Discontinuous Ventilation in a Non-Insect, The Tick Amblyomma Marmoreum (Acari, Ixodidae): Characterization and Metabolic Modulation

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 arthropod 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 arthropods 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 More relevant to natural conditions, in an ambitious experiment similar to Kestler’s (1978, 1980, 1985 and unpublished) pioneering measurements of water loss rate during insect ventilation, Rudolph and Knülle (1979) demonstrated intermittent bouts of rapid mass loss in female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 (Amblyomma variegatum), 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 –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).

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 animal 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.

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 min⁻1 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 production were converted to μl CO₂ h⁻1 for analysis. Areas under CO₂ bursts were determined by integration against time in hours, yielding volume in μl. 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.

Means are accompanied by sample sizes (N) and standard deviations. Regressions were 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 are summarized in Tables 1–4. In all cases, (and ) were very low compared to values expected from similar-sized insects or spiders (see Discussion). The dramatic changes of mass and between life-stages are summarized in Table 5.

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.

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.

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.

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).

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 and DVC frequency [ANCOVA: P (same slope and intercept) >0.05]. However, in both sexes the modulation of burst CO₂ release volume by revealed an interesting discontinuity (Fig. 5). At less than approximately 0.03 μl CO₂ mg⁻1 h⁻1, burst volume fluctuated apparently chaotically, frequently reaching rather large values, and was not correlated with (r²<0.05). Above 0.03 μl CO₂ mg⁻1 h⁻1, however, burst volume increased linearly with while remaining at values well below those characteristic of lower (Fig. 5). In flat nymphs, DVC frequency was also modulated by ⁠, but at a much shallower slope [4.54 vs 8.99mHz(μl CO₂ mg-⁻1 h⁻1)⁻1].

Burst duration was also affected by (Fig. 6). At high ⁠, burst duration was fairly constant at approximately 200s in both males and females, while at low it was usually considerably longer and more variable (see also Table 3). As a consequence of constant burst duration and increasing burst volume at high ⁠, the rate of CO₂ emission during the burst increased at high (Fig. 7). At low however, there were large fluctuations in burst that did not correlate with fluctuations in overall (Fig. 7).

Plainly, the major physiological response to decreased is an decrease in DVC frequency (Fig. 4) and this effect must be eliminated before any more subtle modulation, such as that of burst ⁠, can be characterized. This can be done by examining the relationship between burst-phase and the residuals of the –DVC frequency relationship at low SMR (Fig. 4). Using this approach, a strong trend appears: as the magnitude of unexplained by DVC frequency increases, so does burst (Fig. 8). This demonstrates modulation of burst with overall even at low metabolic rates.

This effect is also revealed by regression analysis of the low- data. By itself, DVC frequency explains only 12% of variance; burst by itself explains only 11%. However, by multiple regression, DVC frequency and burst together explain 47% of variance at low Burst has more explanatory power than DVC frequency in this range (t=893 for burst for DVC frequency), suggesting that, at low SMR, is modulated to a greater extent by burst than by DVC frequency. For all measurements, DVC frequency and burst together explain 88% of variance in total ⁠, but this is not a large increase from the 84% explained by DVC frequency alone over the full range of measured (Fig. 4).

In flat nymphs, burst also increased with overall (slope=4.13, r²=0.67, P<0.0001). Accounting for DVC frequency by multiple regression improved the percentage of explained from 67 to 80%.

As increased in adult flat ticks, inter-burst 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 was so low that it was difficult to measure with confidence (see Table 1).

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 differ from that of insects? And fourth, how widespread is discontinuous ventilation in non-insect arthropods? Not all of these questions can be answered definitively at this stage, but some initial inferences can now be made.

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 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 (Lighton and Wehner, 1993), so CO₂ emission between bursts was detectable. The fact that no unambiguous increase in 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 ant Cataglyphis bicolor, for instance, CO₂ was still released at a rate of 14% of overall during the unambiguous C phase (Lighton and Wehner, 1993; see also Lighton et al. 1993) and at approximately 11% of overall 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 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.

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.

To accommodate changing ⁠, 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 of about 0.03 μl mg⁻1 h⁻1. Below that limit (which could be called the SMR threshold, where SMR is standard metabolic rate), DVC frequency is correlated with but burst volume is not; however, the rate of CO₂ emission during the burst phase significantly increases with increasing after the effects of DVC frequency have been eliminated. Above the SMR threshold, both DVC frequency and burst volume are modulated by (with the latter remaining rather small) but burst 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 . 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 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 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 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.

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 beetles 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.

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

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.