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Veterinary Parasitology, 15 (1984) 5 7 - 6 6 57 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

D E V E L O P M E N T O F H Y A L O M M A L U S I T A N I C U M U N D E R

L A B O R A T O R Y C O N D I T I O N S

H. OUHELLI and V.S. PANDEY*

Inst~tu t Agronomique et V$t~rmaire Hassan II, B.P 704, Rabat-Agdal (Morocco)

(Accepted for publication 10 August 1983)

ABSTRACT

Ouhelli, H. and Pandey, V.S., 1984. Development of Hyalomma lusitanicum under laboratory conditions. Vet. Parasztol., 15 : 57--66.

At the constant temperature of 25°C and relative humidity (RH) of 84%, the average pre-oviposition period of Hyalomma lusitanicum was 47 days, the oviposition lasted an average of 26 days and the total egg production was an average of 6320 per female. At 16°C the females did not lay eggs at all, but those which survived for 1 year and were transferred thereafter to 25°C and 84% RH laid viable eggs. At 35°C, the oviposition was identical at all levels of RH tested (25, 62 and 93%). At 25°C, the pre-oviposition period was shorter at 93% RH, and the number of eggs laid was fewer at 25% RH. The eggs hatched in 32--40 days, the hatching percentage being lower in batches of eggs laid at the beginning and at the end of the oviposition period. The larval and nymphal moultings were not influenced by the type of host. As the temperature increased, the pre-moult period became shorter. The engorged larvae were more sensitive to the low RH than the engorged nymphs, whose moulting percentage was always greater than 72 in all regimes. Low temperature and high humidity had a favourable effect on the survival of unfed nymphs. The female-to-male ratio was 1:2. Hyalornma lusztanicum always behaved as a 3-host tick. The adults did not engorge on rabbits. The female ticks engorged on calves weighed an average 543 mg. Ticks maintained at 25°C and 84% RH and engorged on calves completed the life cycle in 138--196 days, which does not include the period of chitinization of about 30 days. More than half of this period was spent in egg laying and hatching.

INTRODUCTION

H y a l o m m a lus i tanicum is a t i c k speci f ic to t he M e d i t e r r a n e a n reg ion . Af- t e r t he loss o f o r ig ina l s p e c i m e n s s t u d i e d b y K o c h in 1844 , th i s species was c o n f u s e d w i t h H. anato l icum e x c a v a t u m . D e l p y ( 1 9 4 6 ) , w h i l e rev i s ing the g e n u s H y a l o m m a , m e n t i o n e d t h e " v a r i a n t l u s i tan icum" , b u t i t was o n l y in 1 9 6 2 t h a t F e l d m a n - M u h s a m p rec i s e ly r e -desc r ibed H. lus i tanicum based o n s p e c i m e n s f r o m M o r o c c o . I t is i n c r i m i n a t e d in t he t r a n s m i s s i o n o f Theileria

* Present address: Teniersstraat 13, B-1800 Vilvoorde, Belgmm.

0304-4017/84/$03.00 © 1984 Elsevier Science Publishers B.V.

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annulata in the Iberian peninsula (Purnell, 1978). In Morocco, according to Blanc and Bruneau {1956), its larvae are active from May to October, the nymphs from July to September, and adults throughout the year. We have been able to transmit T. annulata by H. lusitanicum experimentally {unpub- lished observations). The precise biological data on the different phases of its life cycle are not available. The present work was therefore undertaken to study the life cycle of this tick under controlled laboratory conditions.

MATERIALS AND METHODS

The original laboratory colony of H. lusitanicum was established from a single engorged female collected in the Gharb region of Morocco, about 100 km north of Rabat. The ticks were fed on 7- 10-month-old calves and on rabbits which had no previous contact with ixodid ticks. The non-parasitic stages were kept at 25°C and 84% relative humidi ty (RH). The engorged females were used in the experiments within 12 h of their detachment from calves. They were weighed and put individually in vials of 7 cm height and 3 cm diameter for _further observations.

The development of non-parasitic stages at 25°C and 84% RH and of parasitic stages on calves and rabbits maintained at ambient temperature (20-- 25°C) and humidi ty (80--100% RH) are arbitrarily designated as standard conditions for development of H. lusitanicum in the present work. The experiments were also carried out at 3 constant temperature regimes (16, 25 or 35°C) which were permutated at 3 relative humidities (25, 62 or 93%), thus giving 9 combinations of temperarature and RH. The relative humidities were obtained by using known concentrations of potassium hydroxide {Solomon, 1951). Standard laboratory incubators were used to maintain the constant temperatures. The tick material used in all the experiments origi- nated from the colony maintained under standard conditions.

Oviposition

To establish a standard oviposition curve, 9 engorged females weighing, between 340 and 730 mg, were kept at 25°C and 84% RH. In 9 other regimes, 3--4 engorged females, weighing between 475 and 618 mg, were used. The pre-oviposition and the oviposition periods were noted for each female. The eggs were collected and counted daffy.

Hatching

To maintain homogeneity in the hatching experiments, batches of 100 eggs laid by single females on their 5th day of egg laying were incubated at 9 regimes of temperature and RH. Four such batches (4 × 100 eggs) were used at each regime. The eggs were observed daffy to determine the first hatching

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(incubation period). At the end of hatching and after microscopical verification that the unhatched eggs were not alive, the larvae were killed by heat, counted and the percentage of hatching noted.

Moulting

In each of the 9 regimes, 32 engorged larvae and 18 engorged nymphs were kept for observation after their detachment from calves. The moulting was noted daily.

Survival of unfed nymphs

In each of the 9 regimes, 16 unfed l(~day-old nymphs were set and observed daily. A nymph was considered dead when it did not react to mechan- ical and physical stimuli.

Statistical methods

The classical statistical methods were used to analyse the results (Dagnelie, 1970; Colton, 1974). The minimum statistical significance was fixed as 95% probabili ty. The comparison of means or proport ions was made b y t-test and by analysis of variance. In some cases, non-parametric methods (distribution- free methods) such as the Wilcoxson rank sum test were utilised.

RESULTS

Oviposition

At standard condit ions (25°C and 84% RH), the pre-oviposition period was 40--60 days (mean 47 days), the oviposition lasted 20--30 days (mean 26 days) and the mean number of eggs laid per female was 6320 (range 4230--7124). The average daily egg ou tpu t is presented in Fig. 1A.

At 16°C the females did no t lay eggs at any of the RH tested. After 1 year of storage at this temperature, many of the females died, but the surviving ones, when transferred to 25°C and 93% RH laid eggs which hatched into larvae. The results for other regimes of temperature and RH are presented in Table I. Statistical analysis showed that at 35°C, RH has a significant influence on the oviposition period (P ~ 0.001) but not on the number of eggs laid and the pre-oviposition period (P~ 0.05). At 25°C, the pre-oviposition period was shorter at 93% RH (9 days) than at 62 and 25% RH (50--51 days). At this temperature, pre-oviposition and oviposition periods and the number of eggs laid were statistically different at 3 RH tested (P ~ 0.001).

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6OO hi d < E ~. 4 0 0 i

< _J ~9 20C L0 Ld

100

8C

6C

£9 z T

4C

-r

20

7 11 13 15 17 19 1

DAYS OF OVIPOSITION

B

1 3

L 7 9 11 13 15 DAYS OF OVIPOSITION

17 19 21 23 25 27

Fig. 1. Course of oviposition (A) and percentage hatching corresponding to the days of oviposition (B) of Hyalomma lusitanicum. I indicates standard deviation.

T A B L E I

Effect o f temperature and relative humidity (RH, %) on the oviposition of l-lyalomma lusitanicum

Factor RH Temperature (°C) (%) 25 35

P r e - o v i p o s i t i o n p e r i o d : 25 5 1 . 7 +- 1 .5 9 . 0 -+ 1 .8 in d a y s ( m e a n +- S .D . ) 62 5 0 . 5 +- 3 .7 P < 0 . 0 0 1 9 .3 +- 4 . 1 P > 0 . 0 5

93 9 .0 ± 2 .0 7 .8 +- 2 .9

O v i p o s i t i o n p e r i o d 25 2 1 . 3 + 1 .5 1 6 . 8 + 2 .9 in d a y s ( m e a n ~ S .D . ) 6 2 2 3 . 5 + 1 .7 P < 0 . 0 5 1 7 . 3 -+ 1 .2 P < 0 . 0 1

93 2 7 . 3 -+ 0 .6 2 3 . 3 +- 3 .5

N u m b e r o f e g g s / f e m a l e 25 3 8 0 8 + 8 1 0 5 4 0 1 +- 8 9 6 ( m e a n ± S.D.) 6 2 6 2 5 0 + 4 7 4 P < 0 . 0 0 1 6 2 3 2 - + 1 8 2 P > 0 . 0 5

- 9 3 7 3 6 4 ± 6 7 0 5 9 1 9 +- 1 1 9 4

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Hatching

T h e i n c u b a t i o n p e r i o d r a n g e d f r o m 32 to 40 days . T h e p e r c e n t a g e h a t c h

d i f f e r e d a c c o r d i n g to t h e d a y o f egg l a y i n g o f t h e f e m a l e (Fig. 1B). T h o s e la id a t t h e b e g i n n i n g a n d a t t h e e n d o f t h e o v i p o s i t i o n p e r i o d h a d a l o w e r

h a t c h i n g p e r c e n t a g e t h a n t h e o thers .

Larval and nymphal moulting

T h e e f f ec t o f h o s t o f e n g o r g e m e n t o n m o u l t i n g o f la rvae a n d n y m p h s is p r e s e n t e d in T a b l e II. T h e p r e - m o u l t i n g p e r i o d a n d p e r c e n t a g e o f m o u l t we re i d e n t i c a l i r r e spec t ive o f t h e h o s t o f e n g o r g e m e n t (P > 0 .05) .

TABLE II

Larval and nymphal moultings of Hyalomma lusitanlcum engorged on calves and rabbits and maintained thereafter at 25°C and 84% RH

Stage Engorged on calves Engorged on rabbits

No. Pre-moulting Percent No. Pre-moulting Percent period (days) moult period (days) moult mean -+ S.D. mean ± S.D.

Larvae 32 13.1 -* 1.0 87 32 13.4 ± 1.5 92 P > 0.05 Nymphs 16 18.2 ± 2.0 100 16 19.1 + 2.3 100 P > 0.05

TABLE III

Effect of temperature and re la t ive humidity on the moulting of larvae and nymphs of Hyalomma lusitanicum engorged on calves

Temper- RH Nymphal moult Larval moult ature (%) (n = 18/regime) (n = 32/regime)

(°C) Pre-moult Percent Pre-moult period moult period (days) (days) m e a n ± S .D . m e a n ± S . D .

Percent moult

16

25

35

25 90.5 ± 2.5 78 62 88.0± 2.0 P > 0.05 8 3 P > 0.05 93 92.0 ± 4.0 72

25 19.5 ± 1.5 83 62 19.5 ± 2.0 P > 0.05 1 0 0 P > 0.05 93 20.5 ± 2.5 89

25 11.0 ± 3.0 100 62 10.0± 2.0 P > 0.05 9 4 P > 0.05 93 11.5 ± 1.5 94

55.5± 5.0 P > 0.05 13 P < 0.01 53.0 ± 3.0 88

13.0 ± 2.0 44 13.5± 1.5 P > 0.05 78 P < 0.05 14.0 ± 3.0 84

8.0 ± 1.1 9 8.2± 2.1 P > 0.05 16 P < 0.01 9.1 ± 2.0 88

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The effect of different temperature and humidi ty regimes on the moulting of larvae and nymphs engorged on calves is presented in Table III. At a given temperature, RH does not influence the pre-moult period of larvae and nymphs (P > 0.05). On the contrary, at a given RH, the pre-moult period decreases very significantly with an increase in temperature (P < 0.01); it is 7--8 times shorter at 35°C compared to 16°C. Between 16 and 25°C, for each degree Centrigrade increase in temperature there was a decrease in the pre- moul t period of 4.5 days for larvae and about 8 days for nymphs, and between 25 and 35°C, the decrease was 0.5 and 0.9 days for larvae and nymphs, respectively, for each degree increase in temperature.

Although RH does not influence the pre-moult period it has an effect on the percentage of moult ing (Table III). At 16°C and 25% RH, larvae did not moult at all. In general, lower RH has an adverse effect on the moulting of larvae (P < 0.05 or 0.01), whereas nymphs seem to be indifferent to the fluc- tuations of RH (P >0.05) .

Survival o f unfed nymphs

Low temperature and high humidi ty have a favourable effect on survival of the nymphs (P < 0.05) (Table IV). In general, with an increase in temperature, the survival period decreases. Conversely, as the RH increases, the survival rate increases.

TABLE IV

Effect of temperature and relative humidity on the survival of unfed nymphs of Hyalomma lusitanlcum

Relative humidity Mean survival in days ± S.D. (n = 16/regime) (%) Temperature 25 62 93 (°C)

16 28+- 3 44-+ 5 89-+ 8 25 12+ 2 19-+ 2 60± 5 P< 0.05 35 8-+2 15-+3 47-+4

Sex ratio

The sex ratio was determined on 118 adult descendants of the females maintained at 25°C and 84% RH and fed on calves. The female-to-male ratio was 1 : 2 .

Parasitic stages

The larvae, nymphs and adult females were fed on calves and rabbits and the engorgement period was determined (Table V). H. lusitanicum behaved

TABLE V

Duration of engorgement of Hyalornma lusitanicum on calves and rabbits

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Stage of engorgement Number Duration (days)

On calves On rabbits Mean Range Mean Range

Larvae 460 4.9 4-- 6 5.2 4-- 8 Nymphs 123 7.3 6--12 7.6 6--11 Adults 34 10.1 8--13 -- --

a lways as a 3 -hos t t ick. Howeve r , the adul ts did n o t engorge on rabbi ts . The we igh t o f f ema les engorged on calves (n = 83) var ied b e t w e e n 218 and 824 m g ( m e a n 543 rag).

Total duration o f life cycle

The du ra t i on o f d i f f e r en t pe r iods in the life cycle o f H. lusitanicum, based on the s t anda rd c o n d i t i o n s (25°C, 84% R H and fed on calves), is p r e sen t ed in Table VI. The t ime t a k e n to c o m p l e t e the life cycle was b e t w e e n 138 and 196 days , m o r e t h a n ha l f o f which is spen t fo r p r o d u c t i o n and ha tch ing o f eggs. The t i m e necessary fo r ch i t in iza t ion o f the y o u n g stages ( the t i m e needed be fo r e the y o u n g f o r m s are able to a t t ach to the hos t ) is n o t inc luded in the table . T h e ch i t in iza t ion pe r iod , u n d e r p re sen t work ing condi t ions , was 10 days fo r each o f the 3 phases ; larva, n y m p h , adul t .

TABLE VI

Data on the different stages of the life cycle of Hyalomma lusitanicum. The non-parasitic stages developed at 25°C and 84% RH, and the parasitic stages on calves maintained at ambient conditions

Stage Duration (days)

Minimum Maximum

Pre-oviposition period 40 Oviposition lasts 20 Incubation + eclosion 32 Engorgement o f larvae 4 Larval pre-moult 12 Engorgement of nymphs 6 Nymphal pre-moult 16 Engorgement o f adult female 8

60 30 40

6 14 12 21 13

Tot~ 138 196

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DISCUSSION

In different species of ticks, temperature is one of the main determining factors in oviposition. Temperature requirements of H. lusitanicum for ovi- posit ion seem to be close to that of H. anatolicum anatolicum (Snow and Arthur, 1966).

Performance of engorged females is influenced by the combined effects of temperature and RH. Data in Table I were analysed as 25°C versus 35°C sep- arately for each of the 3 regimes of RH. At a given RH, no significant differ- ence was found in the number of eggs laid at 25 and 35°C (P > 0.05). For oviposition period, the differences between 25 and 35°C and RH of 25 and 62% were highly significant (P < 0.001). Differences in the oviposition period at the 2 temperatures were significant only at 62% RH (P ~ 0.01). At 93% RH, none of the 3 parameters concerning oviposition are statistically different at 25 and 35°C (P > 0.05) (Table I). These results are quite different from those o f H . a. anatohcum, in which egg laying stops at 75% RH and the oviposition period becomes longer with the increase of RH from 25 to 50% (Snow and Arthur, 1966). In some other ticks such as Rhipicephalus appendiculatus (Branagan, 1973) and Boophilus annulatus (OuheUi et al., 1982), there is no direct relationship between RH and the pre-oviposition and oviposition periods.

The eggs laid after the 23rd day of the start of oviposition did not hatch. According to Londt (1977) and Iwuala and Okpala (1977), this might be due to the exhaustion of nutrients, diminution in the water content and deficien- cy in the fertilization of eggs. Why the hatching percentage of eggs laid in the first 6 days of oviposition is low is not clear.

Water exchange equilibrium (Knulle, 1966), which is affected by RH and nutrients such as glycogen, lipid and protein, which are related to the varia- tions of temperature (Hanumante et al., 1981), are among the main determining factors in the longevity of ticks. In general, the engorged larvae of H. lusitanicum are more vulnerable than the engorged nymphs (Table III). The regime most favourable for the moulting of larvae to nymphs (35°C, 93~ RH) is not the best for the survival of the nymphs (Table IV). For survival, low temperature and high RH are more suitable.

Certain specms of Ixodidae such as H. dromedaril, known to be a 3-host tick in nature, can behave as a 2-host or 3-host tick under laboratory conditions (Bouchalova et al., 1977), but H. lusitanicum behaved invariably as a 3-host tick and on no occasion was the moulting of larvae to nymphs observed on experimental rabbits or calves.

Hyalomma lusitanicum needed 138--196 days to complete the life cycle. By utilizing the more favourable regime, the pre-oviposition period could be reduced by 40 days (Table I), which further reduced the duration of the life cycle. According to Bouchalova et al. (1977), H. dromedarii completes its life-cycle in 3 months, a shorter period than that needed by H. lusitanicum, but Delpy (1937), working under different conditions, found 9 months for

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completion of the life-cycle of H. dromedarii. This demonstrates the difficul- ty in comparing the results of different studies utilising different regimes and experimental conditions.

Hyalomma lusitanicum was able to engorge at larval and nymphal stages on both rabbits and calves. If this behaviour is confirmed under natural conditions, it will have 2 important implications. Firstly, H. lusitanicum will be able to transmit Theileria annulata transstadially, and secondly it will not be easy to control H. lusitanicum by the usual methods, as it can engorge equal- ly well on cattle or rabbits in the larval and nymphal stages. Therefore, one would have to take into consideration the wild rabbit population, if any, where control measures are envisaged in cattle.

There were twice the number of males as females. This numerical domi- nance of males over females would not change the vector role of the tick significantly. Although males do not engorge in the same manner as females, they are shown to transmit the protozoan parasites (Dalgliesh et al., 1978), and it is possible that H. lusitanicum males do transmit the diseases. How- ever, this needs further verification.

ACKNOWLEDGEMENT

This work was undertaken while V.S. Pandey was working under FAO/ UNDP project MOR/78/005 as Professor of Parasitology, and the assistance of FAO, is gratefully acknowledged.

REFERENCES

Blanc, G. and Bruneau, J., 1956. Etude ~pid~miologique dans la ?or~t de Nefifikh. Pres- ence chez le lapin de garence et ses arthropodes piqueurs de virus pathog~ne pour l~omme. Arhc. Inst. Pasteur Maroc, 5: 87--200.

Bouchalova, J., Honzakova, E. and Darnel, M., 1977. Development and survival of the tick Hyalomma dromedarii (Koch) under laboratory conditions. Folia Parasitol. (Prague), 24: 55--62.

Branagan, D., 1973. The developmental period of ixodid tick Rhipicephalus appendiculatus Neum. under laboratory conditions. Bull. Entomol. Res., 63: 155--168.

Colton, T., 1974. Statistics in Medicine. Little Brown and Company, Boston. Dagnehe, P., 1970. Th~orie et M~thodes Statistiques. Vol. II. J. Duculot, Gembloux, Bel-

gium. Dalgliesh, R.J., Stewart, N.P. and Callow, L.L., 1978. Transmission of Babesla bigemma

by transfer of adult male Boophilus annulatus. Aust. Vet. J., 54: 205--206. Delpy, L.P., 1937. Description de Hyalomma dromedari~: morphologie de la larve et de

la nymphe. Ann. Parasitol. Hum. Comp., 15: 481--486. Delpy, L.P., 1946. R~vision par les voies exp~rimentales du genre Hyalomma (Koch,

1884). Note pr~liminaire. Ann. Parasitol. Hum. Comp., 21: 267--293. Feldman-Muhsam, B., 1962. Revision of the genus Hyalomma. III. H lusttan~cum Koch

and H. anatohcum K. Parasitology, 52: 211--219. Hanumante, M.M., Patil, P.M. and Nagabhushanam, R., 1981. Thermobiology of the

ixodid tick Hyalomma anatohcum anatolicum (Koch, 1844). Riv. Parasitol., 42: 67-- 78

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Iwuala, M.O.E. and Okpala, I., 1977. Egg output in the weights and states of engorgement of Amblyomma variegatum (Fabr) and Boophilus annulatus (Say). Folia Parasitol. (Prague), 24: 162--172.

Knulle, W., 1966. Equilibrium humidities and survival of some tick larvae. J. Med. Entomol., 2: 335--338.

Londt, J.G.H., 1977. Oviposition and incubation in Boophilus decoloratus (Koch, 1844). Onderstepoort J. Vet. Res., 44: 13--20.

Ouhelli, H., Pandey, V.S. and Choukri, M., 1982. The effects of temperature, humidity, photoperiod and weight of the engorged female on oviposition of Boophilus annulatus (Say, 1821). Vet. Parasitol., 11: 231--239.

Purnell, R.E., 1978. Theileria annulata as a hazard to cattle in countries on the northern Mediterranean littoral. Vet. Sci. Commun., 2: 3--10.

Snow, K.R. and Arthur, D.R., 1966. Oviposition in Hyalomma anatohcurn anatolicum (Koch, 1844) (Ixodoidea: Ixodidae). Parasitology, 56: 555--568.

Solomon, M.E., 1951. The control of humidity with KOH, H2SO 4 and other solutions. Bull. Entomol. Res., 42: 543--559.

RESUME

Ouhelli, H. and Pandey, V.S., 1984. Developpement de Hyalomma lusitanzcum sous conditions de laboratoire. Vet Parasltol., 15 57--66.

A temperature et humidit~ relative (HR) constante (25°C et 84% HR) la dur4e de pr4- oviposition de Hyalomma lusitanicum est de 40 ~ 60 jours, l 'oviposition dure de 20 ~ 30 jours et le nombre total des oeufs produit par femelle est 6320. A 16°C et quelque soit I 'HR utilis~e, aucune femelle n'a pondue, mais celles qui ont surv~cu et ont 4t4 mise 25°C et 93% HR ont pu pondre des oeufsvlables. A 35°C, I 'HR n'mtervmnt pasde mani~re significative, ni sur les dur~es de prd-oviposition et oviposition, ni sur la production des oeufs. A 25°C, par contre, HR de 93% raccourcit net tement la dur4e de pr~-oviposition. Sauf ,~ 25°C et 25% HR, la production totale d'oeufs ne semble pas ~tre affect~e signifi- cativement par les temperatures et les HR. La dur~e d' incubation d'oeufs est de 32 ~ 40 jours. La pourcentage d'~closion est faible chez les oeufs pondus au d4but et ~ la fin d'oviposition. La dur4e du mue est ind~pendant du hSte; le temps avant la mue diminue avec une augmentation de temperature. Les larves engorg~es sont beaucoup plus sensible

l ' influence de I 'HR que les nymphes engorg~es, qui muent dans tous le s r4gimes ~ un taux sup~rieur ~ 72%. Les falbles temperatures et les hautes HR sont favorables ~ la survie des nymphes d jeun. La proport ion de femelles ~ m~les est de 1:2. Sur le veau ou sur le lapin, H. lusitanwum dvolue comme une tique ~ trois hStes. Les formes adultes n 'ont pas pu s'engorger chez le lapin. Les poids de femelles engorg~es sur le veau varient entre 218 et 824 rag. La dur~e du cycle total de H. lusitanicum est entre 138 et 198 jours, plus du moitid 4tant occup~e par 1 'oviposition et 1 'dclosion.