Veterinary Parasitology, 7 (1980) 255--263 255" Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

THE L I F E CYCLE OF A M B L Y O M M A T H O L L O N I N E U M A N N , 1899 ( A C A R I N A : I X O D I D A E ) U N D E R L A B O R A T O R Y C O N D I T I O N S

R.A.I. NORVAL', J. COLBORNE', J. TANNOCK 2 and P.K.I. MACKENZIE 3

' Veterinary Research Laboratory, P.O. Box 8101, Causeway, Salisbury (Zimbabwe)

2 Chemistry and Soil Research Institute, P. 0. Box 8100, Causeway, Salisbury (Zimbabwe)

3 Wellcome Foundation (Southern Africa) Ltd., Kwanyanga Research Station, P. 0. Box 5034, Greenfields, East London (South Africa)

(Accepted 15 April 1980)

ABSTRACT

Norval, R.A.I., Colborne, J., Tannock, J. and Mackenzie, P.K.I., 1980. The life cycle of Amblyomma tholloni Neumann, 1899 (Acarina: Ixodidae) under laboratory conditions. Vet. Parasitol., 7: 255--263.

The durations of the developmental periods of Amblyomma thoUoni, recorded at 25 ° C and 85--87% RH, were longer than those of most other tick species which have been studied under similar conditions. Fully engorged females laid between 2,871 and 10,347 eggs (mean 5,805). Larvae and nymphs attached rapidly and fed successfully on rabbits and sheep. Adults had to be confined on sheep for as long as 14 days before the first attachment occurred and post-attachment mortality was frequently high. Adult females attached in the absence of males, but did not complete feeding. Prefed males were attrac- ted, both on and off the host, by partially fed females and 2,6-dichlorophenol. Extracts of partially fed females were found to contain 2,6-dichlorophenol, which is a commonly occurring sex pheromone in ixodid ticks.

INTRODUCTION

It has recent ly been shown (Mackenzie and Norval, 1980) tha t the e lephant tick, A m b l y o m m a tholloni , is an eff icient vector o f Cowdria r u m i n a n t i u m (hear twater) and has been implicated in the t ransmission of the disease to domes t ic l ivestock. Despite the wide dis t r ibut ion o f the t ick t h rough t ropical and subtropical Africa, where it occurs in associat ion with the African ele- phant , L o x o d o n t a africana, little is k n o w n a b o u t its b io logy or eco logy (Hoogstraal , 1956).

The present s tudy of the life cycle o f A. thol loni covers the dura t ions o f the deve lopmenta l periods in cont ro l led l abora to ry condi t ions , egg p roduc t iv i ty and a t t a c h m e n t and feeding on l abora to ry hosts. The response o f males to 2 ,6-d ichlorophenol , a sex p h e r o m o n e k n o w n to be emi t ted by engorging females o f a n u m b e r o f species o f ixodid ticks (Berger, 1972; Chow et al., 1975; Sonenshine et al., 1976; Wood et al., 1976) , has also been investigated.

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MATERIALS AND METHODS

Laboratory rearing

A laboratory colony of A..thoUoni was established from the progeny of an engorged female collected from an elephant shot in the Gona-re-zhou National Park (32 ° 00'E, 21°40'S) in southeastern Zimbabwe. All instars were fed on shaved areas on the backs of sheep or, in the case of larvae and nymphs, on the ears of rabbits. In all feeding experiments ticks were confined in canvas bags which were glued to the skin of the host. Engorged ticks were collected daily from hosts and transferred to an incubator (25°C and 85--87 % RH) where the durations of the developmental periods were recorded. Egg batches were removed at 4-day intervals from ovipositing females and the eggs counted.

Extraction of pheromone

Partially fed female ticks were placed in diethyl ether and subjected to ultrasonic vibration. The extract was then evaporated to dryness in a rotary evaporator.

Gas liquid chromatography

A Pye series 104 gas chromatograph fit ted with a 63Ni electron capture detector system was used. The 1.5 m glass column (internal diameter 4 mm) was packed with 5% Carbowax 20 M terephthalic acid on 80--100 mesh Supel- coport. The argon carrier gas flow rate was 60 ml/min and the column and detector oven temperatures were 152°C and 250°C respectively.

Thin layer chromatography

Thin layer chromatography was carried out on silica gel plates developed in chloroform. The 2,6-dichlorophenol spots were visualised by spraying the plates with saturated ethanolic silver nitrate solution followed by irradiation under ultraviolet light.

Bioassay procedure

The petri dish method described by Rechav et al. (1977) was used to test the attractiveness of 2,6-dichlorophenol and partially fed females to prefed and unfed males.

RESULTS

Preo viposition period

The duration of the preoviposition period in 18 fully engorged females was 14--29 days (mean 18.44 days).

257

Oviposition period

In five fully engorged females from which eggs were collected every 4 days, the oviposition period lasted approximately 24--44 days (Fig. 1). Highest numbers of eggs were laid in the first 12 days of oviposition, after which the rate of egg product ion decreased steadily until laying ceased. In eight en- gorged females ranging in weight from 593 to 1,646 mg the total numbers of eggs laid ranged from 2,871 to 10,347 (mean 5,805). The numbers of eggs laid by individual females were significantly correlated with the engorged weights of the ticks (r = 0.963; P < 0.01).

1500 -

10OO -

500 z

4 8 12 16 20 24 28 32 36 40 44 48

TIME (DAYS)

Fig. 1. O v i p o s i t i o n c u r v e s o f f ive f e m a l e s o f Amblyomma tholloni m a i n t a i n e d a t 2 5 ° C a n d 8 5 - - 8 7 % R H .

Incu ba tion period

The incubation period of eggs taken from a single day's laying was 50--59 days.

Premoult periods

In a sample of 616 ticks the nymphal premoult period lasted 16--28 days (mean 20.22 days), while in a sample of 302 ticks the adult premoult period lasted 23--28 days (mean 25.66 days) (Fig. 2). There was no significant dif- ference in the duration of the adult premoult period between males (mean 25.76 days) and females (mean 25.60 days).

258

5 0

2 5 -

123 Ld

o 3 5o E

2 5 -

N Y M P H A L P R E M O U L T PERIOD

i t 1

A D U L T P R E M O U L T PERIOD

16 L 17 I 18 I 19

I i 2 0 i 21 2 2 I 2 3 i 2 4

T I M E (DAYS)

2 5 I 2 6 I 2 7 2 8

Fig. 2. The durations of the nymphal and adult premoult periods of Amblyomma thoUoni at 25 ° C and 85--87%RH.

Larval and nymphal feeding periods

The majority of larvae and nymphs which were confined on both rabbits and sheep attached within the first 24 h. The mean feeding periods of larvae and nymphs on rabbits (6.69 and 6.29 days respectively) were slightly longer than on sheep (6.10 days and 5.42 days respectively). The patterns of detach- ment of bo th instars f rom rabbits and sheep are shown in Fig. 3.

Attachment and feeding of adults

The a t tachment and feeding of adults was observed in batches of equal numbers of males and females which were confined together on the backs of four different sheep and in a batch of males and females which were confined separately on two other sheep (Table I).

Adults did no t at tach readily to the sheep and had to be confined for as long as 14 days before the first a t tachment occurred. Males generally started to attach before females, but with one exception did not achieve maximum at tachment (i.e. maximum numbers attached) until 7--13 days after the females. In one batch maximum at tachment of males only occurred 32 days after the ticks had been introduced onto the host. The longest period re-

259

w I

w c3

100

5 0 -

LARVAE

T : 4958

o 100 T = 590

NYMPHS

l T = 502

RABBITS

T = 1004

SHEEP

5 0 -

9 101 1!

FEEDING PERIOD (DAYS)

Fig. 3. The d u r a t i o n of feeding o f larvae and n y m p h s of Amblyomma tholloni on sheep and rabbi ts . T = to ta l n u m b e r of t icks which c o m p l e t e d feeding.

T A B L E I

A t t a c h m e n t and feeding o f Amblyomma tholloni adul ts c o n f i n e d on sheep. Sheep 1--3 were each in fes ted wi th 30 dd + 3099, sheep 4 was in fes ted wi th 70 dd + 70 QQ, sheep 5 w i th 30 dd and sheep 6 wi th 30 99

Sheep No. Days to first Days to max. Max. % a t t a c h m e n t % 99 c o m p l e t i n g a t t a c h m e n t a t t a c h m e n t e n g o r g e m e n t

dd 99 d 6 99 dd 99

1 3 3 10 10 93.3 93.3 40 .0 2 7 13 26 19 73.3 53.3 40 .0 3 14 15 32 19 86.7 76.7 63.3 4 1 3 22 9 15.7 12.9 12.9 5 4 - - 17 - - 76.7 - - - - 6 - - 5 - - 11 - - 43 .3 0

q u i r e d f o r m a x i m u m a t t a c h m e n t i n f e m a l e s w a s 1 9 d a y s . T h e m a x i m u m

p e r c e n t a g e a t t a c h m e n t v a r i e d c o n s i d e r a b l y b e t w e e n i n f e s t a t i o n s o n d i f f e r e n t

s h e e p , b u t o n i n d i v i d u a l s h e e p w a s m o s t l y h i g h e r i n m a l e s t h a n i n f e m a l e s .

W h e n p l a c e d s e p a r a t e l y o n t h e h o s t t h e r e w a s a t t a c h m e n t o f b o t h m a l e s a n d

260

females, but the females failed to complete engorgement. The ticks detached semi-engorged after 15--20 days at tachment and did not lay eggs. In the presence of males the majority of attached females engorged to completion in 8--18 days and subsequently laid large viable egg masses. The females which did not complete engorgement either died in situ or detached prior to the completion of feeding.

Behaviour o f attached males

It was observed that after feeding for 7--10 days males, confined with females which had fed for a similar period, left their initial sites of attach- ment and re-attached immediately adjacent to the females. This behaviour suggested that the males were responding to a pheromone released by the females.

Response to 2, 6-dichlorophenol

As the female-produced sex pheromone in a number of ixodid tick species is known to be 2,6-dichlorophenol, a preliminary experiment was conducted to test the response of males to the compound. Cork discs (diameter 5 mm) which had been impregnated with 2,6-dichlorophenol and similar untreated control discs were glued to the skin of a sheep in the presence of males which had fed for 10--15 days. Examination after 24 h revealed that there was significant assembly of males around treated discs and no assembly around control discs.

The response of prefed males to 2,6-dichlorophenol and partially fed females was confirmed using the petri dish method (Fig. 4). There was significant assembly in the sector of the petri dish containing either a partially fed female or a filter paper disc (diameter 2 cm) which had been immersed in a solution of 2,6-dichlorophenol in diethyl ether (10 -'o moles/pl). Unfed males showed no significant response to either 2,6-dichlorophenol or partially fed females.

Presence o f 2,6-dichlorophenol in extracts o f partially fed females

The dried extract from three partially fed females was dissolved in 25 ml of hexane. Five pl of this solution was injected into the gas chromatograph and gave a large peak at 1 min 36 secs. An identical retention time was obtained for standard 2,6-dichlorophenol. Co-injection of the extract with standard 2,6-dichlorophenol gave a single peak. Further confirmation of the presence of 2,6-dichlorophenol in the extract was obtained using thin layer chromatography, where it was found that both 2,6-dichlorophenol and the extract gave a dark spot at Rf = 0.67.

261

rfl

Ld

<

75

5 0 -

25

O. 5 0

2 5 -

0__ 75

5 0 -

2 5 -

0

Fed 0~0~/2,6 dichlorophenol F ~ = .

Unfed d'cF/part ia l ly fed

X 2 =12.3 P < 0.1

IL LEk La Fed O~o~/part ial ly fed

1 2 3 4 5 6 7 8

SECTOR OF PETRI DISH

Fig. 4. Average assembly response (% ± SE)of prefed (10---15 days) and unfed males of Arnblyomma tholloni to partially fed females of the same species and filter paper discs impregnated with 2,6-dichlorophenol, placed in sector 8 of a petri dish (diameter 15 cm) which had been divided into 8 equal sectors.

DISCUSSION

As,in Amblyomma hebraeum (Norval, 1977) the durations of the develop- mental periods of A. tholloni are long by comparison with most other ixodid ticks which have been studied in similar conditions. Engorged females of A. tholloni weigh considerably less than the engorged females of other common African Amblyomma species such as A. hebraeurn (Norval, 1974) and Amblyomma variegatum (Iwuala and Okpala, 1977) and consequently lay proportionately fewer eggs.

A knowledge of the host relationships of the different instars of A. tholloni is important in understanding the role of the tick in the transmission of heart- water. Laboratory findings indicate that it is extremely unlikely that adults will attach on ungulates under field condit ions and confirmed earlier obser- vations (Santos Dias, 1948; Theiler, 1962) that the immature stages are less

262

hos t specif ic t han the adults . U n f o r t u n a t e l y it was n o t possible to ob ta in compara t i ve l a b o r a t o r y da t a on the ra tes o f a t t a c h m e n t of adul ts and imma- tures on e lephants . As suggested b y Mackenz ie and Norval (1980) , i t is m o s t l ikely t h a t h e a r t w a t e r t ransmiss ion to dom es t i c animals is b y n y m p h s which have b e c o m e in fec ted in t he larval stage.

Unfed adul t females o f A. tholloni a t t ach to the hos t in the absence of males, b u t as wi th o the r species o f the Metas t r i a ta (Rechav , 1968; Pappas and Oliver, 1972) do n o t c o m p l e t e feeding in the absence of males. In the presence o f p re fed males ma t ing is ensured b y the emiss ion o f a sex phero- m o n e b y par t ia l ly fed females . The u t i l iza t ion o f 2 , 6 -d i ch lo ropheno l as a sex p h e r o m o n e in A. tholloni c o n f o r m s wi th the f indings in a n u m b e r of o t h e r ixod id t i ck species. The wide occu r rence o f this s imple p h e n o l as a t i ck p h e r o m o n e raises t he ques t ion , however , o f acc iden ta l in terspeci f ic ma t i ng oc- curving in the field. In t he case o f A. tholloni the p r o b a b l e occu r r ence o f inter- specif ic ma t i ng m u s t be low, as e lephants are n o t f r e q u e n t l y paras i t ized b y large n u m b e r s of adul ts o f o the r t i ck species (Hoogs t raa l , 1956) .

ACKNOWLEDGEMENTS

We wish to t h a n k Mr. J. M uchuwe for technica l assistance and Dr. J .A. Lawrence for cons t ruc t ive cr i t ic ism o f the manusc r ip t .

REFERENCES

Berger, R.S., 1972. 2,6-dichlorophenol, sex pheromone of the lone star tick. Science, 177: 704--705.

Chow, Y.S., Wang, C.B. and Lin, L.C., 1975. Identification of a sex pheromone of the female brown dog tick, Rhipicephalus sanguineus. Ann. Entomol. Soc. Am., 68: 485-- 488.

Hoogstraal, H., 1956. African Ixodoidea. I. Ticks of the Sudan. Ras. Rep. NM 005 050.29. 07, Dep. Navy, Bur. Med. Surg., Washington D.C., 1101 pp.

Iwuala, M.O.E. and Okpala, I., 1977. Egg output in the weights and states of engorgement of Arnblyomma variegatum (Fabr.) and Boophilus annulatua (Say): (Ixodoidea: Ixodidae). Folia Parasitol., 24: 162--172.

Mackenzie, P.K.I. and Norval, R.A.I., 1980. The transmission of Cowdria ruminantium by Amblyomraa tholloni. Vet. Parasitol., 7: 000---000.

Norval, R.A.I., 1974. The life cycle of Amblyomrna hebraeum Koch, 1844 (Acarina: Ixodidae). J. Entomol. Soc. South. Aft., 37: 357--367.

Norval, R.A.I., 1077. Studies on the ecology of the tick Amblyomma hebraeurn Koch in the Eastern Cape Province of South Africa. J. Parasitol., 63: 740--747.

Pap~as, P.J. and Oliver, J.H., 1972. Reproduction in ticks (Acari: Ixodidae). 2. Analysis of the stimulus for rapid and complete feeding of female Dermacentor variabilia (Say). J. Med. Entomol. , 9: 47--50.

Rechav, Y., 1968. The effect of delayed mating on feeding time and oviposition in Hya- lomma exeavatum (Koch) with a note on parthenogenesis. Refu. Vet., 25: 172--178.

Rechav, Y., Terry, S., Knight, M.M. and Cross, R.H.M., 1977. Chemoreceptor organs used in the detection of pheromone(s) of the tick Amblyomrna hebraeum (Acarina: Ixodi- dae). J. ivied. Entomol. , 14: 395--400.

Santos Dias, J.A.T., 1948. Estudo sobre a biologiado "Amblyomraa thoiloni" Neumann, 1890. Docum. Mocambique, 54: 127--139.

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Sonenshine, D.E., Silverstein, R.M., Plummer, E., West, J.R, and McCullough, T., 1976. 2,6-dichlorophenol, the sex pheromone of the Rocky Mountain wood tick, Dermacen to r anderson i Stiles, and the American dog tick, D e r m a c e n t o r variabilis (Say). J. Chem. Ecol., 2: 201--209.

Theiler, G., 1962. The Ixodoidea parasites of vertebrates in Africa South of the Sahara. Rep. Dir. Vet. Serv. Anita. Ind., Onderstepoort , No. $9958, 260 pp.

Wood, W.F., Leahy, M.G., Galun, R., Prestwich, G., Meinwald, J., PurneU, R.E. and Payne, R.C., 1976. Phenols as pheromones of ixodid ticks: a general phenomenon? J. Chem. Ecol., 1: 501--509.