Laboratory Biology of Ornithodoros (Alectorobius) puertoricensis (Acari: Argasidae) R. G. ENDRIS,14 T. M. HASLETT,2 M. J. MONAHAN,1 AND J. G. PHILLIPS3
J. Med. Entomol. 28(1): 49-62 (1991)
ABSTRACT The laboratory biology of Ornithodoros (Alectorobius) puertoricensis Fox was studied over a 2-yr period. Approximately 100-150 ticks were reared individually at each of four temperatures: 22, 27, 33, and 40°C and 90-95% RH. The mean egg incubation periods at those temperatures were 20.3, 11.1, 7.3, and 6.1 d, respectively. The average larval feeding period was 5.8 ± 1.5 d for 15,875 larvae that fed on guinea pigs. The development times for first to fourth nymphal instars were as follows: 11.7, 48.5, 75.1, and 92.1 d, respectively, at 22°C; 5.8, 18.8, 38.0, and 36.0 d, respectively, at 27°C; 4.2, 10.5, 14.9, and 38.1 d, respectively, at 33°C; and 5.8, 10.7, 21.2, and 35.3 d, respectively, at 40°C. Males usually eclosed after three or four molts, and females usually eclosed after four or five molts. Approximately 10% of all nymphs required more than one blood meal per instar at least once during development. Twenty pairs of adults were held at each of three temperatures (22, 27, and 33°C) for a year to study reproductive behavior. The number of gonotrophic cycles per female per year was 6.9, 9.8, and 10.8 at 22, 27, and 33°C, respectively. The mean duration of the gonotrophic cycle was 42.3 d at 22°C, 25.5 d at 27°C, and 20.5 d at 33°C. Mean egg production per female per gonotrophic cycle was 151 at 22°C, 117 at 27°C, and 130 at 33°C and was not affected by temperature. O. puertoricensis did not exhibit autogeny or parthenogenesis. Hyperparasitism was observed in immatures and adults. KEY WORDS tion
Arachnida, Ornithodoros (Alectorobius) puertoricensis, biology, reproduc-
THE EARLIEST KNOWN COLLECTION of the species now called Ornithodoros (Alectorobius) puertoricensis Fox, 1947 was made by Dunn in Panama in 1931, and he identified the ticks as O. talaje (Guerin-Meneville) (Fairchild et al. 1966). Subsequently, Fox (1947) described O. puertoricensis from larvae collected from rats in San Juan, Puerto Rico. Fairchild et al. (1966) identified the specimens collected by Dunn as O. puertoricensis. A redescription of the species by Endris et al. (1989) provided extensive data on geographic distribution and host relationships. In addition to the original species description, Fox (1947) provided initial observations on the biology by rearing 65 individual ticks on white rats at ambient temperature. Davis (1955) conducted additional studies on the biology and the relationship of O. puertoricensis to relapsing fever spirochetes. He showed that O. puertoricensis was incapable of transmitting spirochetes (Borrelia sp.) from O. talaje and O. dugesi Mazzotti. 1
To date, 167 species in five genera of the family Argasidae, including about 100 species in the genus Ornithodoros alone, have been described (Hoogstraal 1985). Although enough is known of the biology of many species that they can be assigned to subgenera, few detailed studies on the life history and biology of Ornithodoros species have been published. Recent studies on the biology of the following species have been reported: O. erraticus Lucas (El Shoura 1987), O. turicata Duges (Beck et al. 1986), O. rostratus Aragao (Gugliemone & Hadani 1980) and O. gurneyi Warburton (Doube 1975). In this manuscript, we present the results of detailed studies on the life cycle and laboratory biology of O. puertoricensis that were conducted over a 2-yr period. Results of these studies are the cornerstone for subsequent work on the ability of O. puertoricensis to harbor and transmit African swine fever virus and on the effect of infection by that virus on tick populations. Materials and Methods
USDA-ARS, Plum Island Animal Disease Center, P.O. Box 848, Greenport, N.Y. 11944. 2 Long Island Jewish Medical Center, Department of Pharmacy, New Hyde Park, N.Y. 11042. 3 Statistician, USDA-ARS-NAA, 600 Mermaid Lane, Philadelphia, Penn. 19118. * Current address: Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, N.J. 07065.
Ticks. The colony was started with 190 nymphs and adults collected in Haiti (Endris et al. 1989) by using the methods of Butler et al. (1984, 1985). Rearing methods for the colony and for individual ticks have been described previously (Endris et al. 1986).
0022-2585/91/0049-0062$02.00/0 © 1991 Entomological Society of America
50
Vol. 28, no. 1
JOURNAL OF MEDICAL ENTOMOLOGY
Life History Study. Larvae were fed as a group on guinea pigs, Cavia opera porcellus, before individual rearing. When a fed larva detached from the guinea pig, it was placed separately into a 25ml (7 dram) vial with a mesh top. Subsequent feeding of nymphs was accomplished as described by Endris et al. (1986). Suckling mice were placed on each vial for feeding for three 24-h periods per week. Suckling mice were used for blood meals because they were relatively inexpensive, readily acceptable to the ticks, and small enough to facilitate the feeding of individual ticks. Ticks were observed daily to determine if feeding or molting had occurred. One hundred to 150 ticks were reared individually at 90-95% RH at each of four temperatures: 22, 27, 33, and 40°C. A constant 14:10 (L:D) photoperiod was provided with bulbs (Vitalite; Durotest, Bergen, NJ.) that produced light with the spectral composition of sunlight. Oviposition Study. Twenty pairs of unfed virgin ticks were held in 25-ml vials at each of three temperatures: 22, 27, and 33°C. Ticks were offered suckling mice for a 24-h period twice weekly and were observed for feeding. After feeding, females were not offered another blood meal until after they laid eggs. If they failed to lay eggs within 30 d, then a second blood meal was offered until oviposition occurred. Eggs were collected daily, counted, and placed in separate vials. Larvae were collected daily and counted to determine the incubation period and the egg hatch rate. Oviposition studies at each temperature were conducted for at least 1 yr. Single-Mating Egg Production. An experiment was conducted to determine how many eggs a female could produce after a single mating. Fifteen pairs of virgin males and females were fed and held together for 6 d at 33°C. After 6 d, the males were removed and it was assumed that females had been mated once. Females were not dissected to verify that only one spermatophore was present. The study was continued for 212 d. Ticks were observed daily, eggs were collected and counted, and ticks were offered a blood meal for three 24-h periods per week. Eggs were held at 33°C, and 95% RH until they hatched, and the percentage of egg viability was determined for each batch. At the end of the experiment, males were again placed with the females. The ticks were then monitored to verify that fertile eggs were subsequently produced. Parthenogenesis Study. Twenty-two virgin female ticks were held individually at 27°C for >1 yr without exposure to male ticks. The ticks were offered a blood meal twice monthly and were observed for feeding and egg laying weekly. Males were placed with each female after 1 yr. Pairs were offered a blood meal twice per week and were observed for egg laying and egg hatching. Autogeny Study. Thirty-two pairs of virgin, unfed females and fed males were held at 33°C without feeding for >1 yr and were observed weekly for
oviposition. After a year, females and males were fed, and vials were observed for the presence of eggs and subsequent larvae. Sex Ratio Experiment. All larvae were reared on guinea pigs held at 27°C. To determine if the sex ratio of adults was affected by rearing temperature, 200 fed larvae were held at each of three temperatures: 22, 27, and 33°C. Ticks were fed and observed weekly until all had become adults. Statistical Analysis. Data were analyzed by means of the SAS program (SAS Institute 1982). Comparisons for significance were made with the Bonferroni's (Dunn) t test (Miller 1981, 67-70). Bonferroni's t test was used so that all statements of statistical significance hold at the P = 0.05 level simultaneously. In contrast, Duncan's multiple range test was not used, because it only controls P for paired comparisons and does not hold for a collection of treatment comparisons simultaneouslyResults The generalized life cycle of O. puertoricensis is shown in Fig. 1. Larvae require several days on the host to feed to repletion. After dropping off the host, larvae molted to the first instar and then molted again without feeding to the second instar. The subsequent instars fed and after a period of time molted to either the next instar or to an adult. Larval Feeding. The duration of larval feeding on guinea pigs is depicted in Fig. 2. The number of days (x ± SD) needed for 15,875 larvae to feed on 45 guinea pigs was 5.8 ± 1.5. On one occasion, 153 larvae were recovered after having fed on a white rat. The mean duration of feeding for these larvae was 6.2 ± 0.3 d. Larvae were unwilling to feed until « 3 d of age. Older larvae (>7 d) attached to a host more readily than 3-d-old individuals. Development of Immatures. The mean development time (days) for each instar at each temperature is presented in Table 1. Some ticks in an instar exhibited a longer than expected development time. When that occurred, it was usually a result of the ticks' refusal to feed, which resulted in a prolonged prefeeding period. However, once the tick fed, it was physiologically committed to molting to the next instar. This phenomenon is shown by two facts in Table 1: first, for each instar at each temperature, the prefeeding period was generally much longer than the premolting period; second, the standard deviation of the premolting (postfeeding) period was generally much smaller than that of the prefeeding period. Notably, at each temperature, the mean development time of each instar was longer than that of the preceding instar. Statistical comparisons between the four rearing temperatures for mean development times for each development period are shown in Table 1. Development times for fed larvae and first to third instars at 22°C were significantly longer than those at the
January 1991
ENDRIS ET AL.: BIOLOGY OF
Ornithodoros (A.) puertoricensis
Life Cycle of Ornithodoros puertoricensis .^
EGG
51
J. Med. Entomol. 28(1): 49-62 (1991)
ABSTRACT The laboratory biology of Ornithodoros (Alectorobius) puertoricensis Fox was studied over a 2-yr period. Approximately 100-150 ticks were reared individually at each of four temperatures: 22, 27, 33, and 40°C and 90-95% RH. The mean egg incubation periods at those temperatures were 20.3, 11.1, 7.3, and 6.1 d, respectively. The average larval feeding period was 5.8 ± 1.5 d for 15,875 larvae that fed on guinea pigs. The development times for first to fourth nymphal instars were as follows: 11.7, 48.5, 75.1, and 92.1 d, respectively, at 22°C; 5.8, 18.8, 38.0, and 36.0 d, respectively, at 27°C; 4.2, 10.5, 14.9, and 38.1 d, respectively, at 33°C; and 5.8, 10.7, 21.2, and 35.3 d, respectively, at 40°C. Males usually eclosed after three or four molts, and females usually eclosed after four or five molts. Approximately 10% of all nymphs required more than one blood meal per instar at least once during development. Twenty pairs of adults were held at each of three temperatures (22, 27, and 33°C) for a year to study reproductive behavior. The number of gonotrophic cycles per female per year was 6.9, 9.8, and 10.8 at 22, 27, and 33°C, respectively. The mean duration of the gonotrophic cycle was 42.3 d at 22°C, 25.5 d at 27°C, and 20.5 d at 33°C. Mean egg production per female per gonotrophic cycle was 151 at 22°C, 117 at 27°C, and 130 at 33°C and was not affected by temperature. O. puertoricensis did not exhibit autogeny or parthenogenesis. Hyperparasitism was observed in immatures and adults. KEY WORDS tion
Arachnida, Ornithodoros (Alectorobius) puertoricensis, biology, reproduc-
THE EARLIEST KNOWN COLLECTION of the species now called Ornithodoros (Alectorobius) puertoricensis Fox, 1947 was made by Dunn in Panama in 1931, and he identified the ticks as O. talaje (Guerin-Meneville) (Fairchild et al. 1966). Subsequently, Fox (1947) described O. puertoricensis from larvae collected from rats in San Juan, Puerto Rico. Fairchild et al. (1966) identified the specimens collected by Dunn as O. puertoricensis. A redescription of the species by Endris et al. (1989) provided extensive data on geographic distribution and host relationships. In addition to the original species description, Fox (1947) provided initial observations on the biology by rearing 65 individual ticks on white rats at ambient temperature. Davis (1955) conducted additional studies on the biology and the relationship of O. puertoricensis to relapsing fever spirochetes. He showed that O. puertoricensis was incapable of transmitting spirochetes (Borrelia sp.) from O. talaje and O. dugesi Mazzotti. 1
To date, 167 species in five genera of the family Argasidae, including about 100 species in the genus Ornithodoros alone, have been described (Hoogstraal 1985). Although enough is known of the biology of many species that they can be assigned to subgenera, few detailed studies on the life history and biology of Ornithodoros species have been published. Recent studies on the biology of the following species have been reported: O. erraticus Lucas (El Shoura 1987), O. turicata Duges (Beck et al. 1986), O. rostratus Aragao (Gugliemone & Hadani 1980) and O. gurneyi Warburton (Doube 1975). In this manuscript, we present the results of detailed studies on the life cycle and laboratory biology of O. puertoricensis that were conducted over a 2-yr period. Results of these studies are the cornerstone for subsequent work on the ability of O. puertoricensis to harbor and transmit African swine fever virus and on the effect of infection by that virus on tick populations. Materials and Methods
USDA-ARS, Plum Island Animal Disease Center, P.O. Box 848, Greenport, N.Y. 11944. 2 Long Island Jewish Medical Center, Department of Pharmacy, New Hyde Park, N.Y. 11042. 3 Statistician, USDA-ARS-NAA, 600 Mermaid Lane, Philadelphia, Penn. 19118. * Current address: Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, N.J. 07065.
Ticks. The colony was started with 190 nymphs and adults collected in Haiti (Endris et al. 1989) by using the methods of Butler et al. (1984, 1985). Rearing methods for the colony and for individual ticks have been described previously (Endris et al. 1986).
0022-2585/91/0049-0062$02.00/0 © 1991 Entomological Society of America
50
Vol. 28, no. 1
JOURNAL OF MEDICAL ENTOMOLOGY
Life History Study. Larvae were fed as a group on guinea pigs, Cavia opera porcellus, before individual rearing. When a fed larva detached from the guinea pig, it was placed separately into a 25ml (7 dram) vial with a mesh top. Subsequent feeding of nymphs was accomplished as described by Endris et al. (1986). Suckling mice were placed on each vial for feeding for three 24-h periods per week. Suckling mice were used for blood meals because they were relatively inexpensive, readily acceptable to the ticks, and small enough to facilitate the feeding of individual ticks. Ticks were observed daily to determine if feeding or molting had occurred. One hundred to 150 ticks were reared individually at 90-95% RH at each of four temperatures: 22, 27, 33, and 40°C. A constant 14:10 (L:D) photoperiod was provided with bulbs (Vitalite; Durotest, Bergen, NJ.) that produced light with the spectral composition of sunlight. Oviposition Study. Twenty pairs of unfed virgin ticks were held in 25-ml vials at each of three temperatures: 22, 27, and 33°C. Ticks were offered suckling mice for a 24-h period twice weekly and were observed for feeding. After feeding, females were not offered another blood meal until after they laid eggs. If they failed to lay eggs within 30 d, then a second blood meal was offered until oviposition occurred. Eggs were collected daily, counted, and placed in separate vials. Larvae were collected daily and counted to determine the incubation period and the egg hatch rate. Oviposition studies at each temperature were conducted for at least 1 yr. Single-Mating Egg Production. An experiment was conducted to determine how many eggs a female could produce after a single mating. Fifteen pairs of virgin males and females were fed and held together for 6 d at 33°C. After 6 d, the males were removed and it was assumed that females had been mated once. Females were not dissected to verify that only one spermatophore was present. The study was continued for 212 d. Ticks were observed daily, eggs were collected and counted, and ticks were offered a blood meal for three 24-h periods per week. Eggs were held at 33°C, and 95% RH until they hatched, and the percentage of egg viability was determined for each batch. At the end of the experiment, males were again placed with the females. The ticks were then monitored to verify that fertile eggs were subsequently produced. Parthenogenesis Study. Twenty-two virgin female ticks were held individually at 27°C for >1 yr without exposure to male ticks. The ticks were offered a blood meal twice monthly and were observed for feeding and egg laying weekly. Males were placed with each female after 1 yr. Pairs were offered a blood meal twice per week and were observed for egg laying and egg hatching. Autogeny Study. Thirty-two pairs of virgin, unfed females and fed males were held at 33°C without feeding for >1 yr and were observed weekly for
oviposition. After a year, females and males were fed, and vials were observed for the presence of eggs and subsequent larvae. Sex Ratio Experiment. All larvae were reared on guinea pigs held at 27°C. To determine if the sex ratio of adults was affected by rearing temperature, 200 fed larvae were held at each of three temperatures: 22, 27, and 33°C. Ticks were fed and observed weekly until all had become adults. Statistical Analysis. Data were analyzed by means of the SAS program (SAS Institute 1982). Comparisons for significance were made with the Bonferroni's (Dunn) t test (Miller 1981, 67-70). Bonferroni's t test was used so that all statements of statistical significance hold at the P = 0.05 level simultaneously. In contrast, Duncan's multiple range test was not used, because it only controls P for paired comparisons and does not hold for a collection of treatment comparisons simultaneouslyResults The generalized life cycle of O. puertoricensis is shown in Fig. 1. Larvae require several days on the host to feed to repletion. After dropping off the host, larvae molted to the first instar and then molted again without feeding to the second instar. The subsequent instars fed and after a period of time molted to either the next instar or to an adult. Larval Feeding. The duration of larval feeding on guinea pigs is depicted in Fig. 2. The number of days (x ± SD) needed for 15,875 larvae to feed on 45 guinea pigs was 5.8 ± 1.5. On one occasion, 153 larvae were recovered after having fed on a white rat. The mean duration of feeding for these larvae was 6.2 ± 0.3 d. Larvae were unwilling to feed until « 3 d of age. Older larvae (>7 d) attached to a host more readily than 3-d-old individuals. Development of Immatures. The mean development time (days) for each instar at each temperature is presented in Table 1. Some ticks in an instar exhibited a longer than expected development time. When that occurred, it was usually a result of the ticks' refusal to feed, which resulted in a prolonged prefeeding period. However, once the tick fed, it was physiologically committed to molting to the next instar. This phenomenon is shown by two facts in Table 1: first, for each instar at each temperature, the prefeeding period was generally much longer than the premolting period; second, the standard deviation of the premolting (postfeeding) period was generally much smaller than that of the prefeeding period. Notably, at each temperature, the mean development time of each instar was longer than that of the preceding instar. Statistical comparisons between the four rearing temperatures for mean development times for each development period are shown in Table 1. Development times for fed larvae and first to third instars at 22°C were significantly longer than those at the
January 1991
ENDRIS ET AL.: BIOLOGY OF
Ornithodoros (A.) puertoricensis
Life Cycle of Ornithodoros puertoricensis .^
EGG
51
