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The life cycle of Amblyomma tholloni Neumann, 1899 (Acarina: Ixodidae) under laboratory conditions
Veterinary Parasitology, 7 (1980) 255--263 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
255"
THE LIFE CYCLE OF AMBLYOMMA THOLLONI NEUMANN, 1899 (ACARINA: IXODIDAE) UNDER LABORATORY CONDITIONS
R.A.I. NORVAL', J. COLBORNE', J. TANNOCK2 and P.K.I. MACKENZIE3 ' 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 attracted, 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 r e c e n t l y been s h o w n (Mackenzie and Norval, 1 9 8 0 ) t h a t t h e e l e p h a n t tick, A m b l y o m m a tholloni, is an efficient v e c t o r o f Cowdria r u m i n a n t i u m ( h e a r t w a t e r ) a n d has been i m p l i c a t e d in t h e transmission o f t h e disease t o d o m e s t i c livestock. Despite t h e wide d i s t r i b u t i o n o f t h e t i c k t h r o u g h tropical a n d s u b t r o p i c a l Africa, w h e r e it o c c u r s in association with the African elep h a n t , L o x o d o n t a africana, little is k n o w n a b o u t its b i o l o g y or e c o l o g y (Hoogstraal, 1956). The p r e s e n t s t u d y o f the life c y c l e o f A. t h o l l o n i covers t h e d u r a t i o n s o f t h e d e v e l o p m e n t a l periods in c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s , egg p r o d u c t i v i t y and a t t a c h m e n t a n d feeding o n l a b o r a t o r y hosts. The response o f males to 2 , 6 - d i c h l o r o p h e n o l , a sex p h e r o m o n e k n o w n to be e m i t t e d b y engorging females o f a n u m b e r o f species o f i x o d i d ticks (Berger, 1 9 7 2 ; C h o w et al., 1 9 7 5 ; S o n e n s h i n e et al., 1 9 7 6 ; W o o d et al., 1 9 7 6 ) , has also been investigated. 03o4-4017/80/00o0--0000/$02.25 © 1980 Elsevier Scientific Publishing Company
256 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 fitted 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 Supelcoport. 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 m e t h o d 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 production decreased steadily until laying ceased. In eight engorged 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 -
z
500
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 five 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 and 85--87%RH.
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 difference in the duration of the adult premoult period between males (mean 25.76 days) and females (mean 25.60 days).
258 50 NYMPHAL
PREMOULT
PERIOD
25-
i
t
1
123 Ld
o 3 5o E ADULT
PREMOULT
PERIOD
25-
16 L 17 I 18 I 19 i 2 0 i 21
22
I 23
I
i 24
25
I 26 I 27
28
TIME (DAYS)
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 nym phs which were confined on b o t h rabbits and sheep attached within the first 24 h. The mean feeding periods of larvae and n y m p h s on rabbits (6.69 and 6.29 days respectively) were slightly longer t h a n on sheep (6.10 days and 5.42 days respectively). The patterns o f detachm e n t o f b o t h instars f r om rabbits and sheep are shown in Fig. 3.
Attachment and feeding of adults The a t t a c h m e n t and feeding of adults was observed in batches of equal numbers of males and females which were confined t oget her on the backs of four different sheep and in a batch of males and females which were confined separately on two ot he r sheep (Table I). Adults did n o t attach readily to the sheep and had t o be confined for as long as 14 days before the first a t t a c h m e n t occurred. Males generally started to attach before females, but with one exception did n o t achieve m a x i m u m a t t a c h m e n t (i.e. m a x i m u m numbers attached) until 7--13 days after the females. In one batch m a x i m u m a t t a c h m e n t of males only occurred 32 days after the ticks had been introduced o n t o the host. The longest period re-
259 NYMPHS
LARVAE 100
T : 4958
l
T= 502
RABBITS
50-
w I
o 100
T = 590
T = 1004
w c3
SHEEP
50-
9
101 1!
FEEDING PERIOD (DAYS)
Fig. 3. T h e d u r a t i o n o f feeding o f larvae a n d n y m p h s o f A m b l y o m m a tholloni o n s h e e p a n d rabbits. T = t o t a l n u m b e r of ticks w h i c h c o m p l e t e d feeding.
TABLE I A t t a c h m e n t a n d feeding o f A m b l y o m m a tholloni adults c o n f i n e d o n sheep. S h e e p 1--3 were e a c h i n f e s t e d w i t h 30 dd + 3 0 9 9 , s h e e p 4 was i n f e s t e d w i t h 70 dd + 70 QQ, s h e e p 5 w i t h 30 dd a n d s h e e p 6 w i t h 30 99 S h e e p No.
1 2 3 4 5 6
Days t o first attachment
Days t o max. attachment
Max. % a t t a c h m e n t
dd
99
d6
99
dd
99
3 7 14 1 4 --
3 13 15 3 -5
10 26 32 22 17 --
10 19 19 9 -11
93.3 73.3 86.7 15.7 76.7 --
93.3 53.3 76.7 12.9 -43.3
% 99 c o m p l e t i n g engorgement
40.0 40.0 63.3 12.9 -0
quired for maximum attachment in females was 19 days. The maximum percentage attachment varied considerably between infestations on different sheep, but on individual sheep was mostly higher in males than in females. When placed separately on the host there was attachment of both males and
260 females, but the females failed to complete engorgement. The ticks detached semi-engorged after 15--20 days attachment 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 attachment 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 m e t h o d (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 of 2,6-dichlorophenol in extracts of 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
75
Fed 0~0~/2,6 dichlorophenol =
F~
.
50-
25 rfl Ld <
O. 50
Unfed d'cF/partially
X 2 =12.3
P<
fed
0.1
25-
0__ 75
IL
LEk La
F e d O~o~/partially
fed
50-
25-
0 1
2
3
SECTOR
4
5
OF
6
7
8
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 A m b l y o m m a hebraeum (Norval, 1 9 7 7 ) the durations of the developmental 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 c o m m o n African Am bl yom m a species such as A. hebraeurn (Norval, 1 9 7 4 ) and A m b l y o m m a variegatum (Iwuala and Okpala, 1 9 7 7 ) and consequently lay proportionately fewer eggs. A knowledge o f the host relationships of the different instars of A. tholloni is important in understanding the role o f the tick in the transmission of heartwater. Laboratory findings indicate that it is extremely unlikely that adults will attach o n ungulates under field conditions and confirmed earlier observations (Santos Dias, 1948; Theiler, 1 9 6 2 ) that the immature stages are less
262 h o s t specific t h a n t h e adults. U n f o r t u n a t e l y it was n o t possible t o o b t a i n c o m p a r a t i v e l a b o r a t o r y d a t a o n t h e r a t e s o f a t t a c h m e n t o f adults a n d i m m a t u r e s o n e l e p h a n t s . As suggested b y M a c k e n z i e a n d Norval (1980), it is m o s t likely t h a t h e a r t w a t e r t r a n s m i s s i o n t o d o m e s t i c animals is b y n y m p h s w h i c h have b e c o m e i n f e c t e d in t h e larval stage. U n f e d a d u l t f e m a l e s o f A. tholloni a t t a c h t o t h e h o s t in t h e a b s e n c e o f males, b u t as w i t h o t h e r species o f t h e M e t a s t r i a t a ( R e c h a v , 1 9 6 8 ; P a p p a s a n d Oliver, 1 9 7 2 ) d o n o t c o m p l e t e feeding in t h e a b s e n c e o f males. In t h e p r e s e n c e o f p r e f e d m a l e s m a t i n g is e n s u r e d b y t h e e m i s s i o n o f a sex p h e r o m o n e b y partially fed females. T h e u t i l i z a t i o n o f 2 , 6 - d i c h l o r o p h e n o l as a sex p h e r o m o n e in A. tholloni c o n f o r m s w i t h t h e findings in a n u m b e r o f o t h e r i x o d i d t i c k species. T h e w i d e o c c u r r e n c e o f this simple p h e n o l as a t i c k p h e r o m o n e raises t h e q u e s t i o n , h o w e v e r , o f a c c i d e n t a l interspecific m a t i n g occurving in t h e field. In t h e case o f A. tholloni t h e p r o b a b l e o c c u r r e n c e o f interspecific m a t i n g m u s t be low, as e l e p h a n t s are n o t f r e q u e n t l y p a r a s i t i z e d b y large n u m b e r s o f adults o f o t h e r t i c k species ( H o o g s t r a a l , 1 9 5 6 ) . ACKNOWLEDGEMENTS We wish t o t h a n k Mr. J. M u c h u w e f o r t e c h n i c a l assistance a n d Dr. J.A. L a w r e n c e f o r c o n s t r u c t i v e criticism o f t h e m a n u s c r i p 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 Hyalomma 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: Ixodidae). 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.
263 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, D e r m a c e n t o r a n d e r s o n 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.
255"
THE LIFE CYCLE OF AMBLYOMMA THOLLONI NEUMANN, 1899 (ACARINA: IXODIDAE) UNDER LABORATORY CONDITIONS
R.A.I. NORVAL', J. COLBORNE', J. TANNOCK2 and P.K.I. MACKENZIE3 ' 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 attracted, 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 r e c e n t l y been s h o w n (Mackenzie and Norval, 1 9 8 0 ) t h a t t h e e l e p h a n t tick, A m b l y o m m a tholloni, is an efficient v e c t o r o f Cowdria r u m i n a n t i u m ( h e a r t w a t e r ) a n d has been i m p l i c a t e d in t h e transmission o f t h e disease t o d o m e s t i c livestock. Despite t h e wide d i s t r i b u t i o n o f t h e t i c k t h r o u g h tropical a n d s u b t r o p i c a l Africa, w h e r e it o c c u r s in association with the African elep h a n t , L o x o d o n t a africana, little is k n o w n a b o u t its b i o l o g y or e c o l o g y (Hoogstraal, 1956). The p r e s e n t s t u d y o f the life c y c l e o f A. t h o l l o n i covers t h e d u r a t i o n s o f t h e d e v e l o p m e n t a l periods in c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s , egg p r o d u c t i v i t y and a t t a c h m e n t a n d feeding o n l a b o r a t o r y hosts. The response o f males to 2 , 6 - d i c h l o r o p h e n o l , a sex p h e r o m o n e k n o w n to be e m i t t e d b y engorging females o f a n u m b e r o f species o f i x o d i d ticks (Berger, 1 9 7 2 ; C h o w et al., 1 9 7 5 ; S o n e n s h i n e et al., 1 9 7 6 ; W o o d et al., 1 9 7 6 ) , has also been investigated. 03o4-4017/80/00o0--0000/$02.25 © 1980 Elsevier Scientific Publishing Company
256 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 fitted 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 Supelcoport. 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 m e t h o d 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 production decreased steadily until laying ceased. In eight engorged 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 -
z
500
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 five 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 and 85--87%RH.
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 difference in the duration of the adult premoult period between males (mean 25.76 days) and females (mean 25.60 days).
258 50 NYMPHAL
PREMOULT
PERIOD
25-
i
t
1
123 Ld
o 3 5o E ADULT
PREMOULT
PERIOD
25-
16 L 17 I 18 I 19 i 2 0 i 21
22
I 23
I
i 24
25
I 26 I 27
28
TIME (DAYS)
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 nym phs which were confined on b o t h rabbits and sheep attached within the first 24 h. The mean feeding periods of larvae and n y m p h s on rabbits (6.69 and 6.29 days respectively) were slightly longer t h a n on sheep (6.10 days and 5.42 days respectively). The patterns o f detachm e n t o f b o t h instars f r om rabbits and sheep are shown in Fig. 3.
Attachment and feeding of adults The a t t a c h m e n t and feeding of adults was observed in batches of equal numbers of males and females which were confined t oget her on the backs of four different sheep and in a batch of males and females which were confined separately on two ot he r sheep (Table I). Adults did n o t attach readily to the sheep and had t o be confined for as long as 14 days before the first a t t a c h m e n t occurred. Males generally started to attach before females, but with one exception did n o t achieve m a x i m u m a t t a c h m e n t (i.e. m a x i m u m numbers attached) until 7--13 days after the females. In one batch m a x i m u m a t t a c h m e n t of males only occurred 32 days after the ticks had been introduced o n t o the host. The longest period re-
259 NYMPHS
LARVAE 100
T : 4958
l
T= 502
RABBITS
50-
w I
o 100
T = 590
T = 1004
w c3
SHEEP
50-
9
101 1!
FEEDING PERIOD (DAYS)
Fig. 3. T h e d u r a t i o n o f feeding o f larvae a n d n y m p h s o f A m b l y o m m a tholloni o n s h e e p a n d rabbits. T = t o t a l n u m b e r of ticks w h i c h c o m p l e t e d feeding.
TABLE I A t t a c h m e n t a n d feeding o f A m b l y o m m a tholloni adults c o n f i n e d o n sheep. S h e e p 1--3 were e a c h i n f e s t e d w i t h 30 dd + 3 0 9 9 , s h e e p 4 was i n f e s t e d w i t h 70 dd + 70 QQ, s h e e p 5 w i t h 30 dd a n d s h e e p 6 w i t h 30 99 S h e e p No.
1 2 3 4 5 6
Days t o first attachment
Days t o max. attachment
Max. % a t t a c h m e n t
dd
99
d6
99
dd
99
3 7 14 1 4 --
3 13 15 3 -5
10 26 32 22 17 --
10 19 19 9 -11
93.3 73.3 86.7 15.7 76.7 --
93.3 53.3 76.7 12.9 -43.3
% 99 c o m p l e t i n g engorgement
40.0 40.0 63.3 12.9 -0
quired for maximum attachment in females was 19 days. The maximum percentage attachment varied considerably between infestations on different sheep, but on individual sheep was mostly higher in males than in females. When placed separately on the host there was attachment of both males and
260 females, but the females failed to complete engorgement. The ticks detached semi-engorged after 15--20 days attachment 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 attachment 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 m e t h o d (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 of 2,6-dichlorophenol in extracts of 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
75
Fed 0~0~/2,6 dichlorophenol =
F~
.
50-
25 rfl Ld <
O. 50
Unfed d'cF/partially
X 2 =12.3
P<
fed
0.1
25-
0__ 75
IL
LEk La
F e d O~o~/partially
fed
50-
25-
0 1
2
3
SECTOR
4
5
OF
6
7
8
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 A m b l y o m m a hebraeum (Norval, 1 9 7 7 ) the durations of the developmental 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 c o m m o n African Am bl yom m a species such as A. hebraeurn (Norval, 1 9 7 4 ) and A m b l y o m m a variegatum (Iwuala and Okpala, 1 9 7 7 ) and consequently lay proportionately fewer eggs. A knowledge o f the host relationships of the different instars of A. tholloni is important in understanding the role o f the tick in the transmission of heartwater. Laboratory findings indicate that it is extremely unlikely that adults will attach o n ungulates under field conditions and confirmed earlier observations (Santos Dias, 1948; Theiler, 1 9 6 2 ) that the immature stages are less
262 h o s t specific t h a n t h e adults. U n f o r t u n a t e l y it was n o t possible t o o b t a i n c o m p a r a t i v e l a b o r a t o r y d a t a o n t h e r a t e s o f a t t a c h m e n t o f adults a n d i m m a t u r e s o n e l e p h a n t s . As suggested b y M a c k e n z i e a n d Norval (1980), it is m o s t likely t h a t h e a r t w a t e r t r a n s m i s s i o n t o d o m e s t i c animals is b y n y m p h s w h i c h have b e c o m e i n f e c t e d in t h e larval stage. U n f e d a d u l t f e m a l e s o f A. tholloni a t t a c h t o t h e h o s t in t h e a b s e n c e o f males, b u t as w i t h o t h e r species o f t h e M e t a s t r i a t a ( R e c h a v , 1 9 6 8 ; P a p p a s a n d Oliver, 1 9 7 2 ) d o n o t c o m p l e t e feeding in t h e a b s e n c e o f males. In t h e p r e s e n c e o f p r e f e d m a l e s m a t i n g is e n s u r e d b y t h e e m i s s i o n o f a sex p h e r o m o n e b y partially fed females. T h e u t i l i z a t i o n o f 2 , 6 - d i c h l o r o p h e n o l as a sex p h e r o m o n e in A. tholloni c o n f o r m s w i t h t h e findings in a n u m b e r o f o t h e r i x o d i d t i c k species. T h e w i d e o c c u r r e n c e o f this simple p h e n o l as a t i c k p h e r o m o n e raises t h e q u e s t i o n , h o w e v e r , o f a c c i d e n t a l interspecific m a t i n g occurving in t h e field. In t h e case o f A. tholloni t h e p r o b a b l e o c c u r r e n c e o f interspecific m a t i n g m u s t be low, as e l e p h a n t s are n o t f r e q u e n t l y p a r a s i t i z e d b y large n u m b e r s o f adults o f o t h e r t i c k species ( H o o g s t r a a l , 1 9 5 6 ) . ACKNOWLEDGEMENTS We wish t o t h a n k Mr. J. M u c h u w e f o r t e c h n i c a l assistance a n d Dr. J.A. L a w r e n c e f o r c o n s t r u c t i v e criticism o f t h e m a n u s c r i p 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 Hyalomma 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: Ixodidae). 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.
263 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, D e r m a c e n t o r a n d e r s o n 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.