Exp Appl Acarol DOI 10.1007/s10493-015-9888-1
Life cycle of Ornithodoros rostratus (Acari: Argasidae) ticks feeding on mice under laboratory conditions Gabriel Cerqueira Alves Costa • Adriana Coelho Soares • Marcos Hora´cio Pereira • Nelder Figueiredo Gontijo • Maurı´cio Roberto Viana Sant’Anna • Ricardo Nascimento Araujo
Received: 12 September 2014 / Accepted: 17 February 2015 Ó Springer International Publishing Switzerland 2015
Abstract Ornithodoros rostratus Araga˜o is an argasid tick found in Bolivia, Paraguay, Argentina and Brazil. Only limited studies about O. rostratus have been conducted and several aspects of their life cycle differ among studies or remain unexplored. In order to better elucidate the biology of O. rostratus, the present work describes its life cycle when feeding on mice under laboratory conditions. To complete their life cycle on mice, O. rostratus goes through a larval stage, 3–6 nymphal instars (nymph 1–6) and adult male and female. Adults can be originated from nymph 3–6. Nymphs 4 with higher weight after feeding tend to originate adults. Adults originated from early instars tended to be lighter. Females tended to be heavier than males. Larvae needed on average 2.7 days to complete their blood meal whereas other instars ranged from 17.3 to 78.3 min. The capacity to ingest blood was higher in larvae and females in comparison to males. The preecdysis period ranged from 5 to 12.5 days. After one blood meal, females remain on average 15.2 ± 5.8 days laying 276.8 ± 137.2.9 eggs. Females originated from nymph 4 had similar oviposition time, egg incubation and conversion ingested blood/number of eggs produced, but presented lower initial weigh and weigh gain, generating fewer eggs. Our results added novel information on O. rostratus biology and was discussed considering the variability of argasid populations and in context with the differences about their life cycle described in previous works. Keywords Ornithodoros rostratus Argasidae Life cycle Mice Hematophagy Reproduction
G. C. A. Costa A. C. Soares M. H. Pereira N. F. Gontijo M. R. V. Sant’Anna R. N. Araujo (&) Laborato´rio de Fisiologia de Insetos Hemato´fagos, Departamento de Parasitologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Av. Presidente Antoˆnio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil e-mail: [email protected] M. H. Pereira N. F. Gontijo R. N. Araujo Instituto Nacional de Cieˆncia e Tecnologia em Entomologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-909, Brazil
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Introduction Ornithodoros rostratus Araga˜o is an argasid tick found in Bolivia, Paraguay, Argentina and Brazil feeding on humans and several mammal species (Guglielmone et al. 2003; Hoogstraal 1985; Nava et al. 2007; Venzal et al. 2006). The importance of O. rostratus ticks are related to the cutaneous reactions caused by their parasitism. Its bite is painful and itchy, and can cause ecchymotic lesions, inflammation and blisters on the feeding site (Araga˜o 1936; Boero 1957; Estrada-Pena and Jongejan 1999). In addition, they are vectors of the Rocky Mountain spotted fever, caused by the bacteria Rickettsia rickettsii (Hoogstraal 1985) and were described harbouring Coxiella burnetii (Almeida et al. 2012), the causative agent of acute Q fever and endocarditis in humans and animals (Angelakis and Raoult 2010; Griffin et al. 2012). A few years ago, a novel O. rostratus infestation focus was identified in a farm located in the Pantanal region of Mato Grosso do Sul state, Brazil, where domestic animals such as dogs and pigs were reported as hosts (Canc¸ado et al. 2008; Ribeiro et al. 2013). Few studies were conducted concerning the life story of O. rostratus. Most of them showed data from only some developing instars and not many parameters of their life cycle were evaluated when fed on dogs and chickens (Brumpt 1915), guinea pigs (Guglielmone and Hadani 1980), rabbits and reptiles (Venzal and Estrada-Pena 2006). A more detailed analysis was carried out on rabbits (Ribeiro et al. 2013). Nevertheless, aspects of their life cycle differed among studies and others remain unexplored. Ribeiro et al. (2013) reported that O. rostratus larvae complete their blood feed after 33.5 min on average when feeding on rabbits while other studies reported significantly longer feeding periods on mammals that lasted up to 8 days (Brumpt 1915; Guglielmone and Hadani 1980; Venzal and Estrada-Pena 2006). Some studies describe only five nymphal instars (Brumpt 1915; Guglielmone and Hadani 1980) whereas others indicate the existence of a sixth instar (Ribeiro et al. 2013). Although adults may be originated from nymph 3, there is no information about reproductive parameters of females originated from different nymphal instars. Also, the amount of blood ingested considering the initial weigh of each instar was not evaluated in previous works. The present work aims at describing the life cycle of O. rostratus fed on mice (a classic laboratory model) under laboratory conditions. The data generated about their life cycle added novel information on O. rostratus biology highlighting the variability of Argasid populations, also helping to elucidate the differences presented in previous works.
Materials and methods Ticks and colony rearing Ticks used in the experiments were collected in Nhecolaˆndia, Mato Grosso do Sul, Brazil (19° 030 S, 56° 470 W). Collected specimens were taken to the laboratory, identified to the species level (Venzal et al. 2006) and were maintained inside an incubator under semicontrolled conditions of temperature (28 ± 2 °C) and humidity (85 ± 10 %). Ticks were fed on Swiss mice (Mus musculus) every 20 days. Feeding experiments Larvae were fed in Swiss mice using the methodology described by Bouchard and Wikel (2005). Briefly, feeding chambers were made with 1.5 mL polypropylene centrifuge tubes
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that were cut at approximately 1 cm from the top and were then fixed on the mice’s back using nontoxic glue. The caps were pierced with a thin needle to enable air circulation. 25 larvae were put inside the chambers that were closed and maintained until larvae had completed the whole feeding. Chambers were examined daily to record attachment and release. Larvae that had not attached until 24 h after being added to the feeding chamber (first examination of the chamber) were removed and discarded. Ten to 20 day old larvae were used in the experiments. Nymphs and adults were allowed to feed once on anaesthetized mice in a room at 28 ± 2 °C. The specimens used in the experiments had between 25 and 35 days after molt. The feeding area was delimited using a 2 9 1.5 cm cylinder fixed with adhesive tape in the abdomen area of the mice. Each mouse was used as feeding source for a maximum of 10 specimens earlier than nymph 3 or 5 older than nymph 3. All blood feeds were recorded using a digital camera (Canon EOS600D) to measure the number of bites and the total contact time (TCT-period between the bite and the removal of the mouthparts from the host) for each tick. Specimens were weighted before (initial weight—IW) and after feeding (final weight—FW) to measure the weight gain (WG—FW minus IW). After feeding, nymphs were kept individually in rearing containers where they were monitored daily to measure the preecdysis time and the number of nymphs that molted or died. Reproductive experiments To analyze reproductive parameters, couples were paired using heavier females and males that were originated from each nymphal instar. At 25–35 days after molt, couples were fed on mice, weighed before and after feeding and maintained on individual containers. They were then evaluated daily in order to measure the number of eggs layed per female and the period which females remained laying eggs after one single feeding (one gonotrophic cycle). The egg incubation period and percentage of eclosion were also accessed. Statistical analysis The data was analyzed using the GraphPad PrismÒ 5 for Windows. The normality of the data was tested by the Kolmogorov–Smirnov test and Kruskal–Wallis followed by Dunn’s test to analyze pairwise differences between the parameters evaluated. The significance level was set at p \ 0.05. Ethics committee All procedures were in accordance with the manuals for animal experimentation and were approved by the Ethics Committee for Animal Use (CETEA/ICB-UFMG) under the protocol 301/2013.
Results Life stages and molt parameters To complete their life cycle on mice, O. rostratus goes through a larval stage, 3–6 nymphal instars and adult female (Fig. 1) and male (Table 1). When feeding on mice, 68 % of the
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Fig. 1 Dorsal (a) and ventral (b) view of a female of Ornithodoros rostratus
Table 1 Molting parameters of Ornithodoros rostratus fed on mice Stage/ instar
N
Number that molted
Number that molted to
Ratio female:male
Nymph
Female
Male
Preecdysis (days)
Larva
300
204 (68 %)
204 (100 %)
0
0
5.0 ± 1.6a
Nymph 1
156
75 (48 %)
75 (100 %)
0
0
6.8 ± 1.6ab
Nymph 2
68
62 (91 %)
62 (100 %)
0
0
8.2 ± 1.4bc
Nymph 3
125
95 (76 %)
78 (82 %)
0
17 (18 %)
0.0:1.0
11.3 ± 2.5bcd
Nymph 4
78
76 (99 %)
46 (61 %)
11 (14 %)
19 (25 %)
1.0:1.7
13.4 ± 2.3d
Nymph 5
46
41 (89 %)
12 (29 %)
25 (61 %)
4 (10 %)
6.2:1.0
14.0 ± 2.2d
Nymph 6
12
9 (75 %)
7 (78 %)
2 (22 %)
3.5:1.0
12.5 ± 0.8d
0
Different letters indicate statistical difference among preecdysis periods (p \ 0.05; Dunn’s)
larvae molted to nymphs and the percentage of nymphs that molted to the next instar ranged from 48 to 99 %. Adults can be originated from nymph 3–6, with the percentage of nymphs molting to adults (rather than to the next nymphal instar) increasing progressively from 18 to 100 % for third to the sixth nymphal instar, respectively. Among the nymphs that molted to adults, earlier instars (nymph 3 and 4) tended to originate more males while older instars (nymph 5 and 6) originated more females (Table 1). The weight after feeding (FW) of fourth instar nymphs is directly correlated to the instar they will originate. FW of nymphs 4 that originate females are significantly higher (p \ 0.05) than the FW of nymphs that originate males, which is significantly higher (p \ 0.05) than nymphs that originated the next nymphal stage (nymph 5) (Fig. 2). The preecdysis period increases progressively from larvae to nymph 3, showing no significant differences from this nymphal instar to subsequent instars (p [ 0.05) (Table 1).
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Exp Appl Acarol Fig. 2 Final weight of fourth instar nymphs that molted to nymph 5, males and females. Bars indicate mean ± standard deviation
Feeding parameters All data regarding the feeding parameters are shown in Table 2. The number of specimens able to feed on mice was always high, ranging from 83 (nymph 1) to 100 % (nymph 6). The weight of the specimens from 25 to 35 days after molt increased proportionally until the fifth instar, remaining statistically similar to subsequent instars (p [ 0.05). Adults originated from early instars (nymph 3 and 4) tended to be lighter than adults originated from late instars (nymph 5 and 6). Females tended to be heavier than males. The weight gain and the capacity to ingest blood, represented by the ratio WG/IW, varied between instars and gender. Except for males, which ingested lower amounts of blood, WG was proportional to IW. The ratio WG/IW varied among instars, being higher in larvae and females (which were able to ingest 10.7 and 3.7 fold their IW, respectively) and lower in males (WG/IW = 0.6-0.7). Larvae remained on average 2.7 days attached to the host. Other instars fed for significant shorted periods (p \ 0.05) ranging from 17.3 to 78.3 min for first instars nymphs and females originated from fifth instar nymphs, respectively. Reproductive parameters In general, after one blood meal, primary females remained on average 15.2 ± 5.8 days laying 276.8 ± 137.4 eggs. Females originated from nymph 4 had similar oviposition and egg incubation time (p [ 0.05), but had statistically lower IW and WG generating lower number of eggs per female in comparison to females originated from nymph 6 (p \ 0.05) (Table 3). However, conversion ingested blood/number of eggs produced was similar between female groups, with 1 mg of blood generating approximately 2.6 eggs.
Discussion Results shown here add novel information to previous works that described the life cycle and/or biological parameters of O. rostratus under laboratory conditions. For the first time, we showed parameters of their life cycle when feeding on mice, which we concluded are satisfactory hosts once ticks were able to complete their life cycle with high viability (i.e., 300 larvae originated 85 adults). Although mice are not their natural host (Canc¸ado et al. 2008; Hoogstraal 1985; Ribeiro et al. 2013) they were chosen in the present work because
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Exp Appl Acarol Table 2 Feeding parameters of different instars of Ornithodoros rostratus fed on mice Stage/instar (previous stage)
N
Number that fed
Initial weight (mg)
Weight gain (mg)
Ratio WG/IW
Larva
300
264 (88 %)
\0.1a
*1.0 ± 0.4a *
10.7
2.7 ± 1.3**
0.8 ± 0.9ab
1.4
17.3 ± 13.4a
3.1 ± 3.0b
2.8
20.4 ± 10.9a
2.5
26.6 ± 6.4ab
ab
Nymph 1
156
129 (83 %)
0.6 ± 0.2
Nymph 2
68
66 (97 %)
1.1 ± 0.5b
Nymph 3 Nymph 4 Nymph 5 Nymph 6
125 78 46 12
125 (100 %) 76 (97 %) 43 (93 %) 12 (100 %)
3.4 ± 0.8
bc
6.4 ± 2.5
cd
0.7
39.7 ± 16.0
13.7 ± 6.7de
0.7
38.3 ± 9.8ab
15.1 ± 11.9de
0.7
22.3 ± 12.0ab
cde
0.6
58.3 ± 20.8ab
cde
0.6
35.4 ± 10.0ab
Males (Nymph 4)
19
16 (84 %)
22 ± 10.3de 24.4 ± 4.6
cde
27.1 ± 2.5
cde
Females***
43
40 (93 %)
28.1 ± 10.0
Females (Nymph 4)
11
11 (100 %)
23.3 ± 7.0de
Females (Nymph 5)
25
22 (88 %)
28.7 ± 10.1e e
7
7 (100 %)
51.7 ± 4.4b
14,7 ± 9.0
21.2 ± 7.9 18.8 ± 5.4
Females (Nymph 6)
2.3
41,5 ± 19.3
37 (88 %)
2 (100 %)
42.2 ± 13.2ab
de
15 (88 %)
2
2.4
de
17.9 ± 8.9
42
Males (Nymph 6)
37.9 ± 4.9ab
de
17
4 (100 %)
3.4
de
21.4 ± 15.9
cd
17.0 ± 5.8
Males***
4
8.4 ± 7.2
de
Males (Nymph 3) Males (Nymph 5)
bc
Total contact time (min)
33.8 ± 12.2
40,6 ± 28,4
15.3 ± 5.9 17.3 ± 4.3
104.9 ± 48.9
3.7
53.6 ± 21.4
3.2
37.6 ± 8.2ab
111.4 ± 40.4e
3.9
78.3 ± 29.0b
e
3.9
51.5 ± 8.1b
74.4 ± 31.6de 132.6 ± 73.7
Different letters indicate statistical difference (p \ 0.05; Dunn’s) * Final weigh was subtracted from 0.09 ** Period measured in days not in minutes *** Means for all males or females together. Not included in the statistical analysis
they are good experimental animals to be used under laboratorial conditions. Among several advantages, mice are easy to maintain, easy to breed and have several reagents commercially available, which is important considering studies focusing on tick-host interactions. The present work described the presence of six nymphal instars during their life cycle in addition to larvae and adults, as previously observed by Ribeiro et al. (2013). Adults were originated from nymph 3–6, whereas, except from nymph 6 when all molt to adults, fifth instars showed the highest percentage of specimens molting to adults, corroborating with previous findings from Ribeiro et al. (2013) and Guglielmone and Hadani (1980). Ribeiro et al. (2013) stated that probably the most common feature in their cycle would be the presence of five nymphal instars, fact that may justify why previous studies did not observe the presence of a sixth nymphal instar in their life cycle (Brumpt 1915; Guglielmone and Hadani 1980). Ribeiro et al. (2013) observed that females are originated from nymph 5 and 6. Here we show that they can also be originated from nymph 4, although in lower proportion than males as previously observed by Guglielmone and Hadani (1980). The female:male ratio ranged from 0:1 (adults originated from nymph 3) to 6.2:1.0 (adults originated from nymph 5). However, the overall ratio was 1:1, corroborating the observations by Ribeiro et al. (2013). A novel observation was the relation between FW of nymphs 4 and the instar they originate. Nymph 4 was used in the analysis because this is the instar with more specimens
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11
4
2
17
Nymph 4
Nymph 5
Nymph 6
Total
34.5 ± 10.1
46.9 ± 7.4b
42.1 ± 0.9
105.2 ± 61.2
203.2 ± 46.6b
113.8 ± 12.3
ab
72.4 ± 42.8a
26.6 ± 5.3a
ab
Weight gain (mg)
Initial weight (mg)
15.2 ± 5.8
16.5 ± 9.2a
15.8 ± 6.9
a
14.6 ± 4.2a
Oviposition time (days)
* Number of eggs produced with 1 mg of ingested blood
Different letters within a column indicate statistical difference (p \ 0.05; Dunn’s)
N
Previous stage
Table 3 Reproductive parameters of females of Ornithodoros rostratus fed on mice
276.8 ± 137.4
530.0 ± 84.9b
306.0 ± 24.3
ab
187.7 ± 56.9a
Eggs/female
17.2 ± 2,9
19.5 ± 3.5a
16.8 ± 2.8a
16.7 ± 2.6a
Egg incubation (days)
58.6 ± 36.3
94 ± 7.1
48.5 ± 25.4
54.3 ± 40
Eclosion (%)
2.63
2.61
2.69
2.59
Conversion index*
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able to molt to a nymphal instar, female or male. The higher the final weight is, the higher is the chance of a fourth instar nymph to molt to adults, with females being originated from nymphs with higher FW followed by males. Although the relation between FW and the instar they originate is clear, the reasons still remain to be explored. Larvae took 1–5 days (2.7 days on average) to complete their feeding while subsequent feedings made by nymphs and adults were faster and completed in \45 min for most nymphal instars. The feeding time for larvae were longer than the observed by Ribeiro et al. (2013) which reported feedings on rabbits of 33.5 min on average. However it corroborates with findings from Venzal and Estrada-Pena (2006) (which found 2.9 days on average feeding on rabbit) and other reports that showed long feeding periods that lasted up to 8 days (Brumpt 1915; Guglielmone and Hadani 1980), including O. rostratus in the group of argasids which larvae feed ‘‘slowly for many days’’ (Hoogstraal 1985). Such differences were not expected, as the specimens used in our experiments were collected in the same location as tick used by Ribeiro et al. (2013), probably belonging to the same field population. The differences found may be due to experimental conditions (e.g. temperature in the feeding environment) or fasting periods. Larvae fasting for 10–20 days and *15 days were used in this work and in the work done by Venzal and Estrada-Pena (2006), respectively, while larvae used by Ribeiro et al. (2013) had 35–45 days. It was interesting to observe that in the present work, older instars (especially nymph 5 and females) had lower initial weigh and weigh gain in comparison to these same stages described in Ribeiro et al. (2013), another finding that could be explained by the use of different hosts, perhaps suggesting that O. rostratus may feed better on rabbits. The ratio WG/IW (a parameter unexplored in previous work) demonstrated that larvae and females were able to ingest more blood in relation to their IW in comparison to nymphs, which ingest more blood than males. Such findings agree with the aspects of tick physiology; with females needing blood to maturate their ovaries to produce eggs; nymphs needing a good blood feed to grow and molt, while males take blood only to produce sperm to fertilize females. Another unexplored aspect is the female reproductive behavior originated from nymphs 4, 5 and 6. Here we observed that females originated from nymph 6 were significantly heavier (p \ 0.05), being able to ingest more blood from the host and lay more eggs. However the blood conversion was always similar (1 mg diet = *2.6 eggs), indicating that the instar originating females did not interfere with its reproductive performance. In conclusion, O. rostratus can be reared in the laboratory using mice as hosts, allowing the development of a good lab model to study several tick biology aspects, such as the identification of salivary molecules with potential use in the pharmacological industry, different aspects of tick–host interactions and their importance as disease vectors to spread Rickettsia among vertebrate hosts. The results presented here helped to elucidate several aspects of O. rostratus life cycle in laboratory conditions, providing useful information for researchers working in the tick biology field. Acknowledgments We thank Dr M. F. B. Ribeiro (ICB, UFMG) for providing the tick specimens and Dr. P. R. Oliveira (Veterinary School, UFMG) and P. Valente (ICB, UFMG) for the help in the identification of the ticks. The work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Instituto Nacional de Cieˆncia e Tecnologia em Entomologia Molecular (INCT-EM) and Pro-reitoria de Pesquisa (PRPq) da UFMG through the Programa Institucional de Auxı´lio a` Pesquisa de Doutores Rece´m-Contratados. Conflict of interest Authors declare they have no conflict of interest.
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References Almeida AP, Marcili A, Leite RC, Nieri-Bastos FA, Domingues LN, Martins JR, Labruna MB (2012) Coxiella symbiont in the tick Ornithodoros rostratus (Acari: Argasidae). Ticks Tick Borne Dis 3:203–206. doi:10.1016/j.ttbdis.2012.02.003 Angelakis E, Raoult D (2010) Q Fever. Vet Microbiol 140:297–309. doi:10.1016/j.vetmic.2009.07.016 Araga˜o H (1936) Ixodidas brasileiros e de alguns paı´ses limı´trophes. Mem Inst Oswaldo Cruz 31:759–843 Boero JJ (1957) Las Garrapatas de la Repu´blica Argentina (Acarina: Ixodoidea). Departamento Editorial, Universidad de Buenos Aires, Buenos Aires Bouchard KR, Wikel SK (2005) Care, maintenance, and experimental infestation of ticks in the laboratory setting. In: Marquardt WC (ed) Biology of disease vectors, 2nd edn. Elsevier, San Diego, pp 705–712 Brumpt E (1915) Biologia de alguns Ixodideos Brasileiros. Ann Paul Med Cir 4:25–29 Canc¸ado PH, Piranda EM, Mourao GM, Faccini JL (2008) Spatial distribution and impact of cattle-raising on ticks in the Pantanal region of Brazil by using the CO(2) tick trap. Parasitol Res 103:371–377. doi:10.1007/s00436-008-0982-8 Estrada-Pena A, Jongejan F (1999) Ticks feeding on humans: a review of records on human-biting Ixodoidea with special reference to pathogen transmission. Exp Appl Acarol 23:685–715 Griffin AT, Espinosa M, Nakamatsu R (2012) Q fever endocarditis: an unusual presentation. Am J Med Sci 344:480–484. doi:10.1097/MAJ.0b013e3182684979 Guglielmone AA, Hadani A (1980) Ciclo biolo´gico de ‘‘Ornithodoros rostratus’’. Aragao, 1911, bajo condiciones de laboratorio. Rev Med Vet 61:254–257 Guglielmone AA, Estrada-Pena A, Keirans JE, Robbins RG (2003) Ticks (Acari: Ixodida) of the neotropical zoogeographic region. Universiteit Utrecht, J. Bovy-Verbeek (public relations ICTTD), Houten, The Netherlands Hoogstraal H (1985) Argasid and nuttalliellid ticks as parasites and vectors. Adv Parasitol 24:135–238 Nava S et al (2007) The ticks (Acari: Ixodida: Argasidae, Ixodidae) of Paraguay. Ann Trop Med Parasitol 101:255–270. doi:10.1179/136485907X176319 Ribeiro CC, Faccini JL, Cancado PH, Piranda EM, Barros-Battesti DM, Leite RC (2013) Life cycle of Ornithodoros rostratus (Acari: Argasidae) under experimental conditions and comments on the hostparasite relationship in the Pantanal wetland region, Brazil. Exp Appl Acarol. doi:10.1007/s10493-0139669-7 Venzal JM, Estrada-Pena A (2006) Larval feeding performance of two Neotropical Ornithodoros ticks (Acari: Argasidae) on reptiles. Exp Appl Acarol 39:315–320. doi:10.1007/s10493-006-9011-8 Venzal JM, Onofrio VC, Barros-Battesti DM, Arzua M (2006) Famı´lia Argasidae: caracterı´sticas gerais, comenta´rios e chaves para geˆneros e espe´cies. In: Barros-Batestti DM, Arzua M, Bechara GH (eds) Carrapatos de Importaˆncia Me´dico-Veterina´ria da Regia˜o Neotropical. Vox/ICTTD-3/Butantan, Sa˜o Paulo, pp 13–27
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Life cycle of Ornithodoros rostratus (Acari: Argasidae) ticks feeding on mice under laboratory conditions Gabriel Cerqueira Alves Costa • Adriana Coelho Soares • Marcos Hora´cio Pereira • Nelder Figueiredo Gontijo • Maurı´cio Roberto Viana Sant’Anna • Ricardo Nascimento Araujo
Received: 12 September 2014 / Accepted: 17 February 2015 Ó Springer International Publishing Switzerland 2015
Abstract Ornithodoros rostratus Araga˜o is an argasid tick found in Bolivia, Paraguay, Argentina and Brazil. Only limited studies about O. rostratus have been conducted and several aspects of their life cycle differ among studies or remain unexplored. In order to better elucidate the biology of O. rostratus, the present work describes its life cycle when feeding on mice under laboratory conditions. To complete their life cycle on mice, O. rostratus goes through a larval stage, 3–6 nymphal instars (nymph 1–6) and adult male and female. Adults can be originated from nymph 3–6. Nymphs 4 with higher weight after feeding tend to originate adults. Adults originated from early instars tended to be lighter. Females tended to be heavier than males. Larvae needed on average 2.7 days to complete their blood meal whereas other instars ranged from 17.3 to 78.3 min. The capacity to ingest blood was higher in larvae and females in comparison to males. The preecdysis period ranged from 5 to 12.5 days. After one blood meal, females remain on average 15.2 ± 5.8 days laying 276.8 ± 137.2.9 eggs. Females originated from nymph 4 had similar oviposition time, egg incubation and conversion ingested blood/number of eggs produced, but presented lower initial weigh and weigh gain, generating fewer eggs. Our results added novel information on O. rostratus biology and was discussed considering the variability of argasid populations and in context with the differences about their life cycle described in previous works. Keywords Ornithodoros rostratus Argasidae Life cycle Mice Hematophagy Reproduction
G. C. A. Costa A. C. Soares M. H. Pereira N. F. Gontijo M. R. V. Sant’Anna R. N. Araujo (&) Laborato´rio de Fisiologia de Insetos Hemato´fagos, Departamento de Parasitologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Av. Presidente Antoˆnio Carlos 6627, Belo Horizonte, MG 31270-901, Brazil e-mail: [email protected] M. H. Pereira N. F. Gontijo R. N. Araujo Instituto Nacional de Cieˆncia e Tecnologia em Entomologia Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-909, Brazil
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Introduction Ornithodoros rostratus Araga˜o is an argasid tick found in Bolivia, Paraguay, Argentina and Brazil feeding on humans and several mammal species (Guglielmone et al. 2003; Hoogstraal 1985; Nava et al. 2007; Venzal et al. 2006). The importance of O. rostratus ticks are related to the cutaneous reactions caused by their parasitism. Its bite is painful and itchy, and can cause ecchymotic lesions, inflammation and blisters on the feeding site (Araga˜o 1936; Boero 1957; Estrada-Pena and Jongejan 1999). In addition, they are vectors of the Rocky Mountain spotted fever, caused by the bacteria Rickettsia rickettsii (Hoogstraal 1985) and were described harbouring Coxiella burnetii (Almeida et al. 2012), the causative agent of acute Q fever and endocarditis in humans and animals (Angelakis and Raoult 2010; Griffin et al. 2012). A few years ago, a novel O. rostratus infestation focus was identified in a farm located in the Pantanal region of Mato Grosso do Sul state, Brazil, where domestic animals such as dogs and pigs were reported as hosts (Canc¸ado et al. 2008; Ribeiro et al. 2013). Few studies were conducted concerning the life story of O. rostratus. Most of them showed data from only some developing instars and not many parameters of their life cycle were evaluated when fed on dogs and chickens (Brumpt 1915), guinea pigs (Guglielmone and Hadani 1980), rabbits and reptiles (Venzal and Estrada-Pena 2006). A more detailed analysis was carried out on rabbits (Ribeiro et al. 2013). Nevertheless, aspects of their life cycle differed among studies and others remain unexplored. Ribeiro et al. (2013) reported that O. rostratus larvae complete their blood feed after 33.5 min on average when feeding on rabbits while other studies reported significantly longer feeding periods on mammals that lasted up to 8 days (Brumpt 1915; Guglielmone and Hadani 1980; Venzal and Estrada-Pena 2006). Some studies describe only five nymphal instars (Brumpt 1915; Guglielmone and Hadani 1980) whereas others indicate the existence of a sixth instar (Ribeiro et al. 2013). Although adults may be originated from nymph 3, there is no information about reproductive parameters of females originated from different nymphal instars. Also, the amount of blood ingested considering the initial weigh of each instar was not evaluated in previous works. The present work aims at describing the life cycle of O. rostratus fed on mice (a classic laboratory model) under laboratory conditions. The data generated about their life cycle added novel information on O. rostratus biology highlighting the variability of Argasid populations, also helping to elucidate the differences presented in previous works.
Materials and methods Ticks and colony rearing Ticks used in the experiments were collected in Nhecolaˆndia, Mato Grosso do Sul, Brazil (19° 030 S, 56° 470 W). Collected specimens were taken to the laboratory, identified to the species level (Venzal et al. 2006) and were maintained inside an incubator under semicontrolled conditions of temperature (28 ± 2 °C) and humidity (85 ± 10 %). Ticks were fed on Swiss mice (Mus musculus) every 20 days. Feeding experiments Larvae were fed in Swiss mice using the methodology described by Bouchard and Wikel (2005). Briefly, feeding chambers were made with 1.5 mL polypropylene centrifuge tubes
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that were cut at approximately 1 cm from the top and were then fixed on the mice’s back using nontoxic glue. The caps were pierced with a thin needle to enable air circulation. 25 larvae were put inside the chambers that were closed and maintained until larvae had completed the whole feeding. Chambers were examined daily to record attachment and release. Larvae that had not attached until 24 h after being added to the feeding chamber (first examination of the chamber) were removed and discarded. Ten to 20 day old larvae were used in the experiments. Nymphs and adults were allowed to feed once on anaesthetized mice in a room at 28 ± 2 °C. The specimens used in the experiments had between 25 and 35 days after molt. The feeding area was delimited using a 2 9 1.5 cm cylinder fixed with adhesive tape in the abdomen area of the mice. Each mouse was used as feeding source for a maximum of 10 specimens earlier than nymph 3 or 5 older than nymph 3. All blood feeds were recorded using a digital camera (Canon EOS600D) to measure the number of bites and the total contact time (TCT-period between the bite and the removal of the mouthparts from the host) for each tick. Specimens were weighted before (initial weight—IW) and after feeding (final weight—FW) to measure the weight gain (WG—FW minus IW). After feeding, nymphs were kept individually in rearing containers where they were monitored daily to measure the preecdysis time and the number of nymphs that molted or died. Reproductive experiments To analyze reproductive parameters, couples were paired using heavier females and males that were originated from each nymphal instar. At 25–35 days after molt, couples were fed on mice, weighed before and after feeding and maintained on individual containers. They were then evaluated daily in order to measure the number of eggs layed per female and the period which females remained laying eggs after one single feeding (one gonotrophic cycle). The egg incubation period and percentage of eclosion were also accessed. Statistical analysis The data was analyzed using the GraphPad PrismÒ 5 for Windows. The normality of the data was tested by the Kolmogorov–Smirnov test and Kruskal–Wallis followed by Dunn’s test to analyze pairwise differences between the parameters evaluated. The significance level was set at p \ 0.05. Ethics committee All procedures were in accordance with the manuals for animal experimentation and were approved by the Ethics Committee for Animal Use (CETEA/ICB-UFMG) under the protocol 301/2013.
Results Life stages and molt parameters To complete their life cycle on mice, O. rostratus goes through a larval stage, 3–6 nymphal instars and adult female (Fig. 1) and male (Table 1). When feeding on mice, 68 % of the
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Fig. 1 Dorsal (a) and ventral (b) view of a female of Ornithodoros rostratus
Table 1 Molting parameters of Ornithodoros rostratus fed on mice Stage/ instar
N
Number that molted
Number that molted to
Ratio female:male
Nymph
Female
Male
Preecdysis (days)
Larva
300
204 (68 %)
204 (100 %)
0
0
5.0 ± 1.6a
Nymph 1
156
75 (48 %)
75 (100 %)
0
0
6.8 ± 1.6ab
Nymph 2
68
62 (91 %)
62 (100 %)
0
0
8.2 ± 1.4bc
Nymph 3
125
95 (76 %)
78 (82 %)
0
17 (18 %)
0.0:1.0
11.3 ± 2.5bcd
Nymph 4
78
76 (99 %)
46 (61 %)
11 (14 %)
19 (25 %)
1.0:1.7
13.4 ± 2.3d
Nymph 5
46
41 (89 %)
12 (29 %)
25 (61 %)
4 (10 %)
6.2:1.0
14.0 ± 2.2d
Nymph 6
12
9 (75 %)
7 (78 %)
2 (22 %)
3.5:1.0
12.5 ± 0.8d
0
Different letters indicate statistical difference among preecdysis periods (p \ 0.05; Dunn’s)
larvae molted to nymphs and the percentage of nymphs that molted to the next instar ranged from 48 to 99 %. Adults can be originated from nymph 3–6, with the percentage of nymphs molting to adults (rather than to the next nymphal instar) increasing progressively from 18 to 100 % for third to the sixth nymphal instar, respectively. Among the nymphs that molted to adults, earlier instars (nymph 3 and 4) tended to originate more males while older instars (nymph 5 and 6) originated more females (Table 1). The weight after feeding (FW) of fourth instar nymphs is directly correlated to the instar they will originate. FW of nymphs 4 that originate females are significantly higher (p \ 0.05) than the FW of nymphs that originate males, which is significantly higher (p \ 0.05) than nymphs that originated the next nymphal stage (nymph 5) (Fig. 2). The preecdysis period increases progressively from larvae to nymph 3, showing no significant differences from this nymphal instar to subsequent instars (p [ 0.05) (Table 1).
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Exp Appl Acarol Fig. 2 Final weight of fourth instar nymphs that molted to nymph 5, males and females. Bars indicate mean ± standard deviation
Feeding parameters All data regarding the feeding parameters are shown in Table 2. The number of specimens able to feed on mice was always high, ranging from 83 (nymph 1) to 100 % (nymph 6). The weight of the specimens from 25 to 35 days after molt increased proportionally until the fifth instar, remaining statistically similar to subsequent instars (p [ 0.05). Adults originated from early instars (nymph 3 and 4) tended to be lighter than adults originated from late instars (nymph 5 and 6). Females tended to be heavier than males. The weight gain and the capacity to ingest blood, represented by the ratio WG/IW, varied between instars and gender. Except for males, which ingested lower amounts of blood, WG was proportional to IW. The ratio WG/IW varied among instars, being higher in larvae and females (which were able to ingest 10.7 and 3.7 fold their IW, respectively) and lower in males (WG/IW = 0.6-0.7). Larvae remained on average 2.7 days attached to the host. Other instars fed for significant shorted periods (p \ 0.05) ranging from 17.3 to 78.3 min for first instars nymphs and females originated from fifth instar nymphs, respectively. Reproductive parameters In general, after one blood meal, primary females remained on average 15.2 ± 5.8 days laying 276.8 ± 137.4 eggs. Females originated from nymph 4 had similar oviposition and egg incubation time (p [ 0.05), but had statistically lower IW and WG generating lower number of eggs per female in comparison to females originated from nymph 6 (p \ 0.05) (Table 3). However, conversion ingested blood/number of eggs produced was similar between female groups, with 1 mg of blood generating approximately 2.6 eggs.
Discussion Results shown here add novel information to previous works that described the life cycle and/or biological parameters of O. rostratus under laboratory conditions. For the first time, we showed parameters of their life cycle when feeding on mice, which we concluded are satisfactory hosts once ticks were able to complete their life cycle with high viability (i.e., 300 larvae originated 85 adults). Although mice are not their natural host (Canc¸ado et al. 2008; Hoogstraal 1985; Ribeiro et al. 2013) they were chosen in the present work because
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Exp Appl Acarol Table 2 Feeding parameters of different instars of Ornithodoros rostratus fed on mice Stage/instar (previous stage)
N
Number that fed
Initial weight (mg)
Weight gain (mg)
Ratio WG/IW
Larva
300
264 (88 %)
\0.1a
*1.0 ± 0.4a *
10.7
2.7 ± 1.3**
0.8 ± 0.9ab
1.4
17.3 ± 13.4a
3.1 ± 3.0b
2.8
20.4 ± 10.9a
2.5
26.6 ± 6.4ab
ab
Nymph 1
156
129 (83 %)
0.6 ± 0.2
Nymph 2
68
66 (97 %)
1.1 ± 0.5b
Nymph 3 Nymph 4 Nymph 5 Nymph 6
125 78 46 12
125 (100 %) 76 (97 %) 43 (93 %) 12 (100 %)
3.4 ± 0.8
bc
6.4 ± 2.5
cd
0.7
39.7 ± 16.0
13.7 ± 6.7de
0.7
38.3 ± 9.8ab
15.1 ± 11.9de
0.7
22.3 ± 12.0ab
cde
0.6
58.3 ± 20.8ab
cde
0.6
35.4 ± 10.0ab
Males (Nymph 4)
19
16 (84 %)
22 ± 10.3de 24.4 ± 4.6
cde
27.1 ± 2.5
cde
Females***
43
40 (93 %)
28.1 ± 10.0
Females (Nymph 4)
11
11 (100 %)
23.3 ± 7.0de
Females (Nymph 5)
25
22 (88 %)
28.7 ± 10.1e e
7
7 (100 %)
51.7 ± 4.4b
14,7 ± 9.0
21.2 ± 7.9 18.8 ± 5.4
Females (Nymph 6)
2.3
41,5 ± 19.3
37 (88 %)
2 (100 %)
42.2 ± 13.2ab
de
15 (88 %)
2
2.4
de
17.9 ± 8.9
42
Males (Nymph 6)
37.9 ± 4.9ab
de
17
4 (100 %)
3.4
de
21.4 ± 15.9
cd
17.0 ± 5.8
Males***
4
8.4 ± 7.2
de
Males (Nymph 3) Males (Nymph 5)
bc
Total contact time (min)
33.8 ± 12.2
40,6 ± 28,4
15.3 ± 5.9 17.3 ± 4.3
104.9 ± 48.9
3.7
53.6 ± 21.4
3.2
37.6 ± 8.2ab
111.4 ± 40.4e
3.9
78.3 ± 29.0b
e
3.9
51.5 ± 8.1b
74.4 ± 31.6de 132.6 ± 73.7
Different letters indicate statistical difference (p \ 0.05; Dunn’s) * Final weigh was subtracted from 0.09 ** Period measured in days not in minutes *** Means for all males or females together. Not included in the statistical analysis
they are good experimental animals to be used under laboratorial conditions. Among several advantages, mice are easy to maintain, easy to breed and have several reagents commercially available, which is important considering studies focusing on tick-host interactions. The present work described the presence of six nymphal instars during their life cycle in addition to larvae and adults, as previously observed by Ribeiro et al. (2013). Adults were originated from nymph 3–6, whereas, except from nymph 6 when all molt to adults, fifth instars showed the highest percentage of specimens molting to adults, corroborating with previous findings from Ribeiro et al. (2013) and Guglielmone and Hadani (1980). Ribeiro et al. (2013) stated that probably the most common feature in their cycle would be the presence of five nymphal instars, fact that may justify why previous studies did not observe the presence of a sixth nymphal instar in their life cycle (Brumpt 1915; Guglielmone and Hadani 1980). Ribeiro et al. (2013) observed that females are originated from nymph 5 and 6. Here we show that they can also be originated from nymph 4, although in lower proportion than males as previously observed by Guglielmone and Hadani (1980). The female:male ratio ranged from 0:1 (adults originated from nymph 3) to 6.2:1.0 (adults originated from nymph 5). However, the overall ratio was 1:1, corroborating the observations by Ribeiro et al. (2013). A novel observation was the relation between FW of nymphs 4 and the instar they originate. Nymph 4 was used in the analysis because this is the instar with more specimens
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11
4
2
17
Nymph 4
Nymph 5
Nymph 6
Total
34.5 ± 10.1
46.9 ± 7.4b
42.1 ± 0.9
105.2 ± 61.2
203.2 ± 46.6b
113.8 ± 12.3
ab
72.4 ± 42.8a
26.6 ± 5.3a
ab
Weight gain (mg)
Initial weight (mg)
15.2 ± 5.8
16.5 ± 9.2a
15.8 ± 6.9
a
14.6 ± 4.2a
Oviposition time (days)
* Number of eggs produced with 1 mg of ingested blood
Different letters within a column indicate statistical difference (p \ 0.05; Dunn’s)
N
Previous stage
Table 3 Reproductive parameters of females of Ornithodoros rostratus fed on mice
276.8 ± 137.4
530.0 ± 84.9b
306.0 ± 24.3
ab
187.7 ± 56.9a
Eggs/female
17.2 ± 2,9
19.5 ± 3.5a
16.8 ± 2.8a
16.7 ± 2.6a
Egg incubation (days)
58.6 ± 36.3
94 ± 7.1
48.5 ± 25.4
54.3 ± 40
Eclosion (%)
2.63
2.61
2.69
2.59
Conversion index*
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able to molt to a nymphal instar, female or male. The higher the final weight is, the higher is the chance of a fourth instar nymph to molt to adults, with females being originated from nymphs with higher FW followed by males. Although the relation between FW and the instar they originate is clear, the reasons still remain to be explored. Larvae took 1–5 days (2.7 days on average) to complete their feeding while subsequent feedings made by nymphs and adults were faster and completed in \45 min for most nymphal instars. The feeding time for larvae were longer than the observed by Ribeiro et al. (2013) which reported feedings on rabbits of 33.5 min on average. However it corroborates with findings from Venzal and Estrada-Pena (2006) (which found 2.9 days on average feeding on rabbit) and other reports that showed long feeding periods that lasted up to 8 days (Brumpt 1915; Guglielmone and Hadani 1980), including O. rostratus in the group of argasids which larvae feed ‘‘slowly for many days’’ (Hoogstraal 1985). Such differences were not expected, as the specimens used in our experiments were collected in the same location as tick used by Ribeiro et al. (2013), probably belonging to the same field population. The differences found may be due to experimental conditions (e.g. temperature in the feeding environment) or fasting periods. Larvae fasting for 10–20 days and *15 days were used in this work and in the work done by Venzal and Estrada-Pena (2006), respectively, while larvae used by Ribeiro et al. (2013) had 35–45 days. It was interesting to observe that in the present work, older instars (especially nymph 5 and females) had lower initial weigh and weigh gain in comparison to these same stages described in Ribeiro et al. (2013), another finding that could be explained by the use of different hosts, perhaps suggesting that O. rostratus may feed better on rabbits. The ratio WG/IW (a parameter unexplored in previous work) demonstrated that larvae and females were able to ingest more blood in relation to their IW in comparison to nymphs, which ingest more blood than males. Such findings agree with the aspects of tick physiology; with females needing blood to maturate their ovaries to produce eggs; nymphs needing a good blood feed to grow and molt, while males take blood only to produce sperm to fertilize females. Another unexplored aspect is the female reproductive behavior originated from nymphs 4, 5 and 6. Here we observed that females originated from nymph 6 were significantly heavier (p \ 0.05), being able to ingest more blood from the host and lay more eggs. However the blood conversion was always similar (1 mg diet = *2.6 eggs), indicating that the instar originating females did not interfere with its reproductive performance. In conclusion, O. rostratus can be reared in the laboratory using mice as hosts, allowing the development of a good lab model to study several tick biology aspects, such as the identification of salivary molecules with potential use in the pharmacological industry, different aspects of tick–host interactions and their importance as disease vectors to spread Rickettsia among vertebrate hosts. The results presented here helped to elucidate several aspects of O. rostratus life cycle in laboratory conditions, providing useful information for researchers working in the tick biology field. Acknowledgments We thank Dr M. F. B. Ribeiro (ICB, UFMG) for providing the tick specimens and Dr. P. R. Oliveira (Veterinary School, UFMG) and P. Valente (ICB, UFMG) for the help in the identification of the ticks. The work was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Instituto Nacional de Cieˆncia e Tecnologia em Entomologia Molecular (INCT-EM) and Pro-reitoria de Pesquisa (PRPq) da UFMG through the Programa Institucional de Auxı´lio a` Pesquisa de Doutores Rece´m-Contratados. Conflict of interest Authors declare they have no conflict of interest.
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