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Attraction of male Rhipicephalus appendiculatus ticks to females in relation to feeding
Veterinary Parasitology, 1 (1976) 2 4 9 - 2 5 6 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
ATTRACTION TO FEMALES
O F M A L E R H I P I C E P H A L U S A P P E N D I C U L A TUS T I C K S IN RELATION TO FEEDING
M.G. LEAHY*', R. GALUN .2, R.E. PURNELL *.3 and R.C. PAYNE *.4
*International Centre for Insect Physiology and Ecology, Nairobi (Kenya) **East African Veterinary Research Organization, Kikuyu (Kenya) ' Present address: Mount St. Mary's College, Los Angeles, Calif. 90049 (U.S.A.) : Present address: Israel Institute for Biological Research, Ness Ziona (Israel) 3 Present address: ARC Institute for Research on Animal Diseases, Compton, Near Newbury, Berkshire (Great Britain) 4 Present address: The Central Veterinary Laboratory, New Haw, Weybridge, Surrey (Great Britain ) (Received July 14th, 1975)
ABSTRACT Leahy, M.G., Galun, R., Purnell, R.E. and Payne, R.C., 1976. Attraction of male Rhipicephalus appendiculatus ticks to females in relation to feeding. Vet. Parasitol., 1: 249--256. Five experiments were carried out on the movement of partially fed male Rhipicephalus appendiculatus ticks on rabbits towards other ticks of the same species. Male ticks pre-fed for 5 days were attracted towards pre-fed females, slightly attracted towards other pre-fed males, but not attracted towards nymphal ticks or recently relinquished feeding sites of adult or nymphal ticks. Female ticks fed for 5 days or longer were particularly attractive to male ticks which had also fed for 5 days or longer. Eight-week-old female ticks were most attractive to males, and 8-week-old male ticks most attracted to females. It is suggested that the male ticks may be attracted to the females by means of a sex pheromone, as has been shown in other ixodid tick species.
INTRODUCTION A considerable volume of information on sex pheromones of insects has a c c u m u l a t e d o v e r t h e p a s t d e c a d e , b u t i t is o n l y r e c e n t l y t h a t t h e p r o d u c t i o n o f a p h e r o m o n e b y i x o d i d t i c k s h a s b e e n d e m o n s t r a t e d . B e r g e r e t al. ( 1 9 7 1 ) s h o w e d t h a t m a l e A m b l y o m m a americanum, A. maculatum a n d Dermacentor variabilis d i s p l a y b e h a v i o u r a l r e s p o n s e s , c h a r a c t e r i s t i c o f m a t i n g , t o a c h e m i c a l s u b s t a n c e i n e x t r a c t s o f f e m a l e s o f e a c h o f t h e t h r e e s p e c i e s . S t u d i e s o n R. sanguineus a n d Boophilus microplus ( C h o w e t al., 1 9 7 2 ) a l s o i n d i c a t e t h e presence of female pheromones. The sex pheromones of insects have been investigated primarily with a view to controlling the insects themselves, whereas t h e o b j e c t i v e o f c o n t r o l l i n g t i c k s is t o e l i m i n a t e t h e m as v e c t o r s o f a n u m b e r
250 o f economically i m p o r t a n t diseases of man and his domesticated animals. One o f the principal targets of tick c ont r ol measures is R. appendiculatus, the vector of East Coast fever of cattle, the most i m p o r t a n t parasitic disease o f livestock in East Africa. This paper describes preliminary investigations on the possible existence of a female sex p h e r o m o n e of R. appendiculatus and experiments to a t t e m p t to deter min e when peak p r o d u c t i o n of such a substance might occur. MATERIALS AND METHODS The rabbits used in these studies were chinchilla crosses from the East African Veterinary Research Organization (EAVRO) small animal unit. The ticks, R. appendiculatus, have been maintained as a l aborat ory strain at EAVRO for the past 20 years according t o the m et hods described by Bailey (1960).
Experiment 1 The possible at t r a c t i on of feeding male R. appendiculatus ticks to ot her R. appendiculatus ticks or their feeding sites was investigated. Seven h u n d r ed and t w e n t y male ticks and 160 female ticks were selected fr o m a batch which had d r o p p e d as engorged n y m p h s from rabbits approximately 3 m o n t h s prior t o use, and 400 n y m p h s from a batch dropped as engorged larvae 6 weeks prior to use. Groups of 80 male ticks were placed in calico bags on the left ear of each of seven rabbits. Ticks were placed in bags on the right ears of the rabbits as follows -- two rabbits 80 female ticks each, two rabbits 80 male ticks each, two rabbits 200 n y m p h s each, one rabbit no ticks. After 24 h each group of 80 adult ticks was reduced to 50 by discarding u n a t t a c h e d or excess attached ticks. After a furt her 4 days, one group of 50 male ticks, one o f 50 female ticks and one of 200 n y m p h s were removed fr o m the right ears of three of the rabbits. The individual ear-bags were then removed f r o m all rabbits and replaced by larger ear-bags which enveloped b o t h ears o f each rabbit. T he ears were examined 1.5, 3.5 and 24 h later to observe tick migration.
Experiments 2a and 2b T h e effect o f length o f feeding period of male ticks on their attraction towards 6-day-fed females was investigated. In E x p e r i m e n t 2a, 1 100 male ticks and 825 female ticks were selected f r o m a batch which had m oul t ed 9 weeks prior to use. Groups of 100 male ticks were applied in bags to the right ears of each o f 11 rabbits on days 0 to 9. On day 4, groups of 75 female ticks were applied to the left ear o f each rabbit. On day 10, male and female ticks in excess o f 50 per ear were removed. The ears of each rabbit were t hen enveloped in a double ear-bag, and tick migration assessed after 24 h.
251 E x p e r i m e n t 2b was a repeat of 2a e x c e p t t hat l l - w e e k - o l d ticks were used.
Experiments 3a and 3b T h e effect o f length of feeding period of female ticks on t h e k ~bili ty to attract 8-day-fed males was investigated. In E x p e r i m e n t 3a, 975 female ticks and 825 male ticks were selected f r o m a batch which had m o u l t e d 6 weeks prior to use. Groups of 85 female ticks were applied in bags to the left ears of each of 11 rabbits on days 0 t o 10. On day 2, groups of 75 male ticks were applied to th e right ear of each rabbit. On day 10, male and female ticks in excess of 50 per ear were removed. The ears of each rabbit were t hen enveloped in a d o u b le ear-bag, and tick migration assessed after 24 h. E x p e r i m e n t 3b was a repeat of 3a except that l l - w e e k - o l d ticks were used.
Experiment 4 The effect o f the age of female ticks, fed for 6, 8 or 10 days, on their ability to attr act 8-day-fed males was investigated. Three t housand six h u n d r e d female ticks were selected f r o m batches which had moul t ed 2, 4, 6, 8, 12 or 16 weeks previously and 2 700 male ticks f r om a batch which had m oul t ed 8 weeks previously. Thirty-six groups of female ticks were applied in bags to the left ear o f each of 36 rabbits, 12 on day 0, 12 on day 2 and 12 on day 4. On day 2, groups o f 75 male ticks were applied to the right ear of each rabbit. On day 10, male and female ticks in excess of 50 per ear were removed. The ears of each rabbit were t hen enveloped in a double ear-bag, and tick migration assessed after 24 h.
Experiment 5 The effect of t he age of male ticks, fed for 6, 8 or 10 days, on their attraction towards 8-day-fed, 8-week-old females was investigated. Two thousand t w o h u n d r ed and fifty male ticks were selected from batches which had m o ulted 4, 6, 8, 12 or 16 weeks previously, and 3 000 female ticks from a batch which had m o u l t e d 8 weeks previously. T hi rt y groups of 75 male ticks were applied in bags t o the right ear of each of 30 rabbits, 10 on day 0, 10 on day 2 and 10 on day 4. On day 2, groups of 100 female ticks were applied t o t h e left ear o f each rabbit. On day 10, male and female ticks in excess of 50 per ear were removed. The ears of each rabbit were t hen enveloped in a double ear-bag, and tick migration assessed after 24 h. RESULTS
Experiment 1 T h e results of E x p e r i m e n t 1 are shown in Table I. The only considerable
252
TABLE I Migration of 5-day-fed male R. appendiculatus ticks towards o t h e r R. appendiculatus ticks or their feeding sites C o n d i t i o n of right ear
No ticks 200 n y m p h s removed 200 n y m p h s 50 males removed 50 males 50 females removed 50 females
Percentage migration of males, time after ears placed t o g e t h e r 1.5 h
3.5 h
24 h
0 0 0 0 6 0 21
0 2 0 0 10 0 26
2 6 0 0 12 2 45
migration was by male ticks towards females although there was some movement by male ticks towards other males. One female tick transferred within 1.5 h to the ear containing all the male ticks.
Experiments 2a and 2b The results of the experiments are shown in Table II. In Experiment 2a the greatest number of male ticks migrating were those fed for 6 days, although at least half of those fed for 9 or 10 days had also migrated within 24 h. In Experiment 2b, there was less migration, the peak occurring in ticks fed for 8 or 10 days. TABLE II Migration of male R. appendiculatus ticks towards 6-day-fed female ticks after varying pre-feeding periods Pre-feeding period of male ticks (days)
Percentage migration of male ticks after 24 h E x p e r i m e n t 2a
Percentage migration of male ticks after 24 h E x p e r i m e n t 2b
Mean percentage migration of male ticks after 24 h
1 2 3 4 5 6 7 8 9 10
12 6 10 14 42 66 30 12 52 50
4 6 22 22 16 14 6 42 24 44
8 6 16 18 29 40 18 27 38 47
253
E x p e r i m e n t s 3a and 3b
T h e results o f t h e e x p e r i m e n t s are s h o w n in T a b l e III. I n E x p e r i m e n t 3a, t h e g r e a t e s t n u m b e r o f m a l e t i c k s m i g r a t e d t o w a r d s f e m a l e ticks fed f o r 6 days, a l t h o u g h m o r e t h a n h a l f t h e m a l e s m i g r a t e d t o w a r d s f e m a l e t i c k s fed f o r 5, 8 or 10 days. I n E x p e r i m e n t 3b, t h e r e was less m i g r a t i o n , t h e p e a k b e i n g t o w a r d s f e m a l e ticks fed f o r 8 days. TABLE III Migration of male R. appendiculatus ticks towards female ticks fed for varying periods of time Pre-feeding period of female ticks (days)
Percentage migration of male ticks after 24 h Experiment 3a
Percentage migration of male ticks after 24 h Experiment 3b
Mean percentage migration of male ticks after 24 h
0 1 2 3 4 5 6 7 8 9 10
2 6 12 32 20 62 90 38 54 42 68
0 0 14 26 28 24 24 10 48 42 32
1 3 13 24 24 43 57 24 52 42 50
Experiment 4
T h e results o f t h e e x p e r i m e n t are s u m m a r i s e d in T a b l e IV. T h e g r e a t e s t n u m b e r o f m a l e t i c k s m i g r a t e d t o w a r d s 8 - w e e k - o l d females, a l t h o u g h t h e r e was c o n s i d e r a b l e v a r i a t i o n in t h e m i g r a t i o n o f d i f f e r e n t replicates o f m a l e ticks. Statistical analysis, using analysis o f variance, i n d i c a t e d t h a t t h e r e was n o significant d i f f e r e n c e b e t w e e n t h e n u m b e r s o f m a l e ticks a t t r a c t e d t o f e m a l e s o f d i f f e r e n t ages. Experiment 5
T h e results o f t h e e x p e r i m e n t are s u m m a r i s e d in T a b l e V. T h e r e was m o r e m i g r a t i o n b y 8-weekoold m a l e t i c k s t h a n b y o t h e r groups. O n c e again, h o w ever, analysis o f v a r i a n c e i n d i c a t e d t h a t t h e r e was n o significant d i f f e r e n c e b e t w e e n t h e n u m b e r s o f m a l e t i c k s o f d i f f e r e n t ages a t t r a c t e d t o females.
254 TABLE IV Migration of 8-day-fed, 8-week-old male R. appendiculatus ticks towards female R.
appendiculatus ticks of various ages Age of ~male tic ks (weeks)
Percentage migration of male ticksafter 24 h . . . . . . . . Towards Towards Towards 6-day-fed ~males 8-day-fed ~males 10-day-fed ~males
Total percentage migration
2 4 6 8 12 16
54 32 45 40 33 7
29 32 34 46 22 14
26 27 38 44 26 17
6 37 19 53 6 17
TABLE V Migration of male R. appendiculatus ticks of various ages towards 8-day-fed, 8-week-old female R. appendiculatus ticks Age of male ticks (weeks)
Percentage migration of male ticks after 24 h Males fed 6 days
Malesfed 8 days
Malesfed 10 days
Total percentage migration
4 6 8 12 16
25 18 40 51 36
4 22 24 10 18
22 48 40 21 19
17 29 35 27 24
DISCUSSION T h e results o f E x p e r i m e n t 1 i n d i c a t e d t h a t male R. appendiculatus ticks pre-fed f o r 5 d a y s were a t t r a c t e d t o female ticks pre-fed for 5 days, slightly a t t r a c t e d t o o t h e r male ticks similarly fed, b u t n o t a t t r a c t e d t o n y m p h s or to f e e d i n g sites f r o m w h i c h male, f e m a l e or n y m p h a l ticks had been r e m o v e d . T h e results o f E x p e r i m e n t s 2 a n d 3 suggested t h a t female ticks were capable o f a t t r a c t i n g males, a n d males o f being a t t r a c t e d , p a r t i c u l a r l y if the ticks h a d fed for 5 o f m o r e days. T h e results o f E x p e r i m e n t s 4 and 5 suggested t h a t 8-week-old female ticks were r a t h e r m o r e a t t r a c t i v e t o pre-fed male ticks t h a n were female ticks o f o t h e r ages, a n d 8-week-old male ticks r a t h e r m o r e a t t r a c t e d t o the females, a l t h o u g h n e i t h e r result was statistically significant. R. appendiculatus ticks are s h o w n b y these e x p e r i m e n t s t o behave similarly t o o t h e r i x o d i d ticks, in t h a t m o v e m e n t o f male ticks t o w a r d s females after feeding for 5 d a y s is a s s u m e d in o u r e x p e r i m e n t s t o be a p r e c u r s o r o f m a t i n g
255 (Sactor et al., 1948). Gregson (1946) has shown that male Dermacentor andersoni ticks need to feed for 5 days before t h e y will mate with females, and Gladney and D r u m m o n d (1970 a and b) have shown that male A. arnericanum ticks will mate with females only after feeding for at least 5 days, the majority requiring at least 7 days feeding. As discussed by Norval (1974), mate-seeking by adult ixodid ticks is not, however, invariably performed by male ticks. Gladney (1971) has demonstrated t h a t female A. maculatum will only attach to the bovine host in the presence of feeding male A m b l y o m m a ticks, and Norval (1974) has shown that this is also so in the case of A. hebraeum females. Movement of male ticks of three ixodid species has recently been shown to be a behavioural response to a pheromone produced by the female ticks (Berger et al., 1971), and the pheromone of female A. americanum ticks has been identified as 2-6-dichlorophenol (Berger, 1972). The results of these preliminary experiments suggest that a similar pheromone may well be produced by female R. appendiculatus after a feeding period of at least 5 days. Male R. appendiculatus fed for similar periods are thus attracted to the females. In subsequent experiments, we will a t t e m p t to extract the pheromone from 8-week-old pre-fed female ticks, and to use 8-week-old pre-fed male ticks to assess its presence by an in vitro bio-assay. ACKNOWLEDGEMENTS The work described here forms part of the programme of a joint project of the International Centre for Insect Physiology and Ecology (ICIPE) and the East African Veterinary Research Organization (EAVRO). We are most grateful to Dr A.R. Sayers, Head of the Statistics Division at the East African Agriculture and Forestry Research Organization, for carrying out the statistical analyses. This paper is published by kind permission of Professor T.R. Odhiambo, The Director of ICIPE, and Dr G.L. Corry, the Director of EAVRO. This work was supported by the United Nations Development Programme, the Overseas Development Administration of the United Kingdom, the United States Department of Agriculture, the International Atomic Energy Agency and the Pfizer Corporation, in cooperation with the East African Community.
REFERENCES Bailey, K.P., 1960. Notes on the rearing of Rhipicephalus appendiculatus and their infection with Theileria parva for experimental transmission. Bull. Epizoot. Dis. Aft., 8: 33--43. Berger, R.S., 1972. 2-6-Dichlorophenol,sex pheromone of the Lone Star Tick. Science, 177: 704--705. Berger, R.S., Dukes, J.C. and Chow, Y.S., 1971. Demonstration of a sex pheromone in three species of hard ticks. J. Med. Entomol., 8: 84--86. Chow, Y.S., Lu, F.M., Peng, C.T. and Cheng, P.C., 1972. Isolation of lipids and sex pheromone from hard ticks. Bull. Inst. Zool. Acad. Sin., 11: 1--8. Gladney, W.J., 1971. Mate-seekingby female Amblyomma maculatum (Acarina: Ixodidae) on a bovine. Nature (London), 232: 401--402.
256
Gladney, W.J. and Drummond, R.O., 1970 a. Mating behaviour and reproduction of the Lone Star Tick, A m b l y o m m a americanum. Ann. Entomol. Soc. Am., 63: 1036--1039. Gladney, W.J. and Drummond, R.O., 1970 b. Migration of male Lone Star Ticks on the host in relation to mating. J. Econ. Entomol., 63: 1214--1216. Gregson, J.D., 1946. Feeding periods prerequisite to the mating of Dermacentor andersoni (Acarina). Proc. Entomol. Soc. B. C., 43: 3--6. Norval, R.A.I., 1974. The life cycle o f A m b l y o m m a hebraeum Koch, 1844 (Acarina: Ixodidae). J. Entomol. Soc. South. Afr., 37: 357--367. Sactor, B., Hutchinson, M. and Grannett, P., 1948. Biology of the Lone Star Tick in the Laboratory. J. Econ. Entomol., 41: 298--301.
ATTRACTION TO FEMALES
O F M A L E R H I P I C E P H A L U S A P P E N D I C U L A TUS T I C K S IN RELATION TO FEEDING
M.G. LEAHY*', R. GALUN .2, R.E. PURNELL *.3 and R.C. PAYNE *.4
*International Centre for Insect Physiology and Ecology, Nairobi (Kenya) **East African Veterinary Research Organization, Kikuyu (Kenya) ' Present address: Mount St. Mary's College, Los Angeles, Calif. 90049 (U.S.A.) : Present address: Israel Institute for Biological Research, Ness Ziona (Israel) 3 Present address: ARC Institute for Research on Animal Diseases, Compton, Near Newbury, Berkshire (Great Britain) 4 Present address: The Central Veterinary Laboratory, New Haw, Weybridge, Surrey (Great Britain ) (Received July 14th, 1975)
ABSTRACT Leahy, M.G., Galun, R., Purnell, R.E. and Payne, R.C., 1976. Attraction of male Rhipicephalus appendiculatus ticks to females in relation to feeding. Vet. Parasitol., 1: 249--256. Five experiments were carried out on the movement of partially fed male Rhipicephalus appendiculatus ticks on rabbits towards other ticks of the same species. Male ticks pre-fed for 5 days were attracted towards pre-fed females, slightly attracted towards other pre-fed males, but not attracted towards nymphal ticks or recently relinquished feeding sites of adult or nymphal ticks. Female ticks fed for 5 days or longer were particularly attractive to male ticks which had also fed for 5 days or longer. Eight-week-old female ticks were most attractive to males, and 8-week-old male ticks most attracted to females. It is suggested that the male ticks may be attracted to the females by means of a sex pheromone, as has been shown in other ixodid tick species.
INTRODUCTION A considerable volume of information on sex pheromones of insects has a c c u m u l a t e d o v e r t h e p a s t d e c a d e , b u t i t is o n l y r e c e n t l y t h a t t h e p r o d u c t i o n o f a p h e r o m o n e b y i x o d i d t i c k s h a s b e e n d e m o n s t r a t e d . B e r g e r e t al. ( 1 9 7 1 ) s h o w e d t h a t m a l e A m b l y o m m a americanum, A. maculatum a n d Dermacentor variabilis d i s p l a y b e h a v i o u r a l r e s p o n s e s , c h a r a c t e r i s t i c o f m a t i n g , t o a c h e m i c a l s u b s t a n c e i n e x t r a c t s o f f e m a l e s o f e a c h o f t h e t h r e e s p e c i e s . S t u d i e s o n R. sanguineus a n d Boophilus microplus ( C h o w e t al., 1 9 7 2 ) a l s o i n d i c a t e t h e presence of female pheromones. The sex pheromones of insects have been investigated primarily with a view to controlling the insects themselves, whereas t h e o b j e c t i v e o f c o n t r o l l i n g t i c k s is t o e l i m i n a t e t h e m as v e c t o r s o f a n u m b e r
250 o f economically i m p o r t a n t diseases of man and his domesticated animals. One o f the principal targets of tick c ont r ol measures is R. appendiculatus, the vector of East Coast fever of cattle, the most i m p o r t a n t parasitic disease o f livestock in East Africa. This paper describes preliminary investigations on the possible existence of a female sex p h e r o m o n e of R. appendiculatus and experiments to a t t e m p t to deter min e when peak p r o d u c t i o n of such a substance might occur. MATERIALS AND METHODS The rabbits used in these studies were chinchilla crosses from the East African Veterinary Research Organization (EAVRO) small animal unit. The ticks, R. appendiculatus, have been maintained as a l aborat ory strain at EAVRO for the past 20 years according t o the m et hods described by Bailey (1960).
Experiment 1 The possible at t r a c t i on of feeding male R. appendiculatus ticks to ot her R. appendiculatus ticks or their feeding sites was investigated. Seven h u n d r ed and t w e n t y male ticks and 160 female ticks were selected fr o m a batch which had d r o p p e d as engorged n y m p h s from rabbits approximately 3 m o n t h s prior t o use, and 400 n y m p h s from a batch dropped as engorged larvae 6 weeks prior to use. Groups of 80 male ticks were placed in calico bags on the left ear of each of seven rabbits. Ticks were placed in bags on the right ears of the rabbits as follows -- two rabbits 80 female ticks each, two rabbits 80 male ticks each, two rabbits 200 n y m p h s each, one rabbit no ticks. After 24 h each group of 80 adult ticks was reduced to 50 by discarding u n a t t a c h e d or excess attached ticks. After a furt her 4 days, one group of 50 male ticks, one o f 50 female ticks and one of 200 n y m p h s were removed fr o m the right ears of three of the rabbits. The individual ear-bags were then removed f r o m all rabbits and replaced by larger ear-bags which enveloped b o t h ears o f each rabbit. T he ears were examined 1.5, 3.5 and 24 h later to observe tick migration.
Experiments 2a and 2b T h e effect o f length o f feeding period of male ticks on their attraction towards 6-day-fed females was investigated. In E x p e r i m e n t 2a, 1 100 male ticks and 825 female ticks were selected f r o m a batch which had m oul t ed 9 weeks prior to use. Groups of 100 male ticks were applied in bags to the right ears of each o f 11 rabbits on days 0 to 9. On day 4, groups of 75 female ticks were applied to the left ear o f each rabbit. On day 10, male and female ticks in excess o f 50 per ear were removed. The ears of each rabbit were t hen enveloped in a double ear-bag, and tick migration assessed after 24 h.
251 E x p e r i m e n t 2b was a repeat of 2a e x c e p t t hat l l - w e e k - o l d ticks were used.
Experiments 3a and 3b T h e effect o f length of feeding period of female ticks on t h e k ~bili ty to attract 8-day-fed males was investigated. In E x p e r i m e n t 3a, 975 female ticks and 825 male ticks were selected f r o m a batch which had m o u l t e d 6 weeks prior to use. Groups of 85 female ticks were applied in bags to the left ears of each of 11 rabbits on days 0 t o 10. On day 2, groups of 75 male ticks were applied to th e right ear of each rabbit. On day 10, male and female ticks in excess of 50 per ear were removed. The ears of each rabbit were t hen enveloped in a d o u b le ear-bag, and tick migration assessed after 24 h. E x p e r i m e n t 3b was a repeat of 3a except that l l - w e e k - o l d ticks were used.
Experiment 4 The effect o f the age of female ticks, fed for 6, 8 or 10 days, on their ability to attr act 8-day-fed males was investigated. Three t housand six h u n d r e d female ticks were selected f r o m batches which had moul t ed 2, 4, 6, 8, 12 or 16 weeks previously and 2 700 male ticks f r om a batch which had m oul t ed 8 weeks previously. Thirty-six groups of female ticks were applied in bags to the left ear o f each of 36 rabbits, 12 on day 0, 12 on day 2 and 12 on day 4. On day 2, groups o f 75 male ticks were applied to the right ear of each rabbit. On day 10, male and female ticks in excess of 50 per ear were removed. The ears of each rabbit were t hen enveloped in a double ear-bag, and tick migration assessed after 24 h.
Experiment 5 The effect of t he age of male ticks, fed for 6, 8 or 10 days, on their attraction towards 8-day-fed, 8-week-old females was investigated. Two thousand t w o h u n d r ed and fifty male ticks were selected from batches which had m o ulted 4, 6, 8, 12 or 16 weeks previously, and 3 000 female ticks from a batch which had m o u l t e d 8 weeks previously. T hi rt y groups of 75 male ticks were applied in bags t o the right ear of each of 30 rabbits, 10 on day 0, 10 on day 2 and 10 on day 4. On day 2, groups of 100 female ticks were applied t o t h e left ear o f each rabbit. On day 10, male and female ticks in excess of 50 per ear were removed. The ears of each rabbit were t hen enveloped in a double ear-bag, and tick migration assessed after 24 h. RESULTS
Experiment 1 T h e results of E x p e r i m e n t 1 are shown in Table I. The only considerable
252
TABLE I Migration of 5-day-fed male R. appendiculatus ticks towards o t h e r R. appendiculatus ticks or their feeding sites C o n d i t i o n of right ear
No ticks 200 n y m p h s removed 200 n y m p h s 50 males removed 50 males 50 females removed 50 females
Percentage migration of males, time after ears placed t o g e t h e r 1.5 h
3.5 h
24 h
0 0 0 0 6 0 21
0 2 0 0 10 0 26
2 6 0 0 12 2 45
migration was by male ticks towards females although there was some movement by male ticks towards other males. One female tick transferred within 1.5 h to the ear containing all the male ticks.
Experiments 2a and 2b The results of the experiments are shown in Table II. In Experiment 2a the greatest number of male ticks migrating were those fed for 6 days, although at least half of those fed for 9 or 10 days had also migrated within 24 h. In Experiment 2b, there was less migration, the peak occurring in ticks fed for 8 or 10 days. TABLE II Migration of male R. appendiculatus ticks towards 6-day-fed female ticks after varying pre-feeding periods Pre-feeding period of male ticks (days)
Percentage migration of male ticks after 24 h E x p e r i m e n t 2a
Percentage migration of male ticks after 24 h E x p e r i m e n t 2b
Mean percentage migration of male ticks after 24 h
1 2 3 4 5 6 7 8 9 10
12 6 10 14 42 66 30 12 52 50
4 6 22 22 16 14 6 42 24 44
8 6 16 18 29 40 18 27 38 47
253
E x p e r i m e n t s 3a and 3b
T h e results o f t h e e x p e r i m e n t s are s h o w n in T a b l e III. I n E x p e r i m e n t 3a, t h e g r e a t e s t n u m b e r o f m a l e t i c k s m i g r a t e d t o w a r d s f e m a l e ticks fed f o r 6 days, a l t h o u g h m o r e t h a n h a l f t h e m a l e s m i g r a t e d t o w a r d s f e m a l e t i c k s fed f o r 5, 8 or 10 days. I n E x p e r i m e n t 3b, t h e r e was less m i g r a t i o n , t h e p e a k b e i n g t o w a r d s f e m a l e ticks fed f o r 8 days. TABLE III Migration of male R. appendiculatus ticks towards female ticks fed for varying periods of time Pre-feeding period of female ticks (days)
Percentage migration of male ticks after 24 h Experiment 3a
Percentage migration of male ticks after 24 h Experiment 3b
Mean percentage migration of male ticks after 24 h
0 1 2 3 4 5 6 7 8 9 10
2 6 12 32 20 62 90 38 54 42 68
0 0 14 26 28 24 24 10 48 42 32
1 3 13 24 24 43 57 24 52 42 50
Experiment 4
T h e results o f t h e e x p e r i m e n t are s u m m a r i s e d in T a b l e IV. T h e g r e a t e s t n u m b e r o f m a l e t i c k s m i g r a t e d t o w a r d s 8 - w e e k - o l d females, a l t h o u g h t h e r e was c o n s i d e r a b l e v a r i a t i o n in t h e m i g r a t i o n o f d i f f e r e n t replicates o f m a l e ticks. Statistical analysis, using analysis o f variance, i n d i c a t e d t h a t t h e r e was n o significant d i f f e r e n c e b e t w e e n t h e n u m b e r s o f m a l e ticks a t t r a c t e d t o f e m a l e s o f d i f f e r e n t ages. Experiment 5
T h e results o f t h e e x p e r i m e n t are s u m m a r i s e d in T a b l e V. T h e r e was m o r e m i g r a t i o n b y 8-weekoold m a l e t i c k s t h a n b y o t h e r groups. O n c e again, h o w ever, analysis o f v a r i a n c e i n d i c a t e d t h a t t h e r e was n o significant d i f f e r e n c e b e t w e e n t h e n u m b e r s o f m a l e t i c k s o f d i f f e r e n t ages a t t r a c t e d t o females.
254 TABLE IV Migration of 8-day-fed, 8-week-old male R. appendiculatus ticks towards female R.
appendiculatus ticks of various ages Age of ~male tic ks (weeks)
Percentage migration of male ticksafter 24 h . . . . . . . . Towards Towards Towards 6-day-fed ~males 8-day-fed ~males 10-day-fed ~males
Total percentage migration
2 4 6 8 12 16
54 32 45 40 33 7
29 32 34 46 22 14
26 27 38 44 26 17
6 37 19 53 6 17
TABLE V Migration of male R. appendiculatus ticks of various ages towards 8-day-fed, 8-week-old female R. appendiculatus ticks Age of male ticks (weeks)
Percentage migration of male ticks after 24 h Males fed 6 days
Malesfed 8 days
Malesfed 10 days
Total percentage migration
4 6 8 12 16
25 18 40 51 36
4 22 24 10 18
22 48 40 21 19
17 29 35 27 24
DISCUSSION T h e results o f E x p e r i m e n t 1 i n d i c a t e d t h a t male R. appendiculatus ticks pre-fed f o r 5 d a y s were a t t r a c t e d t o female ticks pre-fed for 5 days, slightly a t t r a c t e d t o o t h e r male ticks similarly fed, b u t n o t a t t r a c t e d t o n y m p h s or to f e e d i n g sites f r o m w h i c h male, f e m a l e or n y m p h a l ticks had been r e m o v e d . T h e results o f E x p e r i m e n t s 2 a n d 3 suggested t h a t female ticks were capable o f a t t r a c t i n g males, a n d males o f being a t t r a c t e d , p a r t i c u l a r l y if the ticks h a d fed for 5 o f m o r e days. T h e results o f E x p e r i m e n t s 4 and 5 suggested t h a t 8-week-old female ticks were r a t h e r m o r e a t t r a c t i v e t o pre-fed male ticks t h a n were female ticks o f o t h e r ages, a n d 8-week-old male ticks r a t h e r m o r e a t t r a c t e d t o the females, a l t h o u g h n e i t h e r result was statistically significant. R. appendiculatus ticks are s h o w n b y these e x p e r i m e n t s t o behave similarly t o o t h e r i x o d i d ticks, in t h a t m o v e m e n t o f male ticks t o w a r d s females after feeding for 5 d a y s is a s s u m e d in o u r e x p e r i m e n t s t o be a p r e c u r s o r o f m a t i n g
255 (Sactor et al., 1948). Gregson (1946) has shown that male Dermacentor andersoni ticks need to feed for 5 days before t h e y will mate with females, and Gladney and D r u m m o n d (1970 a and b) have shown that male A. arnericanum ticks will mate with females only after feeding for at least 5 days, the majority requiring at least 7 days feeding. As discussed by Norval (1974), mate-seeking by adult ixodid ticks is not, however, invariably performed by male ticks. Gladney (1971) has demonstrated t h a t female A. maculatum will only attach to the bovine host in the presence of feeding male A m b l y o m m a ticks, and Norval (1974) has shown that this is also so in the case of A. hebraeum females. Movement of male ticks of three ixodid species has recently been shown to be a behavioural response to a pheromone produced by the female ticks (Berger et al., 1971), and the pheromone of female A. americanum ticks has been identified as 2-6-dichlorophenol (Berger, 1972). The results of these preliminary experiments suggest that a similar pheromone may well be produced by female R. appendiculatus after a feeding period of at least 5 days. Male R. appendiculatus fed for similar periods are thus attracted to the females. In subsequent experiments, we will a t t e m p t to extract the pheromone from 8-week-old pre-fed female ticks, and to use 8-week-old pre-fed male ticks to assess its presence by an in vitro bio-assay. ACKNOWLEDGEMENTS The work described here forms part of the programme of a joint project of the International Centre for Insect Physiology and Ecology (ICIPE) and the East African Veterinary Research Organization (EAVRO). We are most grateful to Dr A.R. Sayers, Head of the Statistics Division at the East African Agriculture and Forestry Research Organization, for carrying out the statistical analyses. This paper is published by kind permission of Professor T.R. Odhiambo, The Director of ICIPE, and Dr G.L. Corry, the Director of EAVRO. This work was supported by the United Nations Development Programme, the Overseas Development Administration of the United Kingdom, the United States Department of Agriculture, the International Atomic Energy Agency and the Pfizer Corporation, in cooperation with the East African Community.
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Gladney, W.J. and Drummond, R.O., 1970 a. Mating behaviour and reproduction of the Lone Star Tick, A m b l y o m m a americanum. Ann. Entomol. Soc. Am., 63: 1036--1039. Gladney, W.J. and Drummond, R.O., 1970 b. Migration of male Lone Star Ticks on the host in relation to mating. J. Econ. Entomol., 63: 1214--1216. Gregson, J.D., 1946. Feeding periods prerequisite to the mating of Dermacentor andersoni (Acarina). Proc. Entomol. Soc. B. C., 43: 3--6. Norval, R.A.I., 1974. The life cycle o f A m b l y o m m a hebraeum Koch, 1844 (Acarina: Ixodidae). J. Entomol. Soc. South. Afr., 37: 357--367. Sactor, B., Hutchinson, M. and Grannett, P., 1948. Biology of the Lone Star Tick in the Laboratory. J. Econ. Entomol., 41: 298--301.