Development of Hyalomma lusitanicum under laboratory conditions

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<ul><li><p>Veterinary Parasitology, 15 (1984) 57-66 57 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands </p><p>DEVELOPMENT OF HYALOMMA LUS ITANICUM UNDER LABORATORY CONDIT IONS </p><p>H. OUHELLI and V.S. PANDEY* </p><p>Inst~tu t Agronomique et V$t~rmaire Hassan II, B.P 704, Rabat-Agdal (Morocco) </p><p>(Accepted for publication 10 August 1983) </p><p>ABSTRACT </p><p>Ouhelli, H. and Pandey, V.S., 1984. Development of Hyalomma lusitanicum under labo- ratory conditions. Vet. Parasztol., 15 : 57--66. </p><p>At the constant temperature of 25C 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 16C the females did not lay eggs at all, but those which survived for 1 year and were transferred thereafter to 25C and 84% RH laid viable eggs. At 35C, the oviposition was identical at all levels of RH tested (25, 62 and 93%). At 25C, 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 pe- riod 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 25C 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. </p><p>INTRODUCTION </p><p>Hya lomma lusitanicum is a t ick specif ic to the Med i te r ranean region. Af- ter the loss of or ig inal spec imens s tud ied by Koch in 1844, this species was confused wi th H. anatol icum excavatum. Delpy (1946) , whi le revis ing the genus Hya lomma, ment ioned the "var iant lus i tanicum", but i t was on ly in 1962 that Fe ldman-Muhsam prec ise ly re-descr ibed H. lusitanicum based on spec imens f rom Morocco . It is inc r iminated in the t ransmiss ion of Theileria </p><p>* Present address: Teniersstraat 13, B-1800 Vilvoorde, Belgmm. </p><p>0304-4017/84/$03.00 1984 Elsevier Science Publishers B.V. </p></li><li><p>58 </p><p>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. </p><p>MATERIALS AND METHODS </p><p>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 25C and 84% relative humidity (RH). The engorged fe- males 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. </p><p>The development of non-parasitic stages at 25C and 84% RH and of para- sitic stages on calves and rabbits maintained at ambient temperature (20-- 25C) and humidity (80--100% RH) are arbitrarily designated as standard conditions for development of H. lusitanicum in the present work. The ex- periments were also carried out at 3 constant temperature regimes (16, 25 or 35C) 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. </p><p>Oviposition </p><p>To establish a standard oviposition curve, 9 engorged females weighing, between 340 and 730 mg, were kept at 25C and 84% RH. In 9 other re- gimes, 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. </p><p>Hatching </p><p>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 </p></li><li><p>59 </p><p>(incubation period). At the end of hatching and after microscopical verifica- tion that the unhatched eggs were not alive, the larvae were killed by heat, counted and the percentage of hatching noted. </p><p>Moulting </p><p>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. </p><p>Survival of unfed nymphs </p><p>In each of the 9 regimes, 16 unfed l(~day-old nymphs were set and ob- served daily. A nymph was considered dead when it did not react to mechan- ical and physical stimuli. </p><p>Statistical methods </p><p>The classical statistical methods were used to analyse the results (Dagnelie, 1970; Colton, 1974). The minimum statistical significance was fixed as 95% probability. The comparison of means or proportions was made by 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. </p><p>RESULTS </p><p>Oviposition </p><p>At standard conditions (25C 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 output is presented in Fig. 1A. </p><p>At 16C the females did not 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 25C 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 35C, RH has a significant influ- ence on the oviposition period (P ~ 0.001) but not on the number of eggs laid and the pre-oviposition period (P~ 0.05). At 25C, 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). </p></li><li><p>60 </p><p>6OO hi d &lt; E ~. 400 i </p><p>&lt; _J ~9 20C L0 Ld </p><p>100 </p><p>8C </p><p>6C </p><p>9 z T </p><p>4C </p><p>-r </p><p>20 </p><p>7 11 13 15 17 19 1 </p><p>DAYS OF OVIPOSITION </p><p>B </p><p>1 3 </p><p>L 7 9 11 13 15 DAYS OF OVIPOSITION </p><p>17 19 21 23 25 27 </p><p>Fig. 1. Course of oviposition (A) and percentage hatching corresponding to the days of oviposition (B) of Hyalomma lusitanicum. I indicates standard deviation. </p><p>TABLE I </p><p>Effect of temperature and relative humidity (RH, %) on the oviposition of l-lyalomma lusitanicum </p><p>Factor RH Temperature (C) (%) 25 35 </p><p>Pre -ov ipos i t ion per iod : 25 51.7 +- 1.5 9.0 -+ 1.8 in days (mean +- S.D.) 62 50.5 +- 3.7 P &lt; 0 .001 9.3 +- 4.1 P &gt; 0 .05 </p><p>93 9.0 2.0 7.8 +- 2.9 </p><p>Ov ipos i t ion per iod 25 21.3 + 1.5 16 .8 + 2.9 in days (mean ~ S.D.) 62 23.5 + 1.7 P &lt; 0 .05 17.3 -+ 1.2 P &lt; 0.01 </p><p>93 27.3 -+ 0.6 23.3 +- 3.5 </p><p>Number o f eggs / female 25 3808 + 810 5401 +- 896 (mean S.D.) 62 6250 + 474 P &lt; 0.001 6232-+ 182 P &gt; 0 .05 </p><p>-93 7364 670 5919 +- 1194 </p></li><li><p>61 </p><p>Hatching </p><p>The incubat ion per iod ranged f rom 32 to 40 days. The percentage hatch d i f fered accord ing to the day of egg lay ing of the female (Fig. 1B). Those laid at the beg inn ing and at the end of the ov ipos i t ion per iod had a lower hatch ing percentage than the others. </p><p>Larval and nymphal moulting </p><p>The ef fect of host of engorgement on mou l t ing of larvae and nymphs is p resented in Tab le II. The pre -mou l t ing per iod and percentage of mou l t were ident ica l i r respect ive of the host o f engorgement (P &gt; 0.05). </p><p>TABLE II </p><p>Larval and nymphal moultings of Hyalomma lusitanlcum engorged on calves and rabbits and maintained thereafter at 25C and 84% RH </p><p>Stage Engorged on calves Engorged on rabbits </p><p>No. Pre-moulting Percent No. Pre-moulting Percent period (days) moult period (days) moult mean -+ S.D. mean S.D. </p><p>Larvae 32 13.1 -* 1.0 87 32 13.4 1.5 92 P &gt; 0.05 Nymphs 16 18.2 2.0 100 16 19.1 + 2.3 100 P &gt; 0.05 </p><p>TABLE III </p><p>Effect of temperature and relat ive humidity on the moulting of larvae and nymphs of Hyalomma lusitanicum engorged on calves </p><p>Temper- RH Nymphal moult Larval moult ature (%) (n = 18/regime) (n = 32/regime) </p><p>(C) Pre-moult Percent Pre-moult period moult period (days) (days) mean S.D. mean S.D. </p><p>Percent moult </p><p>16 </p><p>25 </p><p>35 </p><p>25 90.5 2.5 78 62 88.0 2.0 P&gt; 0.05 83P&gt; 0.05 93 92.0 4.0 72 </p><p>25 19.5 1.5 83 62 19.5 2.0 P&gt; 0.05 100P&gt; 0.05 93 20.5 2.5 89 </p><p>25 11.0 3.0 100 62 10.0 2.0 P&gt; 0.05 94P&gt; 0.05 93 11.5 1.5 94 </p><p>55.5 5.0 P&gt; 0.05 13 P&lt; 0.01 53.0 3.0 88 </p><p>13.0 2.0 44 13.5 1.5 P&gt; 0.05 78 P&lt; 0.05 14.0 3.0 84 </p><p>8.0 1.1 9 8.2 2.1 P&gt; 0.05 16 P&lt; 0.01 9.1 2.0 88 </p></li><li><p>62 </p><p>The effect of different temperature and humidity 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 &gt; 0.05). On the contrary, at a given RH, the pre-moult period decreases very significantly with an increase in temperature (P &lt; 0.01); it is 7--8 times shorter at 35C compared to 16C. Between 16 and 25C, for each degree Centrigrade increase in temperature there was a decrease in the pre- moult period of 4.5 days for larvae and about 8 days for nymphs, and be- tween 25 and 35C, the decrease was 0.5 and 0.9 days for larvae and nymphs, respectively, for each degree increase in temperature. </p><p>Although RH does not influence the pre-moult period it has an effect on the percentage of moulting (Table III). At 16C and 25% RH, larvae did not moult at all. In general, lower RH has an adverse effect on the moulting of larvae (P &lt; 0.05 or 0.01), whereas nymphs seem to be indifferent to the fluc- tuations of RH (P &gt;0.05). </p><p>Survival of unfed nymphs </p><p>Low temperature and high humidity have a favourable effect on survival of the nymphs (P &lt; 0.05) (Table IV). In general, with an increase in temper- ature, the survival period decreases. Conversely, as the RH increases, the sur- vival rate increases. </p><p>TABLE IV </p><p>Effect of temperature and relative humidity on the survival of unfed nymphs of Hyalomma lusitanlcum </p><p>Relative humidity Mean survival in days S.D. (n = 16/regime) (%) Temperature 25 62 93 (C) </p><p>16 28+- 3 44-+ 5 89-+ 8 25 12+ 2 19-+ 2 60 5 P&lt; 0.05 35 8-+2 15-+3 47-+4 </p><p>Sex ratio </p><p>The sex ratio was determined on 118 adult descendants of the females maintained at 25C and 84% RH and fed on calves. The female-to-male ratio was 1:2. </p><p>Parasitic stages </p><p>The larvae, nymphs and adult females were fed on calves and rabbits and the engorgement period was determined (Table V). H. lusitanicum behaved </p></li><li><p>TABLE V </p><p>Duration of engorgement of Hyalornma lusitanicum on calves and rabbits </p><p>63 </p><p>Stage of engorgement Number Duration (days) </p><p>On calves On rabbits Mean Range Mean Range </p><p>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 -- -- </p><p>a lways as a 3-host tick. However, the adults did not engorge on rabbits. The weight of females engorged on calves (n = 83) varied between 218 and 824 mg (mean 543 rag). </p><p>Total duration of life cycle </p><p>The durat ion of d i f ferent per iods in the life cycle of H. lusitanicum, based on the standard condi t ions (25C, 84% RH and fed on calves), is presented in Table VI. The t ime taken to complete the life cycle was between 138 and 196 days, more than hal f o f which is spent for p roduct ion and hatching of eggs. The t ime necessary for chit in izat ion of the young stages (the t ime needed before the young forms are able to attach to the host) is not included in the table. The chit in izat ion period, under present work ing condit ions, was 10 days for each of the 3 phases; larva, nymph, adult. </p><p>TABLE VI </p><p>Data on the different stages of the life cycle of Hyalomma lusitanicum. The non-parasitic stages developed at 25C and 84% RH, and the parasitic stages on calves maintained at ambient conditions </p><p>Stage Duration (days) </p><p>Minimum Maximum </p><p>Pre-oviposition period 40 Oviposition lasts 20 Incubation + eclosion 32 Engorgement of larvae 4 Larval pre-moult 12 Engorgement of nymphs 6 Nymphal pre-moult 16 Engorgement of adult female 8 </p><p>60 30 40 </p><p>6 14 12 21 13 </p><p>Tot~ 138 196 </p></li><li><p>64 </p><p>DISCUSSION </p><p>In different species of ticks, temperature is one of the main determining factors in oviposition. Temperature requirements of H. lusitanicum for ovi- position seem to be close to that of H. anatolicum anatolicum (Snow and Arthur, 1966). </p><p>Performance of engorged females is influenced by the combined effects of temperature and RH. Data in Table I were analysed as 25C versus 35C 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 35C (P &gt; 0.05). For oviposition period, the differences between 25 and 35C and RH of 25 and 62% were highly significant (P &lt; 0.001). Differences in the oviposition peri- od 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 35C (P &gt; 0.05) (Table I). These results are quite differ- ent from those ofH. 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. </p><p>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 i...</p></li></ul>

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