J. exp. Biol. 180, 229-245 (1993) 229 Printed in Great Britain © The Company of Biologists Limited 1993

DISCONTINUOUS VENTILATION IN A NON-, THE MARMOREUM (, ): CHARACTERIZATION AND METABOLIC MODULATION

JOHN R. B. LIGHTON Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA LAURA J. FIELDEN Department of Zoology, University of the Witwatersrand, Private Bag 3, Wits 2050, Republic of South Africa and YIGAL RECHAV* Department of Microbiology, Medical University of South Africa, PO Medunsa 0204, Republic of South Africa

Accepted 17 March 1993

Summary We examined and quantified the discontinuous ventilation cycle (DVC) characteristics of unfed nymphs and adults, as well as engorged nymphal and engorged diapausing and non-diapausing female adult life-stages, of the African tortoise tick Amblyomma marmoreum (Koch). All engorged stages ventilated continuously, with little evidence of active spiracular control. Unfed nymphs and adults ventilated discontinuously; at low activity and standard metabolic rate (SMR) levels, mean DVC duration was approximately 0.4h in nymphs (mean mass 0.7mg) and 2.8h in female adults (mean Ϫ1 Ϫ1 mass 70mg). SMR, measured as rate of CO2 production (V˙CO∑; 0.064 ␮l mg h and 0.019 ␮l mgϪ1 hϪ1, respectively), was almost tenfold lower than that estimated for spiders of equivalent mass. In adults, the DVC was modulated to accommodate changing V˙CO∑ chiefly by changes in DVC frequency. Modulation of other DVC characteristics was bimodal; at low V˙ CO∑ (below the ‘SMR threshold’), burst volumes were large and not correlated with V˙CO∑, but the rate of CO2 emission during the burst was modulated by V˙CO∑. Above the SMR threshold, burst volumes were small and tightly correlated with V˙CO∑. No fluttering-spiracle phase could be detected, but CO2 bursts were triggered at low volumes above the SMR threshold, suggesting that hypoxia in addition to hypercapnia may initiate the termination of DVCs in the burst phase, rather than initiating the flutter phase as in . To explain this bimodal modulation of the DVC by V˙CO∑ (and hence V˙O∑) above and below the SMR threshold, we hypothesize that, below the SMR threshold, unfed ixodid – with their very low SMR and large surface area/volume ratio – may obtain significant amounts of O2 by transcuticular or other putative non- spiracular avenues of O2 uptake (larval ticks obtain all their O2 in this way).

*Present address: Department of Biology, University of Bophuthatswana, Private Bag X 2046, Mmabatho 8681, Republic of South Africa.

Key words: Amblyomma marmoreum, tick, ventilation, gas exchange, metabolism. 230 J. R. B. LIGHTON, L. J. FIELDEN and Y. RECHAV

Introduction Apart from their impressive versatility as disease vectors, ticks have interested physiologists for many reasons, foremost among which is their capacity to withstand long periods between moulting and feeding. Ticks are ‘gorging–fasting’ organisms (Wharton, 1978) that usually spend more than 90% of their lives between blood meals, and can survive longer than any other without food or drinking water (for a review, see Needham and Teel, 1991). Their net water loss rates are accordingly very low, probably because of a cuticular lipid barrier (see Sonenshine, 1991) and the ability of many species to sequester water from subsaturated air (Knülle and Devine, 1972; Rudolph and Knülle, 1974). Stringent spiracular control is also presumably important in restricting water loss. Ticks are unusual among tracheate in possessing only one pair of spiracles (see Sonenshine, 1991, for a description). These spiracles are very complex and many aspects of their structure and function remain obscure or controversial, in particular their opening and closing mechanisms (Sonenshine, 1991; Knülle and Rudolph, 1982) and the possible role of the spiracular plate in retarding water loss (Pugh et al. 1988; but see Pugh et al. 1990). However, like insect spiracles, tick spiracles open at high CO2 concentrations (Mellanby, 1935). If the spiracles are forced to stay open in this fashion, overall water loss rates increase considerably (Hefnawy, 1970; Rudolph, 1976). More relevant to natural conditions, in an ambitious experiment similar to Kestler’s (1978, 1980, 1985 and unpublished) pioneering measurements of water loss rate during insect ventilation, Rudolph and Knülle (1979) demonstrated intermittent bouts of rapid mass loss in female ticks (), which they assumed to be synchronous with spiracular opening episodes. They also demonstrated that isolated spiracles held in Ringer’s solution will open and close at predictable CO2 concentrations. This amounts to strong inferential evidence of discontinuous CO2 release, roughly equivalent to the insect discontinuous ventilation cycle or DVC, in a non-hexapod arthropod. In this paper we confirm the existence of discontinuous CO2 release in a tick (Amblyomma marmoreum Koch) by direct measurement and characterize its variation across two of the tick’s three active life stages (the first or larval stage of most ticks lacks tracheae altogether and presumably ventilates by diffusion; Needham and Teel, 1986). Amblyomma marmoreum is a hard-bodied or ixodid tick. Almost all tick disease vectors belong to the family Ixodidae, which differ from soft-bodied or argasid ticks chiefly in possessing a hardened dorsal scutum and the habit of feeding only three times in their lives, with some exceptions. After emerging from the egg, the larval ixodid tick feeds once on a primary host, then drops off and moults into a nymphal tick, which also feeds just once before again dropping off the host. The nymphal tick moults into a ‘flat’ or pre-feeding adult, which searches out prey, generally quite specific to the species (the previous two stages are more catholic in host selection). Once on the host, the adult female tick becomes highly engorged, feeding from a blood pool that gathers beneath her barbed mouthparts, which are set into the host with a secreted cement. Finally, the engorged female drops off, after having consumed as much as 8cm3 of blood (Balashov, 1972), and, after an optional diapause in many species, produces large quantities of eggs – Tick ventilation 231

38000 or more in some Amblyomma species (Dipeolu and Ogunji, 1980) – which are expelled from the anterior of her idiosoma or unsegmented body. She then dies. Amblyomma marmoreum is a typical representative of its genus, being larger than most ixodid ticks and considered to be close to ancestral forms that evolved in the late Paleozoic to early Mesozoic eras, when reptiles first appeared (see Hoogstraal, 1985). Indeed, Amblyomma marmoreum, like many of its congeners, still attacks reptiles. Although larvae and nymphs exhibit wide host preferences (such early hosts may be very mobile and assist in the dispersion of the species) adults are almost invariably ectoparasites of tortoises (Norval, 1975; Fielden et al. 1992). Females may, as in other Amblyomma species, oviposit soon after dropping from the host (typical egg production approximately 12000; Fielden et al. 1992). If temperatures are high, as in mid-summer, a variable delay or diapause may occur before oviposition (Fielden et al. 1992).

Materials and methods Ticks used in this investigation were from laboratory stocks (flat stages) or were collected from or in the vicinity of tortoises (Geochelone pardalis) maintained in an care facility at the Biology Department of the Medical University of South Africa. All appropriate animal care regulations were followed. Ticks were transferred to a laboratory at the Physiology Department of the University of Witwatersrand Medical School in Johannesburg for measurement. Engorged nymphs and non-diapausing engorged adults had dropped from their hosts 2–4 days prior to measurement. Diapausing engorged females had dropped from their hosts approximately 30–45 days before measurement. Respirometry We employed flow-through respirometry utilizing a Sable Systems TR-2 respirometry system (Sable Systems, 476 E South Temple, Salt Lake City, UT 84111), respirometers of 5–20cm3 volume, computer-controlled baselining, and flow rates of 50–100cm3 Ϫ1 min STPD. All H2O and CO2 was scrubbed from the incurrent air stream by a Drierite/Ascarite/Drierite column, and the excurrent air was dried by a low-volume magnesium perchlorate scrubber before passing through the CO2 analyzer. Details of the techniques have been published elsewhere (Lighton, 1990, 1991a,b, 1992; Lighton et al. 1993). We monitored flat ticks for 10–20h at a temporal resolution of 5–10s. Engorged ticks ventilated essentially continuously and could be characterized fully in a single recording of 40min duration at a temporal resolution of 1s. Computer records of CO2 Ϫ1 production (V˙CO∑) were converted to ␮l CO2 h for analysis. Areas under CO2 bursts were determined by integration against time in hours, yielding volume in ␮l. Respiratory quotients (RQ) were determined by a closed-system technique described elsewhere (Lighton, 1991a). All measurements were made in an air-conditioned laboratory at 23±1˚C. Statistics Means are accompanied by sample sizes (N) and standard deviations. Regressions were 232 J. R. B. LIGHTON, L. J. FIELDEN and Y. RECHAV determined by least squares, with significance testing by analysis of variance (ANOVA), and compared by analysis of covariance (ANCOVA). Means were compared using Student’s t-test.

Results

Results are summarized in Tables 1–4. In all cases, V˙CO∑ (and V˙O∑) were very low compared to values expected from similar-sized insects or spiders (see Discussion). The dramatic changes of mass and V˙CO∑ between life-stages are summarized in Table 5.

Ventilation characteristics None of the engorged nymphs or adults ventilated discontinuously (Fig. 1). Ventilation of the engorged stages was somewhat variable, with a coefficient of variation (CV, which is standard deviation divided by mean) of about 0.3 over an interval of approximately 40min. However, variations in ventilation were chaotic and of short duration, and CO2 never dropped below about 25% of the mean value. In contrast, the CV of discontinuously ventilating arthropods is generally in the range of 2–3 (see, Lighton, 1990, Fig. 1) and, between bursts, CO2 release can drop to values almost indistinguishable from zero. V˙CO∑ decreased significantly and CV increased significantly during diapause in engorged adult females (Table 4, Fig. 1), although ventilation remained apparently chaotic and still could not be described as discontinuous.

Table 1. Rate of CO2 emission (V˙CO∑), burst CO2 volume (burst VCO∑), rate of burst CO2 emission (burst V˙CO∑), inter-burst V˙CO∑ (IBV˙CO∑), DVC frequency (DVCF), RQ and estimated rate of O2 uptake (V˙O∑) of flat nymphal and engorged nymphal stages of the tick Amblyomma marmoreum Flat nymphs Engorged nymphs Mass (mg) 0.694±0.052 97.4±21.0* N 6 5 -1 V˙CO∑ (␮lh ) 0.0431±0.0209 12.73±4.33* - 1 - 1 V˙CO∑ (␮l mg h ) 0.0642±0.0362 0.129±0.033* CV 3.197±1.969 0.328±0.162* -1 Burst VCO∑ (␮lg ) 20.81±6.30 - - 1 - 1 Burst V˙CO∑ (␮l mg h ) 0.408±0.182 - Burst length (s) 195.3±45.1 - - 1 IBV˙CO∑ (␮lg ) 0.0143±0.0204 - DVCF (mHz) 0.695±0.264 - RQ - 0.923±0.108 - 1 - 1 V˙O∑ (␮l mg h ) - 0.140±0.036

The sample sizes are the number of ticks measured, each representing the mean of 2–10 measurements, depending on the tick’s life-stage and ventilation characteristics. Note that the nymphs cannot be sexed. DVC variables are not given for engorged nymphs because they ventilated continuously. Significantly different means between columns (P<0.01) are denoted by an asterisk. Tick ventilation 233

Table 2. Rate of CO2 emission (V˙CO∑), burst CO2 volume (burst VCO∑), rate of burst CO2 emission (burst V˙CO∑), inter-burst V˙CO∑ (IBV˙CO∑), DVC frequency (DVCF), RQ and estimated rate of O2 uptake (V˙O∑) of flat adult male and flat adult female stages of the tick Amblyomma marmoreum Flat adult males Flat adult females Mass (mg) 61.0±8.9 70.2±1.9* N 7 5 -1 V˙CO∑ (␮lh ) 1.411±0.743 2.927±1.357* - 1 - 1 V˙CO∑ (␮l mg h ) 0.0234±0.0132 0.0420±0.0198 -1 Burst VCO∑ (␮lg ) 28.7±16.2 40.84±23.0 - 1 - 1 Burst V˙CO∑ (␮l mg h ) 0.252±0.137 0.381±0.125 -1 IBV˙CO∑ (␮lg ) 4.83±2.74 7.82±3.19 Burst length (s) 483.5±261.3 350.1±112.5 DVCF (mHz) 0.318±0.289 0.585±0.486 RQ 0.669±0.074 0.826±0.072* - 1 - 1 V˙O∑ (␮l mg h ) 0.0350±0.0197 0.0508±0.0240

The sample sizes for V˙CO∑ and DVC are the number of ticks measured, each representing the mean of 2–10 measurements. The above means are for all measurements. For measurements corresponding to minimal metabolic rate (see text), see Table 3. Significantly different means between columns (P<0.05) are denoted by an asterisk.

Table 3. Rate of CO2 emission (V˙CO∑), burst CO2 volume (burst VCO∑), rate of burst CO2 emission (burst V˙CO∑), inter-burst V˙CO∑ (IBV˙CO∑), rate of O2 uptake (V˙O∑) and DVC frequency (DVCF) of flat adult male and flat adult female stages of the tick Amblyomma marmoreum Flat adult males Flat adult females Mass (mg) 61.0±8.9 70.2±1.9* N 7 5 -1 V˙CO∑ (␮lh ) 0.823±0.386 1.334±0.228* - 1 - 1 V˙CO∑ (␮l mg h ) 0.0137±0.0061 0.0190±0.0033 -1 Burst VCO∑ (␮lg ) 32.1±15.3 50.2±15.1* - 1 - 1 Burst V˙CO∑ (␮l mg h ) 0.247±0.126 0.419±0.107* -1 IBV˙CO∑ (␮lg ) 3.28±1.87 5.52±1.65 Burst length (s) 542.4±239.4 406.8±63.4 DVCF (mHz) 0.104±0.065 0.137±0.073 - 1 - 1 V˙O∑ (␮l mg h ) 0.0205±0.091 0.0230±0.0040

The sample sizes for V˙CO∑ and DVC are the number of ticks measured, each representing the mean of 1–10 measurements. The above means correspond to minimal metabolic rates (at or below the ‘SMR threshold’; see Discussion). V˙O∑ is estimated from the RQ figures in Table 2. Significantly different means between columns (P<0.05) are denoted by an asterisk. 234 J. R. B. LIGHTON, L. J. FIELDEN and Y. RECHAV

Table 4. Rate of CO2 emission (V˙CO∑), RQ and estimated rate of O2 uptake (V˙O∑) of engorged adult female and diapausing engorged adult female stages of the tick Amblyomma marmoreum Engorged Diapausing engorged adult female adult female Mass (mg) 3273±679 2601±659 N 12 5 -1 V˙CO∑ (␮lh ) 257.9±79.7 141.9±74.6* - 1 - 1 V˙CO∑ (␮l mg h ) 0.0789±0.0173 0.0558±0.0334 RQ 0.841±0.102 - - 1 V˙O∑ (␮lh ) 306.7±94.8 174.3±91.6* - 1 - 1 V˙O∑ (␮l mg h ) 0.0938±0.0206 0.0685±0.0410

The sample sizes for V˙CO∑ and DVC are the number of ticks measured, each representing one recording of approximately 40min duration. DVC variables are not given because the ticks ventilated continuously. V˙O∑ of diapausing ticks was estimated from the RQ of freshly engorged ticks. Significantly different means between columns (P<0.05) are denoted by an asterisk.

0.4

0.3

0.2 0.1

0 0.4

0.3 0.2 0.1

0 0 5 10 15 20 25 30 Time (min) Fig. 1. Ventilation characteristics of an engorged adult female Amblyomma marmoreum, mass 3.496g, 5 days after dropping off its host tortoise (top trace) and of a diapausing engorged Amblyomma marmoreum female, mass 3.374g, 30 days after dropping from its host (bottom Ϫ1 trace). V˙CO∑ of the non-diapausing female is 336.8 ␮lh , with a coefficient of variation (CV) of 0.07; that of the diapausing female is 126.4 ␮lhϪ1, with a CV of 0.28.

All of the flat (i.e. non-engorged) life stages of Amblyomma marmoreum ventilated discontinuously (Figs 2, 3). Ventilation frequency was very low, approximately 0.1mHz at very low activity levels (Table 3), corresponding to a mean DVC duration of approximately 2.8h in flat adult female ticks. Tick ventilation 235

Table 5. Factorial increases in mass, V˙CO∑ and mass-specific V˙CO∑, relative to the preceding unfed (flat) state, of engorged nymphs, engorged adult females and diapausing adult females of Amblyomma marmoreum Engorged Diapausing engorged Engorged nymphs adult females adult females Mass 140.3 46.6 37.1 V˙CO∑ 295.0 193.3 106.4 V˙CO∑ 2.0 4.2 2.9

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 - 0.1 0 1 2 3 4 Time (h)

Fig. 2. Ventilation characteristics of an unfed (flat) nymphal Amblyomma marmoreum, mass Ϫ1 0.71mg. Mean V˙CO∑ is 0.040␮lh , with a CV of 2.53. The DVC frequency is 0.56mHz.

Discontinuous ventilation was maintained even during activity in adult flat ticks (Fig. 3; very fast DVCs, those lasting less than 10min, were not analyzed, as they occurred during activity). V˙CO∑ primarily modulated DVC frequency to accommodate increased demands for CO2 release (Fig. 4). Male and female flat ticks did not differ significantly in the relationship between V˙CO∑ and DVC frequency [ANCOVA: P (same slope and intercept) >0.05]. However, in both sexes the modulation of burst CO2 release volume by V˙CO∑ revealed an interesting discontinuity (Fig. 5). At V˙CO∑ less than approximately Ϫ1 Ϫ1 0.03 ␮l CO2 mg h , burst volume fluctuated apparently chaotically, frequently 2 reaching rather large values, and was not correlated with V˙CO∑ (r <0.05). Above Ϫ1 Ϫ1 0.03 ␮l CO2 mg h , however, burst volume increased linearly with V˙CO∑ while remaining at values well below those characteristic of lower V˙CO∑ (Fig. 5). In flat nymphs, DVC frequency was also modulated by V˙CO∑, but at a much shallower slope [4.54 vs Ϫ1 Ϫ1 Ϫ1 8.99mHz(␮l CO2 mg h ) ]. Burst duration was also affected by V˙CO∑ (Fig. 6). At high V˙CO∑, burst duration was fairly constant at approximately 200s in both males and females, while at low V˙CO∑ it was 236 J. R. B. LIGHTON, L. J. FIELDEN and Y. RECHAV

120 Inactive Active 100

80

60

40

20

0 0 4 8 12 16 20 Time (h) Fig. 3. Ventilation characteristics of a flat adult female Amblyomma marmoreum, mass Ϫ1 70.2mg. V˙CO∑ is 1.58 ␮lh , with a CV of 5.57, while inactive. The DVC frequency while inactive is 0.061mHz. During activity, the DVC frequency increases to 1.22mHz and the Ϫ1 V˙CO∑ increases to 11.04␮lh , with a CV of 1.90.

2.0

1.5

1.0

0.5

0 0 0.03 0.06 0.09 0.12 - 1 - 1 V˙CO∑ (␮lmg h )

Fig. 4. The relationship between V˙CO∑ and DVC frequency in flat adult Amblyomma marmoreum. N=5 females and 7 males, with 2–10 DVCs per individual, total DVCs=62. Males and females do not differ significantly in this relationship (ANCOVA: P>0.05). The overall regression equation is DVCF=Ϫ0.147+18.98V˙CO∑, where DVCF is DVC frequency in Ϫ1 Ϫ1 2 mHz and V˙CO∑ is in ␮l mg h [r =0.84, F (1,60)=308, P<0.0001]. The intercept has a standard error of 0.246 and does not differ significantly from 0. usually considerably longer and more variable (see also Table 3). As a consequence of constant burst duration and increasing burst volume at high V˙CO∑, the rate of CO2 emission during the burst increased at high V˙CO∑ (Fig. 7). At low V˙CO∑, however, there were large fluctuations in burst V˙CO∑ that did not correlate with fluctuations in overall V˙CO∑ (Fig. 7). Tick ventilation 237

0.12

0.09

0.06

0.03

0 0 0.03 0.06 0.09 0.12 - 1 - 1 V˙CO∑ (␮lmg h )

Fig. 5. The relationship between burst CO2 release volume and V˙CO∑ in flat adult Amblyomma marmoreum. N=5 females and 7 males, with 2–10 DVCs per individual, total DVCs=62. Note the abrupt discontinuity in the relationship at V˙CO∑»0.03 ␮l mgϪ1 hϪ1. Below this value, burst volumes are variable and large, and are not correlated with V˙CO∑ (P>0.2). Above this value, burst volumes are tightly correlated with V˙ CO∑ (regression line shown); BV˙CO∑=0.0059+0.103V˙CO∑, where BVCO∑ is in ␮l mgϪ1 and V˙CO∑ is in ␮l mgϪ1 hϪ1 [r2=0.56, F(1, 22)=28.3, P<0.0001].

1200

900

600

300

0 0 0.03 0.06 0.09 0.12 - 1 - 1 V˙CO∑ (␮lmg h )

Fig. 6. The relationship between V˙CO∑ and burst duration in flat adult Amblyomma marmoreum. N=5 females and 7 males, with 2–10 DVCs per individual, total DVCs=62. Note Ϫ1 Ϫ1 the abrupt discontinuity in the relationship at V˙CO∑»0.03 ␮l mg h .

Plainly, the major physiological response to decreased V˙CO∑ is an decrease in DVC frequency (Fig. 4) and this effect must be eliminated before any more subtle modulation, such as that of burst V˙ CO∑, can be characterized. This can be done by examining the relationship between burst-phase V˙CO∑ and the residuals of the V˙CO∑–DVC frequency relationship at low SMR (Fig. 4). Using this approach, a strong trend appears: as the 238 J. R. B. LIGHTON, L. J. FIELDEN and Y. RECHAV

0.8

0.6

0.4

0.2

0 0 0.03 0.06 0.09 0.12 - 1 - 1 V˙CO∑ (␮lmg h )

Fig. 7. The relationship between V˙ CO∑ and rate of CO2 emission during the burst phase in flat adult Amblyomma marmoreum. N=5 females and 7 males, with 2–10 DVCs per individual, Ϫ1 Ϫ1 total DVCs=62. Note the abrupt discontinuity in the relationship at V˙ CO∑»0.03 ␮l mg h .

0.8

0.6

0.4

0.2

0 - 0.02 - 0.01 0 0.01 V˙CO∑ vs DVC frequency residuals

Fig. 8. The relationship between burst rate of CO2 emission and the variation in V˙CO∑ not explained by DVC frequency (equivalent to the residuals of the regression of V˙CO∑ against DVC frequency) in flat adult Amblyomma marmoreum with V˙ CO∑<0.03 ␮l mgϪ1 hϪ1 (i.e. less than the SMR threshold; see text). N=5 females and 7 males, with 2–10 DVCs per individual, total DVCs=62. The relationship reveals an upward modulation of burst CO2 emission rate with increasing V˙CO∑; BV˙ CO∑=0.364+20.2RV˙CO∑, where BV˙CO∑ is burst CO2 emission rate in ␮l mgϪ1 hϪ1 and RV˙CO∑ is the residuals of the regression of V˙ CO∑ against DVC frequency [r2=0.53; F(1, 34)=38.1; P<0.0001].

magnitude of V˙CO∑ unexplained by DVC frequency increases, so does burst V˙CO∑ (Fig. 8). This demonstrates modulation of burst V˙CO∑ with overall V˙CO∑ even at low metabolic rates. This effect is also revealed by regression analysis of the low-V˙CO∑ data. By itself, DVC frequency explains only 12% of V˙ CO∑ variance; burst V˙CO∑ by itself explains only 11%. However, by multiple regression, DVC frequency and burst V˙CO∑ together explain 47% of Tick ventilation 239

0.020

0.015

0.010

0.005

0 0 0.03 0.06 0.09 0.12 - 1 - 1 V˙CO∑ (␮lmg h )

Fig. 9. The relationship between V˙CO∑ and rate of CO2 emission during the inter-burst phase of the DVC in flat adult Amblyomma marmoreum. N=5 females and 7 males, with 2–10 DVCs per individual, total DVCs=62. IBV˙CO∑=0.0029+0.111V˙CO∑, where IBV˙CO∑ is inter-burst rate Ϫ1 Ϫ1 2 of CO2 emission in ␮l mg h and V˙CO∑ has the same units [r =0.59; F(1, 60)=87.3; P<0.0001].

V˙CO∑ variance at low V˙ CO∑. Burst V˙CO∑ has more explanatory power than DVC frequency in this range (t=893 for burst V˙CO∑ vs t=22 for DVC frequency), suggesting that, at low SMR, V˙CO∑ is modulated to a greater extent by burst V˙CO∑ than by DVC frequency. For all V˙CO∑ measurements, DVC frequency and burst V˙CO∑ together explain 88% of variance in total V˙CO∑, but this is not a large increase from the 84% explained by DVC frequency alone over the full range of measured V˙CO∑ (Fig. 4). 2 In flat nymphs, burst V˙CO∑ also increased with overall V˙CO∑ (slope=4.13, r =0.67, P<0.0001). Accounting for DVC frequency by multiple regression improved the percentage of explained V˙CO∑ from 67 to 80%. As V˙CO∑ increased in adult flat ticks, inter-burst V˙CO∑ increased as well (Fig. 9). By ANCOVA, no significant difference existed between the sexes in this relationship. A similar relationship was also found in flat nymphs, but with a higher slope (slope=0.45, r2=0.64, N=19 measurements on six nymphs, P<0.0001). In the latter case, however, inter-burst V˙CO∑ was so low that it was difficult to measure with confidence (see Table 1).

Discussion Discontinuous ventilation We have presented unambiguous proof of discontinuous ventilation in a tracheate, non- insect arthropod. This raises four main questions. First, is the detailed mechanism of discontinuous ventilation in ixodid ticks (assuming Amblyomma marmoreum to be typical of ixodids) different from that in insects? Second, does discontinuous ventilation in ticks serve the same (putative) function of reducing respiratory water loss as it does in insects? Third, how does the modulation of the DVC to accommodate changing V˙CO∑ differ from that of insects? And fourth, how widespread is discontinuous ventilation in 240 J. R. B. LIGHTON, L. J. FIELDEN and Y. RECHAV non-insect arthropods? Not all of these questions can be answered definitively at this stage, but some initial inferences can now be made.

The nature of discontinuous ventilation in ticks

In terms of discontinuous CO2 release, and the presence of a prolonged period during which CO2 is emitted at a very low rate, tick and insect discontinuous ventilation are superficially similar. However, a clear difference appears to exist in terms of the fluttering-spiracle (F) phase. In V˙CO∑ recordings made from most insects, an F phase is unambiguously visible. Such F phases manifest themselves via their diffusive component; even in the presumed presence of a trans-spiracular pressure gradient, the large CO2 concentration gradient across the spiracles leads to significant outward diffusion of CO2 (see Lighton, 1988a,b, 1990). Equally, only inward diffusion of O2 can be detected in a flow-through respirometry system; removal of O2 by bulk flow, which preserves the ratio of O2:N2, cannot be detected because it is functionally equivalent merely to a reduction in flow rate. It is therefore relatively easy to detect an F phase with a significant diffusive component by using flow-through CO2 or (animal size permitting) O2 respirometry. A highly efficient (low-diffusion) F phase, however, is very difficult to detect by flow-through respirometry. A continuous low inter-burst emission of CO2 does occur in ticks, similar to that described in insects during the constricted-spiracle (C) phase, and similarly related to overall V˙ CO∑ (Lighton and Wehner, 1993), so CO2 emission between bursts was detectable. The fact that no unambiguous increase in V˙ CO∑ occurred during the inter-burst period therefore strongly suggests that, if an F phase exists in ticks, it must be highly efficient because it cannot be unambiguously detected via diffusive loss of CO2 through the spiracles. Alternatively, perhaps the entire inter-burst period is equivalent to an F phase. If this is the case, then the F phase in ticks must indeed be unusually efficient; in the xeric Cataglyphis bicolor, for instance, CO2 was still released at a rate of 14% of overall V˙CO∑ during the unambiguous C phase (Lighton and Wehner, 1993; see also Lighton et al. 1993) and at approximately 11% of overall V˙CO∑ during the inter-burst period in ticks. Such efficiency demands the presence of a substantial trans-spiracular pressure gradient, and it seems unlikely that such a gradient can form immediately after the conclusion of the burst phase. On the basis of present evidence, therefore, we conclude that ticks may not exhibit a fluttering-spiracle (F) phase of the kind observed in insects. If they exhibit an analogous phenomenon, then it is remarkably efficient. Possibly, diffusive uptake of O2 through the cuticle between burst phases, in addition to internal stores, may be sufficient for non-active respiration for prolonged periods, although regular release of CO2 is still necessary. Other putative avenues of O2 uptake may include the salivary recirculation implicated in atmospheric water uptake in some ixodid ticks (Needham and Teel, 1986). If some form of low-capacity extra-spiracular O2 uptake (ESOU) is utilized, then the standard metabolic rate of ticks must be remarkably low relative to that of other arthropods (see below). It is perhaps significant in this context that ixodid flat ticks have, by virtue of their flatness, a large surface area compared to a more conventional arthropod of equivalent mass. Although the dorsoventral flattening of unfed ticks may be advantageous with respect to Tick ventilation 241 squashing-resistance and/or the ability to enter suitable microhabitats, it is plainly disadvantageous in terms of increased surface area, and hence increased cuticular water loss rates, relative to a more conventional cylindrical or ovoid body plan. Furthermore, the integument of most ixodid ticks is characterized by very extensive epicuticular folds (Hackman and Filshie, 1982), the function of which is uncertain but may include an enhanced capacity for cuticular expansion after a blood meal. It is clear that, whatever their ultimate function, these folds must significantly increase the surface area of the integument and presumably its capacity for diffusive gas exchange.

Respiratory water loss reduction In view of the measurements of Rudolph and Knülle (1979) on Amblyomma variegatum, and equivalent measurements on insects (Kestler, 1978, 1980, 1985 and unpublished; Lighton, 1992; Lighton et al. 1993), there can be little doubt that discontinuous ventilation in ticks serves an important role in the overall water economy of adult and nymphal ticks during the period – unpredictable and sometimes very long – between moulting and feeding. It is perhaps significant that these are the only life-stages that appear to ventilate discontinuously. Especially in insects with low cuticular permeabilities, peak rates of respiratory water loss during periods of spiracular opening can exceed cuticular water loss rates by twofold or more (Lighton et al. 1993). The same is probably true of ticks. The exact magnitudes of respiratory water loss rates in ticks, and whether ticks differ from insects in terms of CO2 emission efficiency (H2O:CO2 loss ratio) during their burst phase, are currently unknown.

DVC modulation to accommodate V˙ CO∑

To accommodate changing V˙CO∑, adult flat Amblyomma marmoreum primarily modulated DVC frequency (Fig. 4). The same technique is used by some insects, e.g. the xeric ant Cataglyphis bicolor (Lighton and Wehner, 1993). However, other insects modulate both DVC frequency and burst volume (Lighton, 1988b). Interestingly, A. marmoreum appears to switch DVC regulation tactics at a threshold V˙CO∑ of about 0.03 ␮l mgϪ1 hϪ1. Below that limit (which could be called the SMR threshold, where SMR is standard metabolic rate), DVC frequency is correlated with V˙CO∑ but burst volume is not; however, the rate of CO2 emission during the burst phase significantly increases with increasing V˙CO∑ after the effects of DVC frequency have been eliminated. Above the SMR threshold, both DVC frequency and burst volume are modulated by V˙CO∑ (with the latter remaining rather small) but burst V˙CO∑ is not as clearly modulated. Why should ticks have a bimodal ventilation strategy? Clearly, above the SMR threshold the tick is releasing CO2 in far smaller amounts than it can sequester at low SMR (Fig. 5), thus presumably allowing it more control over burst volume as a function of V˙CO∑. This suggests that a factor other than hypercapnia is triggering the burst phase above the SMR threshold. It is probable that this factor is hypoxia. If (and only if) significant quantities of O2 can enter the tick via ESOU, then an increase in V˙O∑ above the SMR threshold will reduce the relative importance of ESOU in the tick’s overall O2 budget to the point where hypoxia precedes hypercapnia. Under these circumstances, the 242 J. R. B. LIGHTON, L. J. FIELDEN and Y. RECHAV tick will open its spiracles primarily to obtain O2 rather than to release CO2. This may explain the abrupt ventilation–modulation switch in burst volumes and rates of CO2 emission above and below the SMR threshold, which may correspond to the V˙O∑ at which the tick can obtain significant quantities of O2 by transcuticular diffusion or other putative mechanisms of ESOU. In insects, hypoxia causes the initiation of the F phase (Levy and Schneiderman, 1966); in ticks, it may instead initiate full spiracular opening (i.e. a burst phase) equivalent to that seen in insects (and ticks; Mellanby, 1935) during hypercapnia. These hypotheses are all readily testable.

Discontinuous ventilation in non-insect arthropods In view of our findings, it could be argued that discontinuous ventilation is likely to be the rule among tracheate arthropods. However, the situation is not so clear. Certainly, many insects ventilate discontinuously, but some do not (e.g. crickets and some and grasshoppers; J. R. B. Lighton, unpublished data; M. Quinlan, personal communication; Hadley and Quinlan, 1993). Several excellent non-insect, tracheate arthropod candidates for discontinuous ventilation exist, but have attracted practically no attention from physiologists to date. Their presence in predominantly xeric areas strongly suggests that they have developed effective control over respiratory water loss rates.

100 Spiders 10

1 Ticks 0.1

0 0.1 1 10 100 1000 Mass (mg)

Fig. 10. A comparison of tick and spider metabolic rates, expressed as V˙O∑ at 25˚C. Spider data (open circles) are from Anderson (1970), Anderson and Prestwich (1982) and Greenstone and Bennett (1980). Where ambient temperatures differed from 25˚C, values were corrected to 25˚C assuming a Q10 of 2.0. Non-feeding (flat) larval, nymphal and adult tick data (filled circles) are from this investigation, and Belozerov (1964, 1966). Raw regression lines are shown. By ANCOVA, the ticks and spiders share a common slope or mass scaling exponent [0.768; F(1, 58)=0.29, P (same slope) >0.3] but the ticks have a far lower intercept or mass scaling coefficient [16.37 vs 145.1 for spiders; F(1, 59)=61.5, P (same intercepts) <0.0001]; 0.768 i.e. the allometric equation relating V˙O∑ to body mass in ticks at 25˚C is 16.37M , while for 0.768 Ϫ1 spiders it is V˙O∑=145.1M , where V˙O∑ is in ␮lh and M is body mass in mg. The difference in intercepts means that ticks have, on average, a standard metabolic rate of only 11.3% that of spiders of equivalent mass. Tick ventilation 243

Plainly, discontinuous ventilation is proving to be more common and more widespread than imagined when it was first described in diapausing lepidopteran pupae.

Standard metabolic rate Our suggestion that ESOU may play a significant role in ixodid tick gas exchange is somewhat heterodox, even though larval ticks (which are admittedly tiny) are thought to employ this technique. We lack the required information on actual tick surface areas, cuticular O2 permeabilities, salivary recirculation volumes, etc., to test this hypothesis using data presented here. However, in view of the fact that adult hexapods of equivalent body mass are not thought to make significant use of ESOU, the putative ability of flat ticks to do so is obviously contingent on a metabolic rate well below that of an equivalent- sized hexapod, in addition to considerations of mechanism. In fact, a highly conservative test of tick metabolic rates is to compare ticks not with hexapods but with spiders which, as sit-and-wait predators, are generally thought to have unusually low metabolic rates (Anderson, 1970; Anderson and Prestwich, 1982; Greenstone and Bennett, 1980). Such a comparison is shown in Fig. 10. The metabolic rates of the flat stages of ticks are highly significantly lower than predicted on the basis of body mass from data on spiders. On average, the metabolic rates of ticks are only 11.3% of that of spiders of equivalent mass. In view of this finding, the role of unorthodox avenues of O2 uptake may indeed be significant in at least some species and tracheate life-stages of ticks.

J.R.B.L. thanks the South African Foundation for Research Development for funding his visit and gratefully acknowledges support from the US NSF (grant BSR 9006265) during data analysis and the writing of this paper; he also thanks Professor Gideon Louw, Professor Duncan Mitchell, Ian and Margaret Grant and especially Dr Rochelle Buffenstein for their kind hospitality in Johannesburg and Dr Frances Duncan for expert assistance with data acquisition.

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