Ixodes rubicundus nymphs are short-day diapause-induced ticks with thermolabile sensitivity and desiccation resistance

Ixodes rubicundus nymphs are short-day diapause-induced ticks with thermolabile sensitivity anddesiccation resistance

L . J . F O U R I E * , V . N . B E L O Z E R O V ² and G . R . N E E D H A M ³

*Department of Zoology and Entomology, University of the Free State, South Africa, ²Laboratory of Entomology, Biological

Research Institute, St. Petersburg State University, St. Petersburg, Russia and ³Acarology Laboratory, Department of

Entomology, the Ohio State University, Columbus, U.S.A.

Abstract. The Karoo Paralysis tick, Ixodes rubicundus Neumann (Acari:

Ixodidae), is a semi-voltine ixodid that survives stressful environmental conditions

using morphogenetic diapause (eggs and engorged nymphs) and desiccation

resistance. Both photoperiod and temperature in¯uence diapause induction in the

engorged nymph. Ixodes rubicundus nymphs are typical long-day photoperiodic

organisms. The critical photoperiod is ~ 13.5 h light, 10.5 h dark, and they display

a thermolabile response. The period between detachment and apolysis in engorged

nymphs is modi®ed by photoperiod; however, apolysis to ecdysis is not affected by

photoperiod. Thus, initiation of development, but not the actual process is

controlled by photoperiod. Most engorged nymphs delayed metamorphosis when

exposed to short-day regimen (LD 12 : 12 h) after feeding. Nymphs exposed to

pre- and post-feeding long-day regimen (LD 14 : 10 h) developed. Times for 50%

of nymphs to apolyse when exposed to photoperiods of LD 14 : 10 h, 13.5 : 10.5 h,

13 : 11 h and 12 : 12 h were 28, 36, 40 and 58 days, respectively. Times for 50%

of engorged nymphs to ecdyse ranged from 38 to 40 days after apolysis. Nymphs

were sensitive to photoperiodic exposures before, during and after feeding. Nymphs

exposed to long day (LD 14 :10 h) before and during feeding, moulted at 20°C;

however, most exposed to 10°C followed by 20°C (post-feeding) went into

diapause. Both short- (10 : 14 h) and long- (14 : 10 h) day exposed engorged

nymphs survived 45 days at 0% r.h. (n = 73), but diapause-destined ticks kept at

13°C lost the least mass (29.5 6 9.5%, SD), while nondiapause ticks at 23°C lost

the most (48.7 6 8.2%, SD). Termination of diapause and transition to

development probably coincides with a de®nite increase of water vapour uptake by

engorged nymphs. Comparatively, I. rubicundus engorged nymphs are more

desiccation tolerant than a North-American counterpart, Amblyomma cajennense

(Fabricius) (Acari: Ixodidae), which is also semi-arid- to xeric-adapted. Diapause

conveys important survival attributes that enable engorged I. rubicundus nymphs to

inhabit a semi-arid environment with great temperature extremes, and to

synchronize their activity periods with seasons and host utilization patterns.

Key words. Ixodes rubicundus, diapause, tick nymphs, desiccation resistance,

South Africa.

Introduction

Ixodes rubicundus Neumann, the Karoo Paralysis tick, is

endemic to the Republic of South Africa (RSA). It is of great

veterinary and economic importance because it paralyses

Correspondence: Professor L. J. Fourie. Department of Zoology and

Entomology, University of the Free State, PO Box 339, Bloemfontein

9300, South Africa. E-mail: [email protected]

ã 2001 Blackwell Science Ltd 335

Medical and Veterinary Entomology (2001) 15, 335±341Medical and Veterinary Entomology (2001) 15, 335±341

domestic stock and wild ungulates (Stampa, 1959; Spickett &

Heyne, 1988; Fourie & Vrahimis, 1989; Fourie et al., 1992a).

The tick is distributed in the more semi-arid and xeric regions

of RSA (Theiler, 1950; Spickett & Heyne, 1988), where it

inhabits hilly or mountainous terrain, occurring in close

association with certain plant species (Stampa, 1959; Fourie

et al., 1991). The distribution range is characterized by winter

or equinoctial rainy seasons. Mean annual rainfall may vary

within 100±600 mm, and mild to severe droughts occur

periodically. Air temperatures show major diel and seasonal

¯uctuations. Maximum temperatures of 41°C may be reached

during summer, whereas minimum winter temperatures can be

down to ± 14°C. A range of 25°C between maximum and

minimum temperatures over a 24-h period is common (Fourie

& Horak, 1994).

The I. rubicundus life cycle, which extends over 2 years, is

synchronized with seasonal climatic changes through devel-

opmental diapause of eggs and engorged nymphs (Fourie &

Horak, 1994). Developmental diapause in ticks is observed as a

photoperiodically induced delay either in embryogenesis,

development of fed larvae or nymphs, or oogenesis of

engorged females (Belozerov, 1982). Temperature is also

important in the induction, modulation or abolishment of tick

diapause (Belozerov, 1982; Pegram et al., 1988; Korotkov &

Kislenko, 1995; Dautel & KnuÈlle, 1998). It was suggested that

temperature together with photoperiod control diapause induc-

tion in I. rubicundus nymphs (Fourie & Horak, 1994). Since

then photoperiod has been con®rmed as a critical element of

diapause induction and termination for nymphs of this tick

(Belozerov et al., 1996). Our study reports the impact of

different photoperiodic and temperature exposure regimes on

the development of engorged I. rubicundus nymphs. Taken

together, diapause and desiccation resistance help to explain

how an ixodid tick can survive harsh environmental conditions

of summer in RSA.

Materials and methods

All the nymphs used were the ®rst laboratory progeny of

engorged females collected from sheep in the south-western

Free State, RSA. Larvae and nymphs were fed on their natural

host, the rock elephant shrew, Elephantulus myurus Thomas &

Schwann (Fourie et al., 1992b) and maintained in the dark at

20°C and 93 6 2% r.h.

Determination of threshold photoperiod

Three- to four-week-old unfed nymphs were exposed for

3 weeks before feeding to one of ®ve different photoperiods

(LD 12 :12 h, 13 :11 h, 13.5 : 10.5 h, 14 : 10 h and

14.5 :9.5 h) at 20°C and 93 6 2% r.h. They were subsequently

fed on the shrews in an environmental room under an aperiodic

light (LD 24 :0 h). Engorged nymphs were divided into three

subgroups, which were either exposed to LD 14 :10 h,

12 : 12 h or total darkness at 20°C and 93 6 2% r.h. The

number of nymphs used varied from 18±23 to 63±64 for each

of the subgroups. Engorged specimens were held in ELISA

plates (3040 Microtest II Tissue Culture plate; 96 ¯at-bottom

wells) for easy observation (Belozerov et al., 1996). Small

ventilation holes were drilled into the lid and bottom of each

well. Nymphs were monitored over a period of 107 days for

the number of nymphs showing apolysis (separation of cuticle

from epidermal cells) and those that had moulted.

Sensitive phase

To determine how long nymphs are sensitive to photoper-

iodic stimuli, unfed nymphs (2±3 weeks old) were exposed to

a long-day photoperiodic regime (LD 14 : 10 h) before feed-

ing (for 2±3 weeks), before and during feeding, only during

feeding, and only after feeding (see Table 1 for exposure

regimens). During non-exposure periods before experiments

they were kept in ELISA plates (see above) in darkness at

20°C and 93 6 2% r.h. An interval of 80 days post-detach-

ment gives nymphs ample time to moult if they are not

diapause destined, so we quanti®ed the number of nymphs that

moulted for each subgroup within this selected period.

Effect of temperature

The possible effect of temperature on diapause induction

was tested on 3±4-week-old unfed nymphs, which were

divided into two groups consisting of ®ve subgroups each.

Groups were exposed to either 10 or 20°C for 14 days before

feeding. Each subgroup was exposed to a different photo-

periodic regime, namely LD 8 : 16 h, 10 : 14 h, 12 : 12 h,

14 : 10 h or 16 : 8 h. Specimens had been subsequently fed on

shrews (6±8 days) under the same temperature and photoper-

iodic conditions. After detachment the engorged nymphs

Table 1. Effect of long- (L) or short-day (S) exposures before,

during and after feeding, on the percentage Ixodes rubicundus, which

moulted 80 days after engorgement (L = LD 14 : 10 h;

S = LD 10 : 14 h; A = aperiodic light condition)

Before

feeding

During

feeding

After

feeding n Percent moult

A S A 40 18

A L A 40 55

S A A 88 8

L A A 49 55

A A S 40 15

A A L 194 64

S S A 36 3

L L A 49 71

A S S 40 9

A L L 172 88

L A L 55 89

ã 2001 Blackwell Science Ltd, Medical and Veterinary Entomology, 15, 335±341

336 L. J. Fourie et al.336 L. J. Fourie et al.

belonging to the different subgroups (n = 48±93) were placed

individually into an ELISA plate and kept in total darkness at

20°C and 93 6 2% r.h. The number of nymphs that moulted

during the 80-day observation period was recorded.

Effect of photoperiod, temperature and low relative humidity

on engorged nymphal mass

Randomly selected engorged nymphs from LD 14 : 10 h,

90 6 2% r.h. and 23°C were placed in ELISA plates then

exposed to either long (LD 14 : 10 h) or short (LD 10 : 14 h)

daylengths, and to either a high (23°C) or low (13°C)

temperature. They were held for 45 days at 0% r.h. (over

silica gel desiccant with colour indicator). Mass readings were

determined initially and on day 45 using a microbalance

(Mettler Toledo UMT2, weighs accurately to 0.1 mg). We are

reporting net mass losses, assuming that much of the loss was

via transpiration. At the end (45 days), individual ticks were

weighed and placed in a 50°C drying oven to determine

individual dry masses. Dry mass for an individual engorged

nymph was subtracted from its initial and ®nal live weights to

estimate net mass loss for that tick. Net loss was divided by

initial mass to obtain a percentage loss over 45 days of

desiccation at either 13 or 23°C. Because of the photoperiod

exposures we are assuming the ticks were destined to either

diapause (short-day length) or to develop (long-day length).

Neither apolysis nor moulting occurred during the experiment.

Some ticks from experiments on threshold photoperiod (see

above) were also monitored for their ability to absorb water

vapour from unsaturated air at 93 6 2% r.h. and 20°C. Ticks

were weighed using a Sartorius R200D balance initially

(5 days after detachment) and 10 weeks afterwards for mass

changes.

Fig. 1. The effect of different photoperiodic exposures before and

after feeding on percentage moult (within 80 days of detachment) of

engorged I. rubicundus nymphs.

Fig. 2. Number of days taken for 50% of engorged nymphs,

maintained at different photoperiods, to undergo apolysis or ecdysis.

Fig. 3. Percentage moult recorded for I. rubicundus nymphs

exposed to 10° and 20°C, respectively, and various photoperiodic

regimens before and during feeding.


Diapause induction in Ixodes rubicundus 337Diapause induction in Ixodes rubicundus 337

Results

Determination of threshold photoperiod

The effects of different photoperiodic exposures on I.

rubicundus nymphal moulting are graphically illustrated

(Fig. 1). Most (> 79%) of the nymphs exposed to a short-day

length (LD 12 : 12 h) after feeding, irrespective of the pre-

feeding photoperiodic regimen, displayed a delay in metamor-

phosis. A similar pattern was evident for nymphs held in

constant darkness after feeding. The only exceptions from this

group (51% moult) were those held in a pre-feeding regimen of

LD 14 : 10 h. Nymphs in a long-day photoperiod

(LD 14 : 10 h) after feeding generally displayed nondiapause

development, except those exposed to a pre-feeding photo-

period of 13 h or less. In the latter case less than 41% of the

nymphs moulted within 80 days (Fig. 1).

The time intervals in days for 50% of the nymphs to apolyse

at long-day photoperiod (LD 14 : 10 h) after engorgement, but

exposed to different light conditions prior to feeding, were: 28

(LD 14 : 10 h), 36 (LD 13.5 : 10.5 h), 40 (LD 13 : 11 h) and

58 (LD 12 : 12 h) days, respectively. The time period from

apolysis to ecdysis in 50% of the nymphs that moulted was 38±

40 days, irrespective of pre-feeding photoperiodic exposures

(Fig. 2). Thus, initiation of development, but not the actual

process, is controlled by photoperiod.

Sensitive phase

Nymphs were sensitive to photoperiodic exposures before,

during and after feeding. Exposures of nymphs to either short-

(LD 10 : 14 h) or long- (LD 14 : 10 h) day photoperiodic

regimes just while feeding (6±8 days) were suf®cient to

in¯uence diapause or nondiapause development (Table 1).

The extent of the diapause or nondiapause state, as re¯ected by

the percentage moult, was in¯uenced by exposures to either

short- or long-day photoperiods before or after feeding

(Table 1). The highest percentage moult (88±89%) was

recorded for nymphs exposed to long days (LD 14 : 10 h)

during and after feeding, or before and after feeding. The latter

fact con®rms that the long-day entrainment received by

nymphs before feeding is maintained during feeding in an

aperiodic regimen of darkness.

Effect of temperature

Results on the percentage moult for nymphs exposed to

various photoperiodic regimes at 10 and 20°C, respectively,

before and during feeding are presented graphically in Fig. 3.

Less than 5% of nymphs moulted when exposed to

photoperiods of less than 13 h (as a photoperiodic threshold)

at 20°C. The majority of nymphs (68%) moulted when

exposed to a LD 14 : 10 h light regime. Those nymphs,

exposed before and during feeding to 10°C and subsequently to

20°C, mostly (> 90%) went into diapause regardless of

photoperiod (Fig. 3). Thus, low temperatures have the

diapause-inducing effect that is similar to that of short

photoperiods. It is worth noting that the mentioned data on

the photoperiodic threshold data for 20°C are in full agreement

with experiments described in the ®rst section of this paper.

Changes in nymphal mass in response to relative humidity

and temperature

There was a trend for short-day (diapause destined) nymphs

to lose less mass than for long-day (nondiapause destined)

nymphs at both 23°C (7.8%) and 13°C (3.9%). Despite being

held at low relative humidity for an extended time there was no

mortality, as ticks remained ambulatory throughout the 45

days.

There was a clear temperature effect on mass loss by

engorged nymphs over 45 days with exposure to low relative

humidity. At 23°C engorged long-day nymphs lost ~14% more

weight (mean = ± 48.7 6 8.24%, n = 17) than those kept at

13°C (mean = ± 34.62 6 6.73%, n = 19). Short-day, high-

temperature specimens (mean = ± 40.9 6 6.97%, n = 18) lost

11.4% more of their mass than those exposed to the low

temperature (mean = ± 29.5 6 9.5%, n = 19).

Of special interest are the results of mass monitoring in ticks

from the critical photoperiod experiments. The engorged

nymphs maintained at 20°C and 93% r.h. were weighed

individually 5 days after detachment (when most defecation

had occurred) and 10 weeks later. The weights probably re¯ect

net changes in water mass and metabolism. Two groups of

nymphs were from short-day post-feeding photoperiods

(LD 12 : 12 h). They retained the diapausing state during

10 weeks, and displayed similar mass losses (± 6.82 6 2.86%

and ± 6.90 6 2.52%, n = 30 in both cases) irrespective of pre-

feeding photoperiods. These losses can be considered mainly

as a result of metabolic processes. But among nymphs from

long-day post-feeding photoperiod (LD 14 : 10 h) were those

that had already moulted within 10 weeks, those that displayed

an apolysis, and those that retained their diapausing state.

Nymphs from the latter group (n = 22) lost 5.62 6 2.44% of

their initial mass, which is similar to short-day diapausing

nymphs (mentioned above), whereas developing nymphs

(n = 18) gained 2.71 6 2.67% from their initial mass over

the same time. This increase in mass could be attributed to

water vapour uptake, and the real gain must be much higher

here due to mass losses (given above) both in developing and

diapausing ticks. The net water gain in developing nymphs

may be calculated therefore as not less than 8.3%

(= 2.7 + 5.6%) of initial mass.

Discussion

Ixodes rubicundus nymphs have a rather typical long-day

photoperiodic reaction because nondiapause development

occurs when they are exposed to daily light periods of

13.5 h or more. Exposure to shorter photoperiods of less than

13.5 h light induced diapause and delayed development. The

critical or threshold photoperiod (Belozerov, 1982) is therefore



LD 13.5 : 10.5 h, and at this exposure the nondiapause to

diapause ratio is 1 : 1. Day-length exposures less than the

critical photoperiod lengthen the interval between host

detachment and apolysis. The next stage of metamorphosis,

from apolysis until ecdysis (adult exit from exuvium), is

independent of photoperiod. Therefore, only the initiation of

development and not the actual processes is controlled by

photoperiod. Similar observations were made for other ixodids,

including larval Ixodes ricinus (Linnaeus) (Belozerov, 1964),

nymphal Hyalomma anatolicum (Koch) (Belozerov & Murad,

1977) and Amblyomma americanum (Linnaeus) (Pound &

George, 1991).

Both diapause induction and abolishment in I. rubicundus

nymphs are under photoperiodic control. Nymphs had a

prolonged interval of sensitivity to photoperiod. That is,

experimental exposures only before feeding, during feeding or

after feeding, to either long- or short-day photoperiods,

signi®cantly in¯uenced development (diapause or nondia-

pause). So an exposure for even the brief feeding period of 6±

8 days was suf®cient to induce either diapause or nondiapause.

The longer the exposure to short-day photoperiods (e.g. before

or after feeding), the greater the proportion of specimens that

diapause, while the shorter the exposure, the more that develop

and moult (and vice versa).

Temperature plays multiple roles in insect diapause

(Danilevsky, 1965; Tyshchenko, 1977; Saunders, 1982;

Tauber et al., 1986; Hodek & Hodkova, 1988; Zaslavski,

1988). Information on how temperature in¯uences tick

diapause is however, meagre. For I. ricinus larvae a

thermolabile photoperiodic response is apparent because the

photoperiod threshold decreases as temperature increases

(Belozerov, 1982). Seasonal ¯uctuations in temperature and

precipitation in May to June are reported to modify the critical

photoperiod in engorged Ixodes persulcatus Schulze larvae and

nymphs (Korotkov & Kislenko, 1995). Low temperatures

terminate diapause for engorged female Dermacentor niveus

Neumann (Zai-jie et al., 1991) and they may also induce

diapause for the larval soft tick Argas re¯exus (Frabicius)

(Dautel & KnuÈlle, 1998).

Our results show a de®nite in¯uence of low temperature on

diapause induction. Nymphs exposed to 10°C prior to and

during feeding (at LD 14 : 10 h) had a marked photoperiodic

insensitivity when compared to nymphs kept at 20°C and

similar photoperiod. There was a major difference in the

percentage that moulted (62%), even though nymphs were

exposed to 10°C prior to and during feeding then kept at 20°C

(LD 14 : 10 h) after engorgement. Low temperature (10°C)

may have a de®nitive inductive in¯uence on diapause that is

only abolished after prolonged exposure to higher temperatures

and a long-day (> 13.5 light). This is indeed the scenario

during South African summer months. The threshold below

which low temperature is inductive still needs resolution. For

A. re¯exus larvae the temperature threshold below which

temperature is inductive actually increases with physiological

age (Dautel & KnuÈlle, 1997).

The engorged nymphs of I. rubicundus were very resilient

when exposed to near-zero relative humidity over an extended

time. None of the specimens perished during the 45-day

experiment, although their mass losses were very high (35±

49%). By contrast, 50% of the Amblyomma cajennense

(Frabicius) adult ticks held at a higher relative humidity

(35% r.h.) and the same temperature (23°C) survived an

average of only 39 and 37 days, for females and males,

respectively (Strey et al., 1997). The Cayenne tick (6±9 mg) is

also approximately twice the mass of the nymphal Karoo

Paralysis tick (3±4 mg), so the latter is at even more of a

disadvantage from a surface area to volume ratio perspective

(Edney, 1977). That is, I. rubicundus should desiccate faster

based on its smaller size, if integumental permeabilities for the

two stages and species were similar. The tough New-World A.

cajennense survives very well in semi-arid to xeric environ-

ments where an average of 410 mm rainfall occurs in the

western fringe of its south-western Texas range (Strey et al.,

1997). This rainfall value is within the range for I. rubicundus

in the Free State (100±600 mm).

We saw a trend that short-day nymphs (diapause destined)

lose less mass than nondiapause destined nymphs. The idea is

that engorged nymphs in diapause are exposed to more

stressful, desiccating conditions of South African summer, and

may have more epicuticular lipids than nondiapausing nymphs.

Certainly diapausing puparia of Sarcophaga bullata Parker

have more lipids than nondiapausing puparis of this ¯esh ¯y

(Yoder & Denlinger, 1992). Although done on fed nondia-

pausing A. americanum nymphs, Yoder et al. (1997) demon-

strated that ticks exposed to severe desiccating conditions prior

to moulting conserved energy by retaining more lipids from

faeces than those kept at a high relative humidity. It would be

instructive to quantify surface lipids, determine integumental

permeability and measure metabolism for diapause and

nondiapause I. rubicundus nymphs.

According to our measurements on weight changes in

engorged I. rubicundus nymphs, maintained at 20°C and 93%

r.h., it is reasonable to conclude that engorged nymphs of this

South African tick maintain their water balance in subsaturated

air during developmental diapause. This is in agreement with

conclusions made by Kahl & KnuÈlle (1988) for the European

forest-inhabiting tick I. ricinus. Thus, the equilibrium period

for water balance is characteristic of diapausing nymphs in

both hygrophilic I. ricinus, and desiccation-resistant I.

rubicundus. Both also display the next phase of net vapour

uptake, which coincides with termination of diapause. It is still

unclear whether developing nymphs after apolysis lose this

capability (Kahl & KnuÈlle, 1988), or if they not only retain but

display an increase during the pharate adult stage (our data). It

would be very interesting to clarify if these differences re¯ect

some species-speci®c peculiarities, or something else.

Diapausing I. rubicundus normally delay their development

for 5±7 months, so the 10-week interval we studied was

obviously insuf®cient for development to commence. Given

that diapausing nymphs are so desiccation tolerant and that

ambient relative humidity is likely to be very low anyway, the

capability to absorb water vapour may not be so crucial to their

summer survival. Rather, they depend more on their capability

to retain moisture while in diapause but regain the ability to

absorb water moisture as development resumes.



The effect of temperature and photoperiod on developmental

diapause in I. rubicundus nymphs should be viewed in the

context of the tick's natural occurrence. In the central and

south-western Free State nymphs are active from April to

November with a peak in seasonal activity during August

(Fourie et al., 1992b). From April to September the daylength

varies from 11.5 to 12 h light per day. During October the

daylength is 13 h and during November 13.5 h. The maximum

daylength to which nymphs (only engorged nymphs) are

exposed is just over 14 h light/day during December (Fourie &

Horak, 1994). Mean monthly temperatures vary from 7.6°C

(June) to 19.8 °C (November). During peak seasonal occur-

rence of nymphs the mean monthly temperature is 10.1°C with

a daily range of 17.7°C. Most of the nymphs are therefore

subjected to mean temperatures of 10°C or less before or

during feeding, which in concert with a short day-length

induces a more consistent diapause state. From a water

conservation and development standpoint the engorged

diapausing nymphs seem very well adapted to survive for an

extended period under very adverse xeric summer conditions

in the Free State of South Africa.

Acknowledgements

We thank Mrs E. M. S. P. van Dalen and Mr F. van der Lingen

for technical assistance. This research was funded by the

National Science Foundation (NSF) and the University of the

Free State.

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Accepted 3 May 2001



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Ixodes rubicundus nymphs are short-day diapause-induced ticks with thermolabile sensitivity and desiccation resistance