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The life cycle of Argas (Argas) africolumbae under constant abiotic and biotic conditions
Veterinary Parasitology, 11 (1982) 365--373 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
365
THE LIFE CYCLE OF ARGAS (ARGAS)AFRICOLUMBAE UNDER CONSTANT ABIOTIC AND BIOTIC CONDITIONS
A. KRAISS and It. GOTHE
Institute for Parasitology, Justus Liebig University, Giessen, Rudolf-Buchheim-Str. 2, 6300 Giessen 1 (W. Germany) (Accepted for publication 4 August 1982)
ABSTRACT Kraiss, A. and Gothe, R., 1982. The life cycle of Argas (Argas) africolumbae under constant abiotic and biotic conditions. Vet. Parasitol., 11: 365--373. The developmental and reproduction pattern as well as the nondiapause behaviour of
Argas (Argas) africolumbae, reared on domestic chickens and held at 27°C and 90% RH, indicate that there may be 2--3 generations annually. The minimum incubation period of eggs requires 16.2 days (mean), irrespective of the day of oviposition. Larvae feed for 5--6 days and moult 10.2 days after detaching to N 1 . Generally, there are only 2 nymphal instars. All N1 develop to N 2 13.7 days after feeding. After repletion, N 2 moult to males and females within 28.3 days with a ratio of 1.12:1.0. Female determined N~ and N 2 are always heavier (1.13 mg and 3.81 rag, respectively) and imbibe larger bloodmeals (4.19 mg and 17.45 mg, respectively) than male N 1 and N 2 (0.9 mg and 2.71 mg unfed weight and 3.04 mg and 12.33 rag bloodmeal size, respectively). Fed females begin to oviposit 9.5 days after engorgement and produce 102.9 eggs during a 14-day oviposition period.
INTRODUCTION
Argas (Argas) africolumbae, described and defined from wild-caught and laboratory-reared adults and immatures (Hoogstraal et al., 1975b) and found in South Africa, Namibia, Tanzania and Kenya (Converse et al., 1975; Hoogstraal et al., 1975b, 1977) as well as in Upper Volta (Gothe et al., 1981a,b) appears to be associated ecologically with wild birds as Hirundo spp. Myrmecocichla cinnamomeiventris subrufipennis, Columba spp., Onychognathus rueppelli, Geronticus calvus and Ptynoprogne fuligula rufigula, nesting in rocky situations and in buildings. In relation to pathogens Pretoria virus, a new African arboviral agent in the tickborne Dera Ghazi Khan group, was isolated from a pool of 2 female ticks, taken near a nest of Columba guinea phaeonata from a house in a Pretoria suburb (Converse et al., 1975; Hoogstraal et al., 1975b). In Upper Volta this tick species is also an efficient natural reservoir and vector of avian
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© 1982 Elsevier Scientific Publishing Company
366 spirochetes, Borrelia anserina, and of Aegyptianella pullorum, the agent of fowl aegyptianellosis, and may even cause tick paralysis in fowls (Gothe et al., 1981a,b). For further research on interrelationships between this tick and its pathogens, however, it is important to determine biological parameters for planning biomedical and veterinary experiments. Therefore, the present investigation was designed to provide essential experimental information on the life cycle of A rgas (Argas) africolum bae applicable to ecological and epidemiological studies, because biological base line data are still lacking. MATERIALS AND METHODS
Argas (Argas) africolumbae adults, originating from chicken-coops, dens and sleeping places in Dedougou, Upper Volta, were used to establish a colony. All ticks were reared in glass tubes in an incubator regulated to 27°C and 90% RH and fed on chickens at room temperature. The avian hosts were always infested once only. Eggs and larvae Eggs laid by 20 females were counted daily, then separately pooled and the numbers of emerging larvae/day of oviposition determined. Eggs of 20 other females were not disturbed until 40 days after first hatching. The hatching percentage was defined by counting larvae and unhatched eggs. 10--14 days after hatching, groups of 20--50, 100--200, 200--300 and more than 400 larvae were placed on the avian hosts to feed. Fully engorged, detached larvae were counted daily and pooled separately/day of repletion, determining the respective premoulting periods.
Nymphs Immediately after moulting each unfed lst-instar n y m p h was thereafter held separately until reaching the adult stage. By weighing single nymphs of each instar before and after feeding, the amounts of bloodmeal were determined and the bloodmeal ratio then calculated. The premoulting periods of fed lst-instar (N1), 2nd-instar (N2) or 3rd-instar (N3) nymphs were evaluated daily, recording the resulting instar or sex.
Adults 42 pairs of newly m o u l t e d male and female adults were placed on chickens for feeding. After copulation each female, isolated in a rearing tube, was checked dally for oviposition. Weights and ratios of bloodmeal of female ticks were measured as for nymphs, and the bloodmeal size was related to the number of eggs deposited. The oviposition was assessed by counting the
367
daily egg output, until egg laying ceased, so quantifying the period and capacity of ovipositions. RESULTS
Eggs and larvae
Minimum incubation period of eggs required 15.9--17.0 days (mean 16.2 days), indepent of the day of oviposition; 51.0% and 96.0% completed embryonic development after 19.0 and 24 days, respectively (Fig. 1). Thus, embryogenesis was temporally unaffected and was always in the same range, irrespective of the date of oviposition. The hatching percentage ranged between 94.5% and 100% (mean 97.7%). The mean feeding period, remained with 5.9, 5.2, 5.3 and 5.4 days unchanged, if the avian hosts were infected with 20--50, 100--200, 200--300 100~
*
80-
~
70.
~ x:
6050-
m e
40-
'~
30"
~-
20' 10-
--//
16 17 ~8 19 2'0 2'1 2'2 23 2'4 2'~ 2'6 2'7 2'8 2'9 3'o ' ' Incubation period (days)
Fig. 1. Cumulative percentage of Argas (Argas) africolumbae larvae hatching. 100-
100-
90"
o'~
90-
"~
80-
~
70-
~
o~ > ~_
8o70-
60-
~
60-
5040-
~
50"
ff
40-
30-
30~-
20' 10.
2010-
I
I
I
Feeding period (days)
I
//
9
~o
~i
I'2
1'3
Premoulting period (daysl
Fig. 2. Cumulative percentage of engorged Argas (Argas) africolumbae larvae dropping from the host. Fig. 3. Cumulative percentage of Argas (Argas) africolumbae N1 emerging from engorged larvae.
368
and more than 400 larvae, respectively. 8.6% of the larvae began to drop on Day 4 after attaching, 64% had detached after 5 days and repletion was completed 8 days p.i. (Fig. 2). Larval premoulting period was 9--12 days, independent of the feeding time. Larvae detached after 5, 6, 7 and 8 days p.i. developed to N1 within (mean) 10.7, 9.9, 10.5 and 10.0 days, respectively. Considering the whole pool of larvae the premoulting phase required (mean) 10.2 days after dropping from the host (Fig. 3).
Nymphs Generally, there were only 2 nymphal instars. Unfed N1 (n = 84) weighed (mean) 0.99 mg and imbibed (mean) 3.65 mg of blood, resulting in a mean bloodmeal ratio of 7.39. All N1 moulted to N2 11--29 days after feeding (mean 13.7 days) (Fig. 4). Unfed and engorged male N~ weighed (mean) I00-
-~
_,~.
j.~../.~
90-
~ ~o-
~
¢v 60~ ~
40302010"
~"
Prernoulting period (days]
Fig. 4. Cumulative percentage of Argas (Argas) africolumbae N2 emerging from engorged N~. 10090-
~ 80~ ¢n
7060-
-=
50-
~ ~
40ao2010-
2
Premoultincj period (days)
Fig. 5. Cumulative percentage of adult Argas (Argas) africolumbae emerging from engorged N 2 . (e • ) females; (•,~) males.
369
0.90 mg and 3.94 mg, respectively, obtaining a meal size of 3.04 mg and having a mean bloodmeal ratio of 6.82, while for female N1 1.13 mg, 5.32 mg, 4.19 mg and 6.35 were determined. The weight of unfed and engorged N2 (n = 79) amounted {mean) 3.14 mg and 18.34 mg, respectively, the mean bloodmeal ratio being 5.86. Of 72 N2, 38 (= 52.8%) developed to males after 18--54 days (mean 28.3 days); and 33 (= 45.8%) to females after 14--59 days {mean 28.3 days); (Fig. 5). Just 1 N2, weighing 1.28 mg and 7.14 mg in the unfed and engorged state, respectively, with a bloodmeal ratio of 5.6 became a 1.79 mg heavy 3rd instar nymph in 14 days and moulted after further infestation, reaching a weight of 14.02 mg, to a female tick within 15 days. The male determined unfed N2 weighed (mean) 2.71 mg and ingested 12.23 mg blood, representing a mean bloodmeal ratio of 5.89; the respective values for female N~ were 3.81 mg, 17.45 mg and 5.98. Adults
The male:female ratio was 1.12:1.0. The amount of blood ingested by recently moulted females was (mean) 31.99 mg and their unfed and engorged weight were 16.15 mg and 46.44 mg, respectively. Fed females (n = 42) began to oviposit 7--18 days (mean 9.54 days) after feeding and copulation. The whole oviposition (n = 42) was 7--22 days (mean 14.0 days) and considering only days of egg-laying, 6--16 days (mean 10.7 days), because females frequently interrupted oviposition for 1--3 days and then continued. Eggs per oviposition numbered 38--163 (mean 102.9 eggs). The mean daily egg output amounted 7.4 and 9.7 eggs, related to the whole oviposition and the actual period of egg production, respectively. In respect to the egg production/day of oviposition this value increased to 59.5% on Day 6 and to 95.6% on day 13 (mean) (Fig. 6). Plotting the egg numbers against the weight or bloodmeal size of engorged females, the relationship was not linear, 100908070-
==so50"
.= •~ 40-
o
30-
-~ 2O~_ 10-
2 3 ~, .5 6 "} 8 9 1~) 1=1 12 1=3 14 115 Whole oviposition period (days) Fig. 6. Cumulative percentage of daily produced eggs during the w h o l e oviposition period of female Argas (Argas) africolumbae.
370 i.e., higher egg counts were not correlated with increased body weights. Females weighing 26.11--46.53 mg in the engorged state and having imbibed 12--35 mg of blood produced 118.2 eggs (mean), while engorged ticks with a weight of 44.45--80.29 mg and a bloodmeal size of 35--62 mg deposited 86.8 eggs (mean) only. DISCUSSION
Argas (Argas) africolumbae, the only species of 21 listed in the subgenus Argas known to be confined to the Ethiopian faunal region (Hoogstraal et al., 1979) and ecologically associated with birds in rocky situations and in buildings (Hoogstraal et al., 1975b, 1977), is closely related to the Palearctic pigeon parasitizing Argas (Argas) hermanni (Hoogstraal and Kohls, 1960b) and to Argas (Argas) reflexus (Hoogstraal and Kohls, 1960a). In addition to the morphological similarities the developmental patterns are also very close. Corresponding with Argas (Argas) hermanni (Khalil and Metwally, 1974) there were no distinct differences with regard to larval and nymphal cycles, sex ratio, fecundity, preoviposition and oviposition periods and nondiapause behaviour between this species and Argas (Argas) africolumbae, if reared under comparable laboratory conditions. The shape of curves, representing the respective developmental phases, and also the values of means, namely, showed convergent, sometimes even identical lines and were equal or at least similar, respectively, confirming also the few records of preliminary biological studies on this species (Hoogstraal et al., 1975a, 1977). A comparable reference to most other species of the subgenus Argas, such as Argas tridentatus, Argas vulgaris, Argas latus and Argas macrostigmatus (Filippova, 1961, 1966), Argas polonicus (Siuda et al., 1979), Argas himalayensis (Hoogstraal and Kaiser, 1973), Argas brevipes (Kohls et al., 1961), Argas dalei (Clifford et al., 1976), Argas cooleyi (Kohls and Hoogstraal, 1960), Argas lagenoplastis (Hoogstraai and Kohls, 1963), Argas falco (Kaiser and Hoogstraal, 1974), Argas rnonachus (Keirans et al., 1973), Argas dulus (Keirans et al., 1971), Argas neghmei (Kohls and Hoogstraal, 1961), Argas moreli (Keirans et al., 1979) and 1 unnamed species (Hoogstraal et al., 1979), is not possible, because biological base line data are altogether lacking so far or are, as for Argas cucumerinus (Clifford et al., 1978) still very preliminary. For Argas reflexus (Mfiller, 1939) and Argas ]aponicus (Uchikawa et al., 1967) only similar developmental patterns may be drawn off, the results of the Palearctic species, however, cannot be compared, because of the very different abiotic conditions used. Principally, there are also some conformities with the persicargasid species Argaspersicus (Khalil, 1979; E1 Kammah and Abdel Wahab, 1980), Argas arboreus (Hafez et al., 1971, 1972), Argas walkerae (Gothe and Koop, 1974), Argas robertsi (Hoogstraal et al., 1975), Argas miniatus (Magalh~es, 1979) and Argas radiatus (Medley and Ahrens, 1970). The developmental and reproduction pattern as well as the nondiapause
371 b e h a v i o u r o f Argas (Argas) africolumbae u n d e r c o n s t a n t abiotic c o n d i t i o n s indicate, t h a t t h e r e m a y be 2--3 generations annually. Thus, dense populations m a y develop, if this tick species is associated with d o m e s t i c a t e d birds, as c o u l d be d e m o n s t r a t e d for fowls in U p p e r Volta, because it has t h e n an ample source o f n o u r i s h m e n t and an e x c e p t i o n a l l y p r o t e c t e d e n v i r o n m e n t y e a r r o u n d . In c o n s e q u e n c e o f this c a p a c i t y in these ecological niches Argos (Argas) africolumbae is t h e n o f considerable epidemiological i m p o r t a n c e for the p o u l t r y i n d u s t r y , because o f its high natural i n f e c t i o n - p o t e n t i a l as vectors o f Borrelia anserina and Aegyptianella pullorurn. B o t h disease agents c o u l d be isolated with a respective m a x i m u m incidence in every wild-derived tick p o p u l a t i o n f r o m U p p e r Volta. T h e natural i n f e c t i o n - p o t e n t i a l o f every tick p o p u l a t i o n was equally strong and c o u l d c o n s t a n t l y be p r o v e n b y transstadial transmission, since t h r o u g h infestation o f highly susceptible chickens with n y m p h s a n d / o r adults f r o m every original tick h a b i t a t b o t h agents were m i c r o s c o p i c a l l y d e m o n s t r a b l e in Giemsa-stained b l o o d smears. A transovarial passage o f the agents o n t o the n e x t tick-generation was also possible, h o w e v e r , it did n o t o c c u r s i m u l t a n e o u s l y with the h o r i z o n t a l transmission. The infectivity r e m a i n e d qualitatively and q u a n t i t a t i v e l y u n c h a n g e d in all p o p u l a t i o n s involved and persisted during the entire investigation p e r i o d o f -nine m o n t h s u p t o the third r e p l e t i o n series. T h e rate o f m o r b i d i t y and m o r t a l i t y a m o u n t e d t o 100% in every r e p l e t i o n , the infested animals were always i n f e c t e d with b o t h agents s i m u l t a n e o u s l y and died in a l m o s t equal p r o p o r t i o n s o f 52.4% and 47.6% o f a borreliosis and aegyptianellosis, respectively ( G o t h e et al., 1981a). F u r t h e r m o r e , it was d e m o n s t r a t e d t h a t all populations o f this tick species f r o m U p p e r V o l t a also display a paralysis-inducing c a p a c i t y ( G o t h e et al., 1 9 8 1 b ) .
REFERENCES Clifford, C.M., Keirans, J.E., Hoogstraal, H. and Corwin, D., 1976. Observations on the subgenus Argos (Ixodoidea: Argasidae: Argas). A. (A.) dalei, new species, parasitizing the burrowing owl in Peru. Ann. Entomol. Soc. Am., 69: 917--925. Clifford, C.M., Hoogstraal, H., Keirans, J.E., Rice, R.C.A. and Dale, W.E., 1978. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argos). 14. Identity and biological observations of Argos (A.) cucumerinus from Peruvian seaside cliffs and a summary of the status of the subgenus in the neotropieal faunal region. J. Med. Entomol., 15: 57--73. Converse, J.D., Hoogstraal, H., Moussa, M.I., Casals, J. and Kaiser, M.N., 1975. Pretoria virus: a new African agent in the tickborne Dera Ghazi Khan (DGK) group and antigenic relationships within the DGK group. J. Med. Entomol., 12: 202--205. El Kammah, K.M. and Abdel Wahab, K.S., 1980. Argas (Persicargas) persicus life cycle under controlled and outdoor conditions. Acarologia, 21: 163--172. Filippova, N.A., 1961. Materials on ticks belonging to the subfamily Argasinae. Part I. Adult ticks and larvae of the genus Argas Latr., group reflexus. Zool. Zh., 40: 1815--1826. Filippova, N.A., 1966. Argasid ticks (Argasidae). Fauna SSSR, Paukoobraznye, 4: 255.
372 Gothe, R. and Koop, E., 1974. Zur biologischen Bewertung der Validit~t von Argas (Persicargas) persicus (Oken, 1818), Argas (Persicargas) arboreus Kaiser, Hoogstraal und Kohls, 1964 und Argas (Persicargas) walkerae Kaiser und Hoogstraal, 1969. I. Untersuchungen zur Entwicklungsbiologie. Z. Parasitenkd., 44: 299--317. Gothe, R., Buchheim, C. and Schrecke, W., 1981a. Argas (Persicargas) persicus und Argas (Argas) africolumbae als natilrliche Llbertr~iger von Borrelia anserina und Aegyptianella pullorum in Obervolta. Berl. MUnch. Tier~rtzl. Wochenschr., 94: 280--285. Gothe, R., Buchheim, C. and and Schrecke, W., 1981b. Zur Paralyse-induzierenden Kapazit~t wildst~mmiger Argas (Persicargas) persicus- und Argas (Argas) #fricolumbaePopulationen aus Obervolta. Berl. MQnch. Tier~rtzl. Wochenschr., 94: 299---302. Hafez, M., Abdel-Malek, A.A. and Guirgis, S.S., 1971. The subgenus Persicargas (Ixodoidea, Argasidae, Argas). 12. Biological studies on the immature stages of A. (P.) arboreus Kaiser, Hoogstraal & Kohls in Egypt. J. Med. Entomol., 8: 421--429. Hafez, M., Abdel-Malek, A.A. and Guirgis, S.S., 1972. The subgenus Persicargas (Ixodoidea, Argasidae, Argas). 14. Biological studies on the adult stage of A. (P.) arboreus Kaiser, Hoogstraal & Kohls in Egypt. J. Med. Entomol., 9: 19--29. Hoogstraal, H. and Kaiser, M.N., 1973. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 7. A. (A.) himalayensis, new species, parasitizing the snow partridge, Lerwa lerwa, in Nepal. Ann. Entomol. Soc. Am., 66: 1--3. Hoogstraal, H. and Kohls, G.M., 1960a. Observations on the subgenus Argas (Ixodoidea, Argasidae, Argas). 1. Study of A. reflexus reflexus (Fabricius, 1794), the European bird argasid. Ann. Entomol. Soc. Am., 53: 611--618. Hoogstraal, H. and Kohls, G.M., 1960b. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 3. A biological and systematic study of A. reflexus hermanni Audouin, 1827 (revalidated), the African bird argasid. Ann. Entomol. Soc. Am., 53: 743--755. Hoogstraal, H. and Kohls, G.M., 1963. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 6. Redescription and biological notes on A. lagenoplastis Frogatt, 1906, of Australian fairy martins, Hylochelidon ariel (Gould). Ann. Entomol. Soc. Am., 56: 577--582. Hoogstraal, H., Kaiser, M.N. and McClure, H.E., 1975a. The subgenus Persicargas (Ixodoidea: Argasidae: Argas). 20. A. (P.) robertsi parasitizing nesting wading birds and domestic chickens in the Australian and Oriental regions, viral infections, and host migration. J. Med. Entomol., 11: 513--524. Hoogstraal, H., Kaiser, M.N., Walker, J.B., Ledger, J.A., Converse, J.D. and Rice, R.C,A., 1975b. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 10. A. (A.) africolumbae, n. sp., a Pretoria virus-infected parasite of birds in Southern and Eastern Africa. J. Med. Entomol., 12: 194--201. Hoogstraal, H., Wassef, H.Y., Easton, E.R. and Dixon, J.E.W., 1977. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 12. Argas (A.) africolumbae: variation, bird hosts, and distribution in Kenya, Tanzania, and South and South-West Africa. J. Med. Entomol., 13: 441--445. Hoogstraal, H., Clifford, C.M., Keirans, J.E. and Wassef, H.Y., 1979. Recent developments in biomedical knowledge of Argas ticks (Ixodoidea: Argasidae). In: J.G. Rodriguez (Editor), Recent Advances in Acarology. Vol. 2. Academic Press, New York, San Francisco, London, pp. 269--278. Kaiser, M.N. and Hoogstraal, H., 1974. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 9. A. (A.) falco, new species, parasitizing kestrels in Western Australia. Ann. Entomol. Soc. Am., 67: 5--10. Keirans, J.E., Clifford, C.M. and Maldonado Capriles, J., 1971. Argas (Argas) dulus, new species (Ixodoidea: Argasidae), from nests of the palm chat Dulus dominicus in the Dominican Republic. Ann. Entomol. Soc. Ann., 64: 1410--1413.
373 Keirans, J.E., Radovsky, F.J. and Clifford, C.M., 1973. Argas (Argas) monachus, new species (Ixodoidea: Argasidae), from nests of the monk parakeet, Myiopsitta monachus, in Argentina. J. Med. Entomol., 10: 511--516. Keirans, J.E., Hoogstraal, H. and Clifford, C.M., 1979. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 16. Argas (A.) moreli, new species, and keys to neotropical species of the subgenus. J. Med. Entomol., 15: 246--252. Khalil, G.M., 1979. The subgenus Persicargas (Ixodoidea: Argasidae: Argas). 31. The life cycle of A. (P.) persicus in the laboratory. J. Med. Entomol., 16: 200--206. Khalil, G.M. and Metwally, S.A., 1974. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 8. The life cycle of A. (.4.) hermanni. J. Med. Entomol., 11: 355--362. Kohls, G.M. and Hoogstraal, H., 1960. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 2. A. cooleyi, new species, from Western North American birds. Ann. Entomol., Soc. Am., 53: 625--631. Kohls, G.M. and Hoogstraal, H., 1961. Observations on the subgenus Argas (Ixodoidea, Argasidae, Argas). 4. A. neghmei, new species, from poultry houses and human habitations in Northern Chile. Ann. Entomol. Soc. Am., 54 : 844--851. Kohls, G.M., Hoogstraal, H. and Clifford, C.M., 1961. Observations on the subgenus Argas (Ixodoidea, Argasidae, Argas). 5. Study of A. brevipes Banks, 1908, from birds in Arizona and California, U.S.A., and Baja California, Mexico. Ann. Entomol. Soc. Am., 54: 869--877. Magalha'es de, F.E.P., 1979. Novos aspectos morfol6gicos, biol6gicos e t6xicos de Argas (Persicargas) miniatus Koch, 1844 (Ixodoidea -- Argasidae) no estado do Rio de Janeiro. Tese, apresentada ~ Universidade Federal Rural do Rio de Janeiro para o b t e n ~ o do grau de "Magister Seientiae". Medley, J.G. and Ahrens, E., 1970. Life history and bionomics of two American species of fowl ticks (Ixodoidea, Argasidae, Argas) of the subgenus Persicargas. Ann. Entomol. Soc. Am., 63: 1591--1594. M~ller, K.H., 1939. Zur Biologie der Taubenzecke Argas columbarum. Inaugural-Dissertation, Berlin. Siuda, K., Hoogstraal, H., Clifford, C.M. and Wassef, H.Y., 1979. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 17. Argas (A.) polonicus sp. n. parasitizing domestic pigeons in Krakow, Poland. J. Parasitol., 65: 170--181. Uchikawa, K., Sato, A. and Kugimoto, M., 1967. Studies on the argasid infesting the Japanese house-martin, Delichon urbica. Med. J. Shinshu Univ., 12: 141--155.
365
THE LIFE CYCLE OF ARGAS (ARGAS)AFRICOLUMBAE UNDER CONSTANT ABIOTIC AND BIOTIC CONDITIONS
A. KRAISS and It. GOTHE
Institute for Parasitology, Justus Liebig University, Giessen, Rudolf-Buchheim-Str. 2, 6300 Giessen 1 (W. Germany) (Accepted for publication 4 August 1982)
ABSTRACT Kraiss, A. and Gothe, R., 1982. The life cycle of Argas (Argas) africolumbae under constant abiotic and biotic conditions. Vet. Parasitol., 11: 365--373. The developmental and reproduction pattern as well as the nondiapause behaviour of
Argas (Argas) africolumbae, reared on domestic chickens and held at 27°C and 90% RH, indicate that there may be 2--3 generations annually. The minimum incubation period of eggs requires 16.2 days (mean), irrespective of the day of oviposition. Larvae feed for 5--6 days and moult 10.2 days after detaching to N 1 . Generally, there are only 2 nymphal instars. All N1 develop to N 2 13.7 days after feeding. After repletion, N 2 moult to males and females within 28.3 days with a ratio of 1.12:1.0. Female determined N~ and N 2 are always heavier (1.13 mg and 3.81 rag, respectively) and imbibe larger bloodmeals (4.19 mg and 17.45 mg, respectively) than male N 1 and N 2 (0.9 mg and 2.71 mg unfed weight and 3.04 mg and 12.33 rag bloodmeal size, respectively). Fed females begin to oviposit 9.5 days after engorgement and produce 102.9 eggs during a 14-day oviposition period.
INTRODUCTION
Argas (Argas) africolumbae, described and defined from wild-caught and laboratory-reared adults and immatures (Hoogstraal et al., 1975b) and found in South Africa, Namibia, Tanzania and Kenya (Converse et al., 1975; Hoogstraal et al., 1975b, 1977) as well as in Upper Volta (Gothe et al., 1981a,b) appears to be associated ecologically with wild birds as Hirundo spp. Myrmecocichla cinnamomeiventris subrufipennis, Columba spp., Onychognathus rueppelli, Geronticus calvus and Ptynoprogne fuligula rufigula, nesting in rocky situations and in buildings. In relation to pathogens Pretoria virus, a new African arboviral agent in the tickborne Dera Ghazi Khan group, was isolated from a pool of 2 female ticks, taken near a nest of Columba guinea phaeonata from a house in a Pretoria suburb (Converse et al., 1975; Hoogstraal et al., 1975b). In Upper Volta this tick species is also an efficient natural reservoir and vector of avian
0304-4017/82/0000--0000/$02.75
© 1982 Elsevier Scientific Publishing Company
366 spirochetes, Borrelia anserina, and of Aegyptianella pullorum, the agent of fowl aegyptianellosis, and may even cause tick paralysis in fowls (Gothe et al., 1981a,b). For further research on interrelationships between this tick and its pathogens, however, it is important to determine biological parameters for planning biomedical and veterinary experiments. Therefore, the present investigation was designed to provide essential experimental information on the life cycle of A rgas (Argas) africolum bae applicable to ecological and epidemiological studies, because biological base line data are still lacking. MATERIALS AND METHODS
Argas (Argas) africolumbae adults, originating from chicken-coops, dens and sleeping places in Dedougou, Upper Volta, were used to establish a colony. All ticks were reared in glass tubes in an incubator regulated to 27°C and 90% RH and fed on chickens at room temperature. The avian hosts were always infested once only. Eggs and larvae Eggs laid by 20 females were counted daily, then separately pooled and the numbers of emerging larvae/day of oviposition determined. Eggs of 20 other females were not disturbed until 40 days after first hatching. The hatching percentage was defined by counting larvae and unhatched eggs. 10--14 days after hatching, groups of 20--50, 100--200, 200--300 and more than 400 larvae were placed on the avian hosts to feed. Fully engorged, detached larvae were counted daily and pooled separately/day of repletion, determining the respective premoulting periods.
Nymphs Immediately after moulting each unfed lst-instar n y m p h was thereafter held separately until reaching the adult stage. By weighing single nymphs of each instar before and after feeding, the amounts of bloodmeal were determined and the bloodmeal ratio then calculated. The premoulting periods of fed lst-instar (N1), 2nd-instar (N2) or 3rd-instar (N3) nymphs were evaluated daily, recording the resulting instar or sex.
Adults 42 pairs of newly m o u l t e d male and female adults were placed on chickens for feeding. After copulation each female, isolated in a rearing tube, was checked dally for oviposition. Weights and ratios of bloodmeal of female ticks were measured as for nymphs, and the bloodmeal size was related to the number of eggs deposited. The oviposition was assessed by counting the
367
daily egg output, until egg laying ceased, so quantifying the period and capacity of ovipositions. RESULTS
Eggs and larvae
Minimum incubation period of eggs required 15.9--17.0 days (mean 16.2 days), indepent of the day of oviposition; 51.0% and 96.0% completed embryonic development after 19.0 and 24 days, respectively (Fig. 1). Thus, embryogenesis was temporally unaffected and was always in the same range, irrespective of the date of oviposition. The hatching percentage ranged between 94.5% and 100% (mean 97.7%). The mean feeding period, remained with 5.9, 5.2, 5.3 and 5.4 days unchanged, if the avian hosts were infected with 20--50, 100--200, 200--300 100~
*
80-
~
70.
~ x:
6050-
m e
40-
'~
30"
~-
20' 10-
--//
16 17 ~8 19 2'0 2'1 2'2 23 2'4 2'~ 2'6 2'7 2'8 2'9 3'o ' ' Incubation period (days)
Fig. 1. Cumulative percentage of Argas (Argas) africolumbae larvae hatching. 100-
100-
90"
o'~
90-
"~
80-
~
70-
~
o~ > ~_
8o70-
60-
~
60-
5040-
~
50"
ff
40-
30-
30~-
20' 10.
2010-
I
I
I
Feeding period (days)
I
//
9
~o
~i
I'2
1'3
Premoulting period (daysl
Fig. 2. Cumulative percentage of engorged Argas (Argas) africolumbae larvae dropping from the host. Fig. 3. Cumulative percentage of Argas (Argas) africolumbae N1 emerging from engorged larvae.
368
and more than 400 larvae, respectively. 8.6% of the larvae began to drop on Day 4 after attaching, 64% had detached after 5 days and repletion was completed 8 days p.i. (Fig. 2). Larval premoulting period was 9--12 days, independent of the feeding time. Larvae detached after 5, 6, 7 and 8 days p.i. developed to N1 within (mean) 10.7, 9.9, 10.5 and 10.0 days, respectively. Considering the whole pool of larvae the premoulting phase required (mean) 10.2 days after dropping from the host (Fig. 3).
Nymphs Generally, there were only 2 nymphal instars. Unfed N1 (n = 84) weighed (mean) 0.99 mg and imbibed (mean) 3.65 mg of blood, resulting in a mean bloodmeal ratio of 7.39. All N1 moulted to N2 11--29 days after feeding (mean 13.7 days) (Fig. 4). Unfed and engorged male N~ weighed (mean) I00-
-~
_,~.
j.~../.~
90-
~ ~o-
~
¢v 60~ ~
40302010"
~"
Prernoulting period (days]
Fig. 4. Cumulative percentage of Argas (Argas) africolumbae N2 emerging from engorged N~. 10090-
~ 80~ ¢n
7060-
-=
50-
~ ~
40ao2010-
2
Premoultincj period (days)
Fig. 5. Cumulative percentage of adult Argas (Argas) africolumbae emerging from engorged N 2 . (e • ) females; (•,~) males.
369
0.90 mg and 3.94 mg, respectively, obtaining a meal size of 3.04 mg and having a mean bloodmeal ratio of 6.82, while for female N1 1.13 mg, 5.32 mg, 4.19 mg and 6.35 were determined. The weight of unfed and engorged N2 (n = 79) amounted {mean) 3.14 mg and 18.34 mg, respectively, the mean bloodmeal ratio being 5.86. Of 72 N2, 38 (= 52.8%) developed to males after 18--54 days (mean 28.3 days); and 33 (= 45.8%) to females after 14--59 days {mean 28.3 days); (Fig. 5). Just 1 N2, weighing 1.28 mg and 7.14 mg in the unfed and engorged state, respectively, with a bloodmeal ratio of 5.6 became a 1.79 mg heavy 3rd instar nymph in 14 days and moulted after further infestation, reaching a weight of 14.02 mg, to a female tick within 15 days. The male determined unfed N2 weighed (mean) 2.71 mg and ingested 12.23 mg blood, representing a mean bloodmeal ratio of 5.89; the respective values for female N~ were 3.81 mg, 17.45 mg and 5.98. Adults
The male:female ratio was 1.12:1.0. The amount of blood ingested by recently moulted females was (mean) 31.99 mg and their unfed and engorged weight were 16.15 mg and 46.44 mg, respectively. Fed females (n = 42) began to oviposit 7--18 days (mean 9.54 days) after feeding and copulation. The whole oviposition (n = 42) was 7--22 days (mean 14.0 days) and considering only days of egg-laying, 6--16 days (mean 10.7 days), because females frequently interrupted oviposition for 1--3 days and then continued. Eggs per oviposition numbered 38--163 (mean 102.9 eggs). The mean daily egg output amounted 7.4 and 9.7 eggs, related to the whole oviposition and the actual period of egg production, respectively. In respect to the egg production/day of oviposition this value increased to 59.5% on Day 6 and to 95.6% on day 13 (mean) (Fig. 6). Plotting the egg numbers against the weight or bloodmeal size of engorged females, the relationship was not linear, 100908070-
==so50"
.= •~ 40-
o
30-
-~ 2O~_ 10-
2 3 ~, .5 6 "} 8 9 1~) 1=1 12 1=3 14 115 Whole oviposition period (days) Fig. 6. Cumulative percentage of daily produced eggs during the w h o l e oviposition period of female Argas (Argas) africolumbae.
370 i.e., higher egg counts were not correlated with increased body weights. Females weighing 26.11--46.53 mg in the engorged state and having imbibed 12--35 mg of blood produced 118.2 eggs (mean), while engorged ticks with a weight of 44.45--80.29 mg and a bloodmeal size of 35--62 mg deposited 86.8 eggs (mean) only. DISCUSSION
Argas (Argas) africolumbae, the only species of 21 listed in the subgenus Argas known to be confined to the Ethiopian faunal region (Hoogstraal et al., 1979) and ecologically associated with birds in rocky situations and in buildings (Hoogstraal et al., 1975b, 1977), is closely related to the Palearctic pigeon parasitizing Argas (Argas) hermanni (Hoogstraal and Kohls, 1960b) and to Argas (Argas) reflexus (Hoogstraal and Kohls, 1960a). In addition to the morphological similarities the developmental patterns are also very close. Corresponding with Argas (Argas) hermanni (Khalil and Metwally, 1974) there were no distinct differences with regard to larval and nymphal cycles, sex ratio, fecundity, preoviposition and oviposition periods and nondiapause behaviour between this species and Argas (Argas) africolumbae, if reared under comparable laboratory conditions. The shape of curves, representing the respective developmental phases, and also the values of means, namely, showed convergent, sometimes even identical lines and were equal or at least similar, respectively, confirming also the few records of preliminary biological studies on this species (Hoogstraal et al., 1975a, 1977). A comparable reference to most other species of the subgenus Argas, such as Argas tridentatus, Argas vulgaris, Argas latus and Argas macrostigmatus (Filippova, 1961, 1966), Argas polonicus (Siuda et al., 1979), Argas himalayensis (Hoogstraal and Kaiser, 1973), Argas brevipes (Kohls et al., 1961), Argas dalei (Clifford et al., 1976), Argas cooleyi (Kohls and Hoogstraal, 1960), Argas lagenoplastis (Hoogstraai and Kohls, 1963), Argas falco (Kaiser and Hoogstraal, 1974), Argas rnonachus (Keirans et al., 1973), Argas dulus (Keirans et al., 1971), Argas neghmei (Kohls and Hoogstraal, 1961), Argas moreli (Keirans et al., 1979) and 1 unnamed species (Hoogstraal et al., 1979), is not possible, because biological base line data are altogether lacking so far or are, as for Argas cucumerinus (Clifford et al., 1978) still very preliminary. For Argas reflexus (Mfiller, 1939) and Argas ]aponicus (Uchikawa et al., 1967) only similar developmental patterns may be drawn off, the results of the Palearctic species, however, cannot be compared, because of the very different abiotic conditions used. Principally, there are also some conformities with the persicargasid species Argaspersicus (Khalil, 1979; E1 Kammah and Abdel Wahab, 1980), Argas arboreus (Hafez et al., 1971, 1972), Argas walkerae (Gothe and Koop, 1974), Argas robertsi (Hoogstraal et al., 1975), Argas miniatus (Magalh~es, 1979) and Argas radiatus (Medley and Ahrens, 1970). The developmental and reproduction pattern as well as the nondiapause
371 b e h a v i o u r o f Argas (Argas) africolumbae u n d e r c o n s t a n t abiotic c o n d i t i o n s indicate, t h a t t h e r e m a y be 2--3 generations annually. Thus, dense populations m a y develop, if this tick species is associated with d o m e s t i c a t e d birds, as c o u l d be d e m o n s t r a t e d for fowls in U p p e r Volta, because it has t h e n an ample source o f n o u r i s h m e n t and an e x c e p t i o n a l l y p r o t e c t e d e n v i r o n m e n t y e a r r o u n d . In c o n s e q u e n c e o f this c a p a c i t y in these ecological niches Argos (Argas) africolumbae is t h e n o f considerable epidemiological i m p o r t a n c e for the p o u l t r y i n d u s t r y , because o f its high natural i n f e c t i o n - p o t e n t i a l as vectors o f Borrelia anserina and Aegyptianella pullorurn. B o t h disease agents c o u l d be isolated with a respective m a x i m u m incidence in every wild-derived tick p o p u l a t i o n f r o m U p p e r Volta. T h e natural i n f e c t i o n - p o t e n t i a l o f every tick p o p u l a t i o n was equally strong and c o u l d c o n s t a n t l y be p r o v e n b y transstadial transmission, since t h r o u g h infestation o f highly susceptible chickens with n y m p h s a n d / o r adults f r o m every original tick h a b i t a t b o t h agents were m i c r o s c o p i c a l l y d e m o n s t r a b l e in Giemsa-stained b l o o d smears. A transovarial passage o f the agents o n t o the n e x t tick-generation was also possible, h o w e v e r , it did n o t o c c u r s i m u l t a n e o u s l y with the h o r i z o n t a l transmission. The infectivity r e m a i n e d qualitatively and q u a n t i t a t i v e l y u n c h a n g e d in all p o p u l a t i o n s involved and persisted during the entire investigation p e r i o d o f -nine m o n t h s u p t o the third r e p l e t i o n series. T h e rate o f m o r b i d i t y and m o r t a l i t y a m o u n t e d t o 100% in every r e p l e t i o n , the infested animals were always i n f e c t e d with b o t h agents s i m u l t a n e o u s l y and died in a l m o s t equal p r o p o r t i o n s o f 52.4% and 47.6% o f a borreliosis and aegyptianellosis, respectively ( G o t h e et al., 1981a). F u r t h e r m o r e , it was d e m o n s t r a t e d t h a t all populations o f this tick species f r o m U p p e r V o l t a also display a paralysis-inducing c a p a c i t y ( G o t h e et al., 1 9 8 1 b ) .
REFERENCES Clifford, C.M., Keirans, J.E., Hoogstraal, H. and Corwin, D., 1976. Observations on the subgenus Argos (Ixodoidea: Argasidae: Argas). A. (A.) dalei, new species, parasitizing the burrowing owl in Peru. Ann. Entomol. Soc. Am., 69: 917--925. Clifford, C.M., Hoogstraal, H., Keirans, J.E., Rice, R.C.A. and Dale, W.E., 1978. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argos). 14. Identity and biological observations of Argos (A.) cucumerinus from Peruvian seaside cliffs and a summary of the status of the subgenus in the neotropieal faunal region. J. Med. Entomol., 15: 57--73. Converse, J.D., Hoogstraal, H., Moussa, M.I., Casals, J. and Kaiser, M.N., 1975. Pretoria virus: a new African agent in the tickborne Dera Ghazi Khan (DGK) group and antigenic relationships within the DGK group. J. Med. Entomol., 12: 202--205. El Kammah, K.M. and Abdel Wahab, K.S., 1980. Argas (Persicargas) persicus life cycle under controlled and outdoor conditions. Acarologia, 21: 163--172. Filippova, N.A., 1961. Materials on ticks belonging to the subfamily Argasinae. Part I. Adult ticks and larvae of the genus Argas Latr., group reflexus. Zool. Zh., 40: 1815--1826. Filippova, N.A., 1966. Argasid ticks (Argasidae). Fauna SSSR, Paukoobraznye, 4: 255.
372 Gothe, R. and Koop, E., 1974. Zur biologischen Bewertung der Validit~t von Argas (Persicargas) persicus (Oken, 1818), Argas (Persicargas) arboreus Kaiser, Hoogstraal und Kohls, 1964 und Argas (Persicargas) walkerae Kaiser und Hoogstraal, 1969. I. Untersuchungen zur Entwicklungsbiologie. Z. Parasitenkd., 44: 299--317. Gothe, R., Buchheim, C. and Schrecke, W., 1981a. Argas (Persicargas) persicus und Argas (Argas) africolumbae als natilrliche Llbertr~iger von Borrelia anserina und Aegyptianella pullorum in Obervolta. Berl. MUnch. Tier~rtzl. Wochenschr., 94: 280--285. Gothe, R., Buchheim, C. and and Schrecke, W., 1981b. Zur Paralyse-induzierenden Kapazit~t wildst~mmiger Argas (Persicargas) persicus- und Argas (Argas) #fricolumbaePopulationen aus Obervolta. Berl. MQnch. Tier~rtzl. Wochenschr., 94: 299---302. Hafez, M., Abdel-Malek, A.A. and Guirgis, S.S., 1971. The subgenus Persicargas (Ixodoidea, Argasidae, Argas). 12. Biological studies on the immature stages of A. (P.) arboreus Kaiser, Hoogstraal & Kohls in Egypt. J. Med. Entomol., 8: 421--429. Hafez, M., Abdel-Malek, A.A. and Guirgis, S.S., 1972. The subgenus Persicargas (Ixodoidea, Argasidae, Argas). 14. Biological studies on the adult stage of A. (P.) arboreus Kaiser, Hoogstraal & Kohls in Egypt. J. Med. Entomol., 9: 19--29. Hoogstraal, H. and Kaiser, M.N., 1973. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 7. A. (A.) himalayensis, new species, parasitizing the snow partridge, Lerwa lerwa, in Nepal. Ann. Entomol. Soc. Am., 66: 1--3. Hoogstraal, H. and Kohls, G.M., 1960a. Observations on the subgenus Argas (Ixodoidea, Argasidae, Argas). 1. Study of A. reflexus reflexus (Fabricius, 1794), the European bird argasid. Ann. Entomol. Soc. Am., 53: 611--618. Hoogstraal, H. and Kohls, G.M., 1960b. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 3. A biological and systematic study of A. reflexus hermanni Audouin, 1827 (revalidated), the African bird argasid. Ann. Entomol. Soc. Am., 53: 743--755. Hoogstraal, H. and Kohls, G.M., 1963. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 6. Redescription and biological notes on A. lagenoplastis Frogatt, 1906, of Australian fairy martins, Hylochelidon ariel (Gould). Ann. Entomol. Soc. Am., 56: 577--582. Hoogstraal, H., Kaiser, M.N. and McClure, H.E., 1975a. The subgenus Persicargas (Ixodoidea: Argasidae: Argas). 20. A. (P.) robertsi parasitizing nesting wading birds and domestic chickens in the Australian and Oriental regions, viral infections, and host migration. J. Med. Entomol., 11: 513--524. Hoogstraal, H., Kaiser, M.N., Walker, J.B., Ledger, J.A., Converse, J.D. and Rice, R.C,A., 1975b. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 10. A. (A.) africolumbae, n. sp., a Pretoria virus-infected parasite of birds in Southern and Eastern Africa. J. Med. Entomol., 12: 194--201. Hoogstraal, H., Wassef, H.Y., Easton, E.R. and Dixon, J.E.W., 1977. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 12. Argas (A.) africolumbae: variation, bird hosts, and distribution in Kenya, Tanzania, and South and South-West Africa. J. Med. Entomol., 13: 441--445. Hoogstraal, H., Clifford, C.M., Keirans, J.E. and Wassef, H.Y., 1979. Recent developments in biomedical knowledge of Argas ticks (Ixodoidea: Argasidae). In: J.G. Rodriguez (Editor), Recent Advances in Acarology. Vol. 2. Academic Press, New York, San Francisco, London, pp. 269--278. Kaiser, M.N. and Hoogstraal, H., 1974. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 9. A. (A.) falco, new species, parasitizing kestrels in Western Australia. Ann. Entomol. Soc. Am., 67: 5--10. Keirans, J.E., Clifford, C.M. and Maldonado Capriles, J., 1971. Argas (Argas) dulus, new species (Ixodoidea: Argasidae), from nests of the palm chat Dulus dominicus in the Dominican Republic. Ann. Entomol. Soc. Ann., 64: 1410--1413.
373 Keirans, J.E., Radovsky, F.J. and Clifford, C.M., 1973. Argas (Argas) monachus, new species (Ixodoidea: Argasidae), from nests of the monk parakeet, Myiopsitta monachus, in Argentina. J. Med. Entomol., 10: 511--516. Keirans, J.E., Hoogstraal, H. and Clifford, C.M., 1979. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 16. Argas (A.) moreli, new species, and keys to neotropical species of the subgenus. J. Med. Entomol., 15: 246--252. Khalil, G.M., 1979. The subgenus Persicargas (Ixodoidea: Argasidae: Argas). 31. The life cycle of A. (P.) persicus in the laboratory. J. Med. Entomol., 16: 200--206. Khalil, G.M. and Metwally, S.A., 1974. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 8. The life cycle of A. (.4.) hermanni. J. Med. Entomol., 11: 355--362. Kohls, G.M. and Hoogstraal, H., 1960. Observations on the subgenus Argas (Ixodoidea: Argasidae, Argas). 2. A. cooleyi, new species, from Western North American birds. Ann. Entomol., Soc. Am., 53: 625--631. Kohls, G.M. and Hoogstraal, H., 1961. Observations on the subgenus Argas (Ixodoidea, Argasidae, Argas). 4. A. neghmei, new species, from poultry houses and human habitations in Northern Chile. Ann. Entomol. Soc. Am., 54 : 844--851. Kohls, G.M., Hoogstraal, H. and Clifford, C.M., 1961. Observations on the subgenus Argas (Ixodoidea, Argasidae, Argas). 5. Study of A. brevipes Banks, 1908, from birds in Arizona and California, U.S.A., and Baja California, Mexico. Ann. Entomol. Soc. Am., 54: 869--877. Magalha'es de, F.E.P., 1979. Novos aspectos morfol6gicos, biol6gicos e t6xicos de Argas (Persicargas) miniatus Koch, 1844 (Ixodoidea -- Argasidae) no estado do Rio de Janeiro. Tese, apresentada ~ Universidade Federal Rural do Rio de Janeiro para o b t e n ~ o do grau de "Magister Seientiae". Medley, J.G. and Ahrens, E., 1970. Life history and bionomics of two American species of fowl ticks (Ixodoidea, Argasidae, Argas) of the subgenus Persicargas. Ann. Entomol. Soc. Am., 63: 1591--1594. M~ller, K.H., 1939. Zur Biologie der Taubenzecke Argas columbarum. Inaugural-Dissertation, Berlin. Siuda, K., Hoogstraal, H., Clifford, C.M. and Wassef, H.Y., 1979. Observations on the subgenus Argas (Ixodoidea: Argasidae: Argas). 17. Argas (A.) polonicus sp. n. parasitizing domestic pigeons in Krakow, Poland. J. Parasitol., 65: 170--181. Uchikawa, K., Sato, A. and Kugimoto, M., 1967. Studies on the argasid infesting the Japanese house-martin, Delichon urbica. Med. J. Shinshu Univ., 12: 141--155.