Biology and life cycle of Amblyomma incisum(Acari: Ixodidae)

Matias Pablo J. Szabo Æ Lucas de F. Pereira Æ Marcio B. Castro ÆMarcos V. Garcia Æ Gustavo S. Sanches Æ Marcelo B. Labruna

Received: 19 September 2008 / Accepted: 30 December 2008 / Published online: 8 January 2009� Springer Science+Business Media B.V. 2009

Abstract Amblyomma incisum Neumann is a major tick species in the Atlantic Forest of

Brazil. Tapir is the main host for adult ticks and a high aggressiveness of nymphs to

humans has been reported. In this work data on the biology and life cycle of this tick

species is presented for the first time. It was shown that horse is a suitable host for

A. incisum adults and rabbit for larvae and nymphs. It was also shown that A. incisum is a

big tick species (mean engorged female weight of 1.96 g) with a long life cycle which lasts

262.3 days when maintained at 27�C and 85% RH. These laboratory conditions were,

however, inappropriate and egg hatching rate (1.2%) was very low. Nevertheless egg

hatching of ticks in a forest patch increased considerably (72.2%) indicating that this

A. incisum population is highly dependent on a forest-like environment.

Keywords Amblyomma incisum � Horse � Rabbit � Ixodidae � Life cycle �Atlantic rain forest

M. P. J. Szabo (&)Faculdade de Medicina Veterinaria da Universidade Federal de Uberlandia, Av. Para, 1720,Campus Umuarama-Bloco 2T, Uberlandia, MG CEP 38400-902, Brazile-mail: szabo@famev.ufu.br

L. de F. PereiraUniversidade de Franca, Franca, SP, Brazil

M. B. CastroFaculdade de Agronomia e Medicina Veterinaria, Universidade Federal de Brasılia, Brasılia, DF,Brazil

M. V. Garcia � G. S. SanchesFaculdade de Ciencias Agrarias e Veterinarias, Universidade Estadual Paulista, Jaboticabal, SP, Brazil

M. B. LabrunaFaculdade de Medicina Veterinaria e Zootecnia, Universidade de Sao Paulo, Sao Paulo, SP, Brazil

123

Exp Appl Acarol (2009) 48:263–271DOI 10.1007/s10493-008-9234-y

Introduction

The tick fauna of Brazil is estimated at 61 species from which 33 belong to the genus

Amblyomma (Barros-Battesti 2006). Because the great majority of Brazilian Amblyommaspecies have seldom been associated with livestock, pets or humans, very little is known

about their life histories (Labruna et al. 2005). At the same time, many Brazilian natural

areas are shrinking; meanwhile our knowledge of the numerous animal species inhabiting

them, parasitic and otherwise, remains fragmentary.

While conducting projects on ticks of deer and on tick ecology in the Atlantic rainforest

of the Intervales State Park, Ribeirao Grande County, state of Sao Paulo, Brazil (Szabo

et al. 2006a; Szabo et al. 2009), it was observed that the most abundant tick species host-

seeking on vegetation in animal trails was Amblyomma incisum Neumann. This species has

been reported in the Amazon region, with additional records from non-Amazonian areas of

Argentina, Paraguay, and southern Brazil (Labruna et al. 2005; Szabo et al. 2006a). The

biosystematic status of this tick and data on its hosts were recently reviewed, and it was

shown that the South American tapir, Tapirus terrestris L., is the main host for A. incisumadults (Labruna et al. 2005). Even tough nymphs have been reported on small red-brocket

deer (Mazama bororo Duarte) in southern Brazil (Szabo et al. 2006a) and a high

aggressiveness of A. incisum nymphs to human beings has also been reported (Szabo et al.

2006b), the life cycle and host range for its immature stages are largely unknown. This

paper presents data on the biology and life cycle of A. incisum obtained in the laboratory,

with additional data obtained under field conditions.

Materials and methods

Ticks and original environment

Unfed adult ticks were collected in the Intervales State Park (24�180S, 48�240W), Sao

Paulo, Brazil. The park is a natural reserve of 42,000 ha of Atlantic rain forest in the

southeastern part of the State of Sao Paulo. The annual rainfall of the region is 2,000–

3,000 mm, the park’s altitude varies from 60 to 1,065 m. The mean temperature at 790 m

of altitude is of 22�C in summer (February) and of 14.3�C in winter (July). The mean

relative humidity per month inside the reserve is always above 80% and daily levels bellow

40% are rarely measured.

Due to the difficulties associated with the maintenance of a laboratory colony of

A. incisum (very long pre-oviposition and oviposition periods, and lack of or very low egg

hatching), adult ticks were supplied for the experiments through repeated field collection in

the Intervales Park. For this purpose, ticks were collected from the vegetation at animal

trails, placed immediately in dry plastic vials containing small pieces of dry absorbent

paper, and stoppered with a cork perforated with several minute holes. These vials were

placed in another larger vial lined with moistened cotton, which was maintained at room

temperature for 1–3 days. In the laboratory unfed adult ticks were maintained in an

incubator at 20�C and 85% RH until used for infestations. Adult ticks were identified as

A. incisum according to Labruna et al. (2005). Voucher tick specimens collected during this

study have been deposited in the CNC-FMVZ/USP National Tick Collection, University of

Sao Paulo, SP, Brazil (accession numbers: CNC-305, 354, 595, 521, 523, 595, 606) and

CC-FAMEV/UFU Tick Collection, Federal University of Uberlandia, MG, Brazil

(accession numbers: 328–341).

264 Exp Appl Acarol (2009) 48:263–271

123

Hosts

Ten adult mixed-bred horses (Equus caballus L.) were used as experimental hosts. The

choice for horses was based on the knowledge that tapirs are the primary host for

A. incisum under natural conditions (Guimaraes et al. 2001; Labruna et al. 2005) and

among possible experimental hosts, horses are phylogenetically the closest to tapirs. All

horses used in the experiments were generously donated by either neighboring farms

(n = 4) or by a highway maintenance company (Autovias S/A, Sao Paulo) (n = 6). Horses

were taken to Franca University’s Veterinary Hospital stalls, in Franca Municipality, state

of Sao Paulo, Brazil. Many of these animals, initially in poor conditions, were held through

a recovery period before submitted to the experimental infestations. Horses were fed with

commercial pellets (Techhorse, Purina), hay (Coast-cross, tifton 85) and water ad libitum

through both pre-experimental and experimental periods. All horses had been recently

infested by Dermacentor nitens Neumann and Amblyomma cajennense (Fabricius) ticks.

These horses were used as hosts for the adult stage of A. incisum.

Tick-bite naive New Zealand white rabbits [Oryctolagus cuniculus (Linnaeus)] were

used as hosts for larvae and nymphs of A. incisum. The choice of this medium-sized host

relied in two reasons: (1) immature stages of ticks are known to display a much broader

host range than the adult stage (Balashov 1972); (2) laboratory costs to perform artificial

infestation on rabbits are much lower than on horses. Rabbits were obtained from the Sao

Paulo State University’s animal house (Botucatu-SP, Brazil). Water and commercial pel-

lets were available ad libitum for rabbits.

Experimental infestation procedures

For each experimental infestation with adult ticks, a feeding infestation chamber was glued

to the shaved back of each horse with an adhesive glue (Brascoplast�, Brascola Ltd,

Brazil) as described elsewhere (Castagnolli et al. 2003). Briefly, a feeding chamber con-

sisted of a plastic tube (8 cm in diameter and 3 cm in height) with a rubber and cloth base.

Chambers were glued on the thoracic region of the horses, near the vertebral midline. Neck

collars were worn to horses during each infestation to prevent grooming on the infestation

chamber. Each chamber received at once five pairs of adult ticks.

Infestation conditions of rabbits were similar to those of horses except that each rabbit

was infested either with 200 larvae or 28 nymphs. Due to low number of available nymphs

and larvae, only four and two rabbits were infested once with larvae and nymphs,

respectively.

Biological parameters

The life cycle of A. incisum was evaluated by the analysis of feeding and reproductive data

of the parasite as described elsewhere (Castagnolli et al. 2003). Briefly, chambers were

inspected daily and detached engorged ticks were collected and maintained in an incubator

at 27�C, 85% RH, and a photoperiod regimen 00:24 (light:darkenss). Detached engorged

females were placed into individual plastic vials and larvae and nymphs stored separately

in daily batches. The following biological parameters were recorded: tick yield, engorged

female, larval, nymphal and egg mass weights, feeding, premolt, and pre-oviposition

periods, molting and oviposition rates, and larval percent hatch. Egg mass was weighed

50 days after tick detachment. Feeding period was assumed to be the time elapsed from

the liberation of ticks on the hosts till their detachment, partially or fully engorged;

Exp Appl Acarol (2009) 48:263–271 265

123

pre-oviposition was the number of days from detachment to beginning of oviposition;

premolt period was assumed as the number of days between detachment from host and first

tick to molt from that daily batch; molting and oviposition rates were the proportion of

engorged ticks that molted or oviposited, respectively. The larval hatching rate for each

female offspring was the mean value of visual evaluation performed by three different

persons. For the measurement of the metabolic activity of females for the oviposition

process, the index of egg production efficiency (EPE) was determined using the formula:

weight of eggs/weight of the engorged female 9 100 (Bennett 1974).

Oviposition and larval hatching under natural conditions

Mean larval hatching in the incubator was very low. Considering that in the Atlantic forest

this tick species lives in very humid conditions and in vegetation under constant shadow it

was supposed that the conditions in incubator were not adequate for larval hatching.

Moreover, previous unpublished laboratory trials at 27�C and 99% RH failed to produce

larvae because most females and eggs became moldy during the long pre-oviposition and

ovipostion periods. For this reason, a new trial was tried placing 15 engorged females

individually inside stainless wire-gauze cylindrical tubes (60 mesh/cm2, 61 mm long,

17 mm diameter), which were closed with rubber corks and taken to a humid forest patch

15 km from Franca city, where they were placed horizontally in the soil surface at the roots

of vegetation, close to a stream, under the dense vegetation. Females were released in the

field in March (21/03/2004, n = 9; 5/03/2005, n = 3) and April (14/04/2005, n = 3).

Humidity of the forest was naturally provided by the dense vegetation and the stream. For

these ticks, oviposition and larval hatching were observed. Local range of temperature and

humidity were recorded through this field experiment.

Oviposition period

It is difficult to determine ovipostion period of ticks because minute quantities of eggs are

usually laid during many days after the oviposition of several thousands of eggs. To

determine this period more precisely, 15 engorged females that began oviposition were

incubated in individual vials at 27�C and 85% RH. Thirty days after the onset of oviposition

egg mass was collected every second day to observe additional egg laying until the death of

the female. The initial 30 day-egg mass, as well as, the additional laid every 2 days then off

were weighed, in order to obtain the total egg mass weight laid by each female.

Number of eggs per gram

To determine the number of A. incisum eggs per gram of egg mass, the number of eggs in a

sample of 10 mg of eggs laid by each of eight females were counted under a stereomi-

croscope. Then, the proportion of eggs present in a gram of eggs was estimated based on

the mean number of eggs per 10 mg of eggs.

Results

Biological parameters for A. inicisum larvae, nymphs, females, and eggs are presented in

Table 1. During horse infestations, most adult ticks attached readily to horses after the

266 Exp Appl Acarol (2009) 48:263–271

123

Ta

ble

1B

iolo

gic

alp

aram

eter

so

fA

mb

lyo

mm

ain

cisu

mu

nd

erla

bora

tory

con

dit

ion

so

f2

7�C

and

85

%R

H(f

or

larv

ae,n

ym

phs,

fem

ales

and

egg

s)o

ru

nd

erfi

eld

con

dit

ion

s(f

or

fem

ales

and

eggs)

Fac

tor

Tic

kst

age

Lar

vae

Ny

mp

hF

emal

esan

deg

gs

Lab

ora

tory

Fie

ldco

nd

itio

ns

Ho

st(n

)R

abbit

s(4

)R

abbit

s(2

)H

ors

es(1

0)

Ho

rses

(3)

No

.o

fex

po

sed

tick

s8

00

56

50

15

No

.o

fre

cov

ered

tick

s(%

)1

92

(24

.0)

45

(80

.3)

44

(88

.0)

15

(10

0)

Fee

din

gp

erio

d(d

ays)

a6

.5±

0.5

(5–

11)

7.7

±1

.8(5

–1

5)

11

.0±

1.7

(7–

14)

11

.2±

2.4

(9–

15)

Wei

gh

to

fen

go

rged

tick

(mg

)a2

.2±

0.3

(1.1

–2.7

)4

0.0

±2

.0(2

0–

58

)1

,96

0±

71

3(3

26

–3

,09

2)

1,8

50

±5

09

(68

7–2

,42

6)

No

.o

fm

olt

edo

ro

vip

osi

ted

(%)

70

(36

.4)

33

(73

.3)

27

(61

.4)

12

(80

)

Pre

mo

lto

rp

reo

vip

osi

tio

np

erio

d(d

ays)

a3

1.6

±2

.7(2

8–

36

)5

0.0

±0

.49

(49

–51

)3

5.1

±2

8.8

(20

–15

2)

94

.2±

23

.8(7

1–

14

8)

Wei

gh

to

fd

epo

site

deg

gs

(mg

)a–

–9

17

±3

67

(17

0–1

,481

)N

ot

det

erm

ined

Eg

gin

cub

atio

np

erio

d(d

ays)

a–

–1

20

.4±

9.8

(11

2–1

40

)2

11

.1±

33

.8(1

52

–2

56

)

Eg

gh

atch

(%)a

––

1.2

±0

.6(1

–3

)7

2.2

±3

5.2

(10

–10

0)

Eg

gp

rod

uct

ion

effi

cien

cy(E

PE

)a–

–4

4.9

±1

9.1

(6.1

–80

.1)

No

td

eter

min

ed

aV

alu

esp

rese

nte

das

mea

n±

SE

.R

ange

of

val

ues

issh

ow

nb

etw

een

bra

cket

s

Exp Appl Acarol (2009) 48:263–271 267

123

release in the chamber and the females fully engorged (Fig. 1) in a mean of 11 days. Mean

preoviposition and egg incubation periods for ticks held in the incubator were 35.1 and

120.4 days, respectively, and mean engorged female and egg mass weights were 1,960 and

917 mg, respectively. However, 38.6% of females did not lay eggs (data not included in

the calculation of mean egg mass weights). Only 1.2% of the eggs in the incubator hatched.

Fifteen A. incisum females fed on horses were taken to the forest patch to evaluate the

non-parasitic parameters of this tick species under natural conditions. Egg mass weight

was not recorded for these ticks. During the observation period temperature range was

11.2–27.0�C, whereas RH was 69.3–91.2%. Three females did not oviposit and ticks from

three egg masses did not hatch. In the forest, mean preoviposition and egg incubation

periods were, respectively, almost three (94.2 days) and two (211.1 days) times longer

than those recorded in the incubator. On the other hand, egg hatching success increased

from 1.2% in the incubator to 72.2% in the forest.

By the 30th day of oviposition most off the egg mass was already laid by 15 engorged

females. At this day the mass had a mean weight of 896 mg (range 403–1,222 mg). The

first female completed oviposition by day 34 and the last by day 88, before its death. From

the 30th to the 88th days, several females interrupted oviposition for a few days and started

it again later but in very small amounts. A mean number of 65.4 ± 6.6 eggs (range 56–74)

were counted in 10 mg of eggs laid by each of eight females. These values give a mean

number of 6,540 eggs per gram of egg batch, or a mean weight of 0.153 mg per an

individual egg.

Discussion

Adult A. incisum from nature attached readily to horses and fed until engorgement indi-

cating the suitability of this host species. This observation is particularly relevant if one

considers that such horses were not tick-bite naıve. It also indicates that horses that have

access to Atlantic rainforest or Amazonian areas (the natural habitat of A. incisum) could

be naturally infested by this tick. However, by our knowledge, there has been no report of

infestation by A. incisum on horse.

Laboratory data on female feeding and oviposition showed that A. incisum is a huge tick

species with a long life cycle lasting a minimum of 262.3 days under controlled conditions

Fig. 1 Engorged and unfed Amblyomma incisum female

268 Exp Appl Acarol (2009) 48:263–271

123

of 27�C, 85% RH, and a photoperiod regimen 00:24 (light:darkenss). Although different

laboratory conditions and host species used in other studies should be taken into account,

generally the mean engorged female weight was the highest and pre-oviposition and

incubation periods were the longest when compared to other Neotropical Amblyommaspecies studied so far (mostly under 27�C likely the present study), such as Amblyommaaureolatum (Pallas) (Rodrigues et al. 2002; Pinter et al. 2004); A. cajennense (Sanavria

et al. 1996; Castagnolli et al. 2003; Chacon et al. 2004); Amblyomma brasiliense (Sanches

et al. 2008) Amblyomma dubitatum Neumann (Chacon et al. 2004; Labruna et al. 2004);

Amblyomma parvum Aragao (Guglielmone et al. 1991); Amblyomma pseudoconcolorAragao (Chacon et al. 2004); Amblyomma tigrinum Koch (Labruna et al. 2002), and

Amblyomma triste Koch (Labruna et al. 2003). At the same time, mean EPE and egg

hatching rate were very low, suggesting inappropriate off-host conditions for A. incisumspecies in the laboratory. In contrast, egg hatching rate under natural conditions improved

considerably, showing that the forests patch provided a more suitable environment for the

development of this parasite species. In this regard, we speculate that in nature, higher

humidity, lower temperature and natural mold control were key features. Photoperiod is

another feature that might have had an effect on egg hatching in nature. Engorged females

in the laboratory were maintained permanently in the dark whereas in the forest patch some

daylight may have reached the ticks. Such amount of light, however, was restricted by the

relatively dark forest and by the leaves that covered the tick tubes. Finally, oviposition and

incubation periods were even longer in the forest, reassuring that A. incisum has a long life

cycle indeed.

In the comparison of the development of A. incisum immatures in laboratory to previous

studies on other Amblyomma species found in Brazil (Guglielmone et al. 1991; Daemon

and Ishizuka 1995; Prata et al. 1996, 1997; Labruna et al. 2002, 2003, 2004; Castagnolli

et al. 2003; Pinter et al. 2004; Aguirre et al. 2005; Sanches et al. 2008) it was observed that

larvae and nymphs fed fairly well on rabbits and, again, a big tick species with a long life

cycle is suggested. Both engorged larvae and nymphs had mean weights much higher than

any other Neotropical Amblyomma larvae and nymphs previously studied, as well as longer

mean feeding and premolting periods. Tick yield of A. incisum immatures in the laboratory

fell within the wide range of results of the literature, meanwhile molting rate of both

A. incisum larvae and nymphs were generally low, once more indicating inappropriate

conditions for the development in the laboratory.

Data on natural infestations by A. incisum immatures have been restricted to deer and

humans (Szabo et al. 2006a, b). In humans, nymphs were shown to feed well, able to molt

to the adult stage thereafter (Szabo et al. 2006b). A recent field study with A. incisumshowed that larvae and nymphs host-quest mostly above 30 cm height on the vegetation

(Szabo et al. 2009). These facts indicate that different medium to large-sized mammals

may be involved in the maintenance of A. incisum immatures in nature, including the tapir,

the primary host for the adult stage.

A recent 3-year study has suggested that A. incisum is capable of completing one

generation every 12 months under the field conditions of Intervales state park, with larvae

predominating during autumn, nymphs during winter, and adults during spring–summer

months (Szabo et al. 2009). A very similar pattern of one generation/year was also dem-

onstrated by A. cajennense in Brazil, Interestingly, the life cycle of A. cajennense can be

completed in less then 90 days under laboratory conditions (27�C for free-living devel-

opmental stages), but under natural conditions in the state of Sao Paulo, it was shown that

larval behavioral diapause during spring–summer was considered to be the main factor

responsible for synchronizing the life cycle to 12 months (Labruna et al. 2003). The

Exp Appl Acarol (2009) 48:263–271 269

123

present study shows that the life cycle of A. incisum would require almost 365 days to be

completed under laboratory conditions (27�C for free-living developmental stages), if we

consider summing the mean feeding, premolt, preoviposition and egg incubation values

listed in Table 1, plus a regular pre-feeding period of 30 days for larvae, nymphs, and

adults. Thus, it is likely that the 1 year generation pattern suggested for A. incisum in

Intervales State Park (Szabo et al. 2009) is controlled by naturally long developmental

stages of A. incisum, which are primarily regulated by temperature, with no indication of

occurrence of diapause.

During our observations of engorged females and their eggs under field conditions,

preoviposition and egg incubation periods were much longer (their sum [250 days) than

under laboratory conditions. However, it should be noted that these engorged females were

released in the field at end of March and April, a period corresponding to the very end of

summer and autumn. During this observation period, temperature ranged from 11.2 to

27.0�C, much lower the temperature inside the incubator, which might have accounted for

the longer tick-developmental periods. Since Szabo et al. (2009) showed that adults of

A. incisum are mostly active during spring–summer months, one would expect that under

natural conditions, preoviposition and egg incubation periods would be much shorter than

those observed in Franca municipality forest patch, during autumn–winter months.

On the whole, data obtained in this study indicates a high dependency of A. incisum on

the environment. In this regard, and on a global view, Klompen et al. (1996) have high-

lighted the importance of ecological specificity of ticks determining their host-association

patterns. In the case of A. incisum differing laboratory conditions should be tried to

determine ideal off-host requirement of this tick species. However, this is a long lasting

task in view of the extended periods demanded for the development of each stage. Nev-

ertheless, the requirement for high humidity, even though speculative at this point, might

explain the association with or even dependency for rainforests of A. incisum.

Acknowledgments Financial support was given by FAPESP and FUNADESP. Authors would like tothank Parque Estadual Intervales (institution and staff) for logistical support. We are also indebted toAparecido Dias de Oliveira for guiding researchers inside the park during tick collection. Part of this workhas been facilitated through the International Consortium on Ticks and Tick-borne Diseases (ICTTD-3)Coordination Action financed by the INCO program of the European Commission Project No. 510561.

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