of adult males and nymphs (0.007 ll/suction). À1 (CO2 llh ) (A), and between the blood meal size (mg) and the amount (mg) of Previous reports indicated that ventilation patterns in unfed coxal fluid excreted (B) in nymph and adult Ornithodoros turicata fed using a ixodid ticks are discontinuous (Fielden et al., 1994; Zheng et al., Ò Parafilm based membrane feeding system. 2013). The reduction in metabolic rate by starved ticks is consid- ered a strategy for conserving energy and minimizing water loss

Table 3 when hosts are not readily available (Needham and Teel, 1986), Weight loss of unfed and fed stages, and amount of coxal ﬂuid excretion of unfed and and DGEP was generally thought to be an adaptation to lower fed O. turicata (mean ± SD) recorded during 24 h respirometry period. metabolic rate (Lighton, 1998; Chown et al., 2006). Our ﬁndings

Measurement* Nymphs Females Males of alternating discontinuous and continuous gas exchange patterns corresponding to daytime and nighttime in O. turicata may reflect Weight loss (mg)a the subtle differences in host finding and feeding behaviors Unfed 0.3 ± 0.1 0.4 ± 0.3 0.2 ± 0.1 Fed 10.8 ± 9.4 29.7 ± 23.0 9.7 ± 4.1 between Argasid and Ixodid tick species. The observation of diurnal b Coxal fluid (mg) 8.7 ± 7.1 27.0 ± 21.0 7.7 ± 3.4 CO2 release patterns in O. turicata in our study may be indicative of Proportion of coxal fluid to blood meal 27.0 ± 5.9 23.4 ± 10.8 26.7 ± 7.4 daily activity patterns for this nidicolous tick species. Argas cooleyi, (%) a nocturnally active Argasid species, exhibited little locomotion * n = 5 for all measurements. activity during daylight (George, 1987). This activity pattern may a Measurements of water loss through coxal fluid excretion as determined by be highly beneficial to soft ticks parasitizing diurnal hosts, which direct measurement of body weight reduction. likely bed down or stay in their burrows at night. It is worth noting b Measurements of water loss through coxal fluid excretion as determined by that fed O. turicata, unlike fed ixodid ticks, tended to ventilate dis- using the CO2/H2O analyzer software. continuously, with a few exceptions. Given the multiplicity of nymphal instars as well as the multi-gonotrophic cycle of soft Beck et al. (1986) reported blood feeding for forth instar ticks, this strategy may be favorable for conserving energy when nymphs, adult females and males of O. turicata lasting an average the next blood meal is unpredictable. of 30.5, 68.8 and 34.9 min, respectively when fed on suckling mice. Higher SMR was observed in fed ticks due to the increase in Blood feeding was much longer than that observed by membrane metabolic expenditure relating to digestion of the blood meal. feeding and may be explained by additional time needed to pene- This conclusion is supported by the positive correlation observed trate host skin and secrete bioactive salivary factors to overcome between SMR and the blood meal size. The increase in CO2 output hemostasis and other host defense mechanisms to obtain a blood was achieved by an increase in gas exchange cycle frequency while meal (Waladde and Rice, 1982; Wikel, 2013). The combined use the burst volume remained virtually unchanged. In insects, the Ò of a Parafilm membrane with defibrinated blood may have accumulation of CO2 in hemolymph to a threshold level triggered enabled faster feeding by O. turicata in our in vitro feeding system. initiation of the burst phase and thus cycle frequency (Harrison H. Zheng et al. / Journal of Insect Physiology 75 (2015) 73–79 79 et al., 1995). Likewise in fed O. turicata, increase in metabolic George, J.E., 1987. Field observations on the life cycle of Ixodes baergi and some expenditure may also have resulted in a rapid CO accumulation seasonal and daily activity cycles of Oeciacus vicarius (Hemiptera: Cimicidae), 2 Argas cooleyi (Acari: Argasidae), and Ixodes baergi (Acari: Ixodidae). J. Med. in hemolymph, leading to an increase in the frequency of CO2 Entomol. 24, 683–688. release cycle. SMR values were significantly higher in fed males Hadley, N.F., 1994. Water Relations of Terrestrial Arthropods. Academic Press, than in the unfed counterparts. California, pp. 356. Harrison, J., Hadley, N., Quinlan, M., 1995. Acid-base status and spiracular control All post-larval stages of soft ticks excrete coxal fluid during or during discontinuous ventilation in grasshoppers. J. Exp. Biol. 198, 1755–1763. soon after blood meal to eliminate excess water and ions and to Hess, W.R., 1981. African swine fever: a reassessment (Virus strains). Adv. Vet. Sci. maintain internal osmotic equilibrium (Kaufman and Sauer, Comp. Med. 25, 39–69. Hess, W.R., Endris, R.G., Haslett, T.M., Monahan, M.J., McCoy, J.P., 1987. Potential 1982). We found all fed O. turicata initiated coxal fluid excretion arthropod vectors of African swine fever virus in North America and the within a few minutes after engorgement and produced copious Caribbean basin. Vet. Parasitol. 26, 145–155. amounts of coxal fluid in the first two hours. Estimates of coxal Jongejan, F., Uilenberg, G., 2004. The global importance of ticks. Parasitology 129, S3–S14. fluid excreted as a proportion of blood meal ingested (23.4– Kaufman, R.W., 2010. Ticks: physiological aspects with implications for pathogen 27.0%) in O. turicata are consistent with results reported in several transmission. Ticks Tick Borne Dis 1, 11–22. other soft ticks species, including Ornithodoros papillipes, 17–48% Kaufman, W.R., Sauer, J.R., 1982. Ion and water balance in feeding ticks: (Balashov, 1972); Ornithodoros moubata, 35% (Kaufman et al., mechanisms of tick excretion. In: Obenchain, F.D., Galun, R. (Eds.), Physiology of Ticks. Pergamon Press, Oxford, pp. 213–244. 1981); Ornithodoros erraticus, 20.0% (Khalil et al., 1986), and O. Kaufman, S.E., Kaufman, W.R., Phillips, J.E., 1981. Fluid balance in the argasid tick, hermsi, 24.1–35.8 (McCoy et al., 2010). The positive correlation Ornithodorus moubata, fed on modified blood meals. J. Exp. Biol. 93, 225–242. between blood meal size and coxal fluid suggested that a larger Kestler, P., 1985. Respiration and respiratory water loss. In: Hoffmann, K.H. (Ed.), Environmental Physiology and Biochemistry of Insects. Springer, pp. 137–186. blood meal increased water loss through coxal fluid secretion dur- Khalil, G.M., Helmy, N., Hoogstraal, H., El-Said, A., 1986. Female Ornithodoros ing this phase of osmoregulation. The only other likely source of (Pavlovskyella) erraticus (Acari: Ixodoidea: Argasidae): effect of feeding. J. Med. water vapor loss after a blood meal was respiratory water loss Entomol. 23, 380–383. Kleiboeker, S.B., Burrage, T.G., Scoles, G.A., Fish, D., Rock, D.L., 1998. African swine through spiracles. However, the amount water vapor loss from fever virus infection in the argasid host, Ornithodoros porcinus porcinus. J. Virol. spiracles was trivial compared to the volume of coxal fluid 72, 1711–1724. excreted. Lighton, J.R., 1998. Notes from underground: towards ultimate hypotheses of cyclic, discontinuous gas-exchange in tracheate arthropods. Am. Zool. 38, 483–491. Lopez, J.E., McCoy, B.N., Krajacich, B.J., Schwan, T.G., 2011. Acquisition and Acknowledgements subsequent transmission of Borrelia hermsii by the soft tick Ornithodoros hermsi. J. Med. Entomol. 48, 891–895. McCoy, B.N., Raffel, S.J., Lopez, J.E., Schwan, T.G., 2010. Blood meal size and The authors thank Darci Burchers for helping maintain the tick spirochete acquisition of Ornithodoros hermsi (Acari: Argasidae) during feeding. colony and two anonymous reviewers for critical review of the J. Med. Entomol. 47, 1164–1172. Needham, G.R., Teel, P.D., 1986. Water balance in ticks between blood meals. In: manuscript. H.-Y. Zheng was supported by a scholarship from Sauer, J.R., Heir, J.A. (Eds.), Morphology, Physiology and Behavioral Biology of China Scholarship Council. A.Y. Li and A.A. Pérez de León were Ticks. Ellis Horwood, Chichester, pp. 100–151. funded by United States Department of Agriculture (USDA), Ravaomanana, J., Jori, F., Andriatsimahavandy, A., Roger, F., Albina, E., Vial, L., 2010. 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