الفهرس 
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Abstract
Argasidae are soft bodied and multi-host ticks with several nymphal stages and repeatedly feeding adults. They infest caves, nests, crevices or burrows or hide in soils or cracks of tree bark or wood nearby host-occupied sheltered sites. They play an important role in the transmission of certain pathogens that cause heavy economic losses to poultry industry. Massive infestations by Argas spp. on poultry can cause anemia, paralysis and death by exsanguinations. The soft tick, Argas (Persigargas) persicus, is the most important ectoparasite of chicken and other domestic fowl, as well as humans.
The entomopathogenic fungus, Beauveria bassiana (Balsamo) (Ascomycota: Hypocreales), is distributed worldwide and can be isolated from soil. This mycoinsecticide fungus species has adapted successfully to control a number of important pests and is proposed as a biological control agent due to its non toxic nature towards non target animals and humans. Beauveria bassiana has a broad host range of approximately 700 insect species. Among insects, growth of the fungus inside them causes death by exhaustion and by disruption of their physiological process.
7.1. Biological studies:
The present study is carried out to assess the effect of different sprayed concentrations of B. bassiana (106, 107, 108, 109 and 1010 conidia/ml) suspended in triton X100, separately, on some biological parameters of the unfed and fed adult tick A. persicus including mobility and viability, sexual behavior, ability of feeding, amounts of ingested blood and emitted coxal fluid, blood digestion and fertility. Two control groups were used; +ve control group sprayed with 0.01% triton X100 and -ve control one not sprayed.
The study revealed a more or less similar pattern of response in adults treated before or after feeding. A marked increase of immobile and dead male or female ticks following a single treatment with the above concentrations of the fungus, particularly the higher ones was observed. It also caused a decrease in the percentage of fed ticks, amounts of ingested blood and emitted coxal fluid and sexual response while it completely inhibited oviposition.
The present biological study revealed that spraying B. bassiana on A. persicus ticks away from its host is sufficient to support its efficiency in controlling the tick.
7.2. Histological studies:
from the above observations we selected 107 conidia/ml B. bassiana as a sublethal concentration sprayed after feeding to be followed histologically on two target organs namely integument and ovary in the female A. persicus.
7.2.1. Integument:
Using scanning electron microscopy, the surface morphology of female A. persicus revealed that the dorsal integument contained circular dermal discs that are surrounded with irregular mammillae-like structures, some bearing setae.
Histological examination using light and transmission electron microscopy showed that the integument consists of the cuticle, underlined by the epidermis. The cuticle is distinguished into a thin outer epicuticle and a thick inner procuticle. The epicuticle is formed of cement, wax, cuticulin and a dense homogenous layer. The latter is penetrated by wax canals that open at the surface. The procuticle is differentiated into outer and inner procuticle, and an underlying sub-cuticular layer. Both outer and inner procuticle contain laminae and pore canals which are connected to the wax canals at the interface with the epicuticle.
The epidermal layer is a single layer of flattened epithelial cells. Their cytoplasm is filled with ribosomes, RER, mitochondria and lysosomes, and carry microvilli at their apical surface. Dermal glands found in this layer are connected to the exterior via a duct. Each gland consists of 2-5 large polygonal cells with irregular nuclei and prominent nucleoli.
Scanning electron microscopy revealed that B. bassiana caused extensive alterations in the surface morphology of A. persicus integument. The ticks showed external symptoms of fungal adhesion, mucilage formation and integumental erosion. Four days after treatment, germination is evident on the surface and conidia differentiate into germ tubes, some forming appressoria exerting mechanical pressure leading to penetration. Repenetration of the fungus from the interior to the surface of the integument is observed 3 days after death on the cadavers which appeared encompassed with a network of hyphae.
Light and transmission electron microscopy revealed that the fungal penetration destructed the epicuticle and procuticle leaving empty canals. The procuticle lost its lamellar pattern and became spongy-like due to the dilated pore canals that were invaded by the fungus.
Fungal penetration caused degeneration of the epidermal cell organelles, their nuclei appeared karyorrhexed and karyolysed, absence of microvilli and rupture of the plasma membrane. The organelles of dermal gland cells were also highly damaged.
The changes observed in the integument of the tick A. persicus after treatment with the fungus B. bassiana facilitated the entrance of the fungus into the circulating haemolymph thus exerting its effect and reaching other organs.
7.2.2. Ovary:
The scanning electron microscopy revealed that the ovary of fed female A. persicus appeared as cluster of grape-like structure to which oocytes at different developmental stages are attached. The egg shell covering the vitrellogenic oocytes exhibited a characteristic rough tuberculated pattern on its surface.
Histological studies using light and transmission electron microscopy revealed that the ovarian wall carried 3 developmental stages of oocytes namely young, previtellogenic and vitellogenic. After feeding and mating, the 3 stages markedly increased in size along the examined period and their cytoplasmic organelles were highly developed.
Beauveria bassiana caused deterioration of growth and development of all oocytes. Scanning electron microscopy revealed that the ovary became stretched considerably carrying fewer highly deformed oocytes. The ovarian wall became transparent, distended and filled with a fluid. No signs of vitellogenic oocytes could be observed.
Light and transmission electron microscopy revealed that the ovarian wall appeared ruptured. Previtellogenic oocytes exhibited thick tunica propria, masses of dense bodies peripheral basophilia and damaged surface microvilli. Cytoplasmic organelles of all remaining oocyte, interstitial cells and funicle cells appeared degenerated. All oocytes revealed a decrease in size when compared to untreated one. Conidia were observed in all oocytes and funicle cells.
The histopathological study of the ovary confirm that B. bassiana possesses a toxic potential that interferes with reproduction and control the reproductive system either directly by entering the oocytes or indirectly by producing toxins that interfere with feeding and vitellogenin production and release into the haemolymph. Beauveria bassiana contributes to the tickʼs control in a way safer to its host and to the environment.
7.3. Biochemical and molecular studies:
Argas persicus females were sprayed with 107 conidia/ml B. bassiana as a sublethal concentration after feeding to study its effect on tick resistance and genotoxicity using biochemical and molecular analyses for both mobile and immobile individuals.
7.3.1. Total proteins:
In the present study, the expression of a band with molecular size ≈ 65 KDa increased in mobile ticks after one week of treatment, decreased after 2 weeks and finally disappeared after 4 weeks compared with control ones. However, in immobile ones it increased after 2 weeks and then decreased after 4 weeks. A band with molecular size ≈ 14.5 KDa was not affected in mobile ticks after one or two weeks of treatment and this band was slightly expressed in immobile ones. This expression was higher in mobile than immobile ones after 4 weeks. On the other hand, the band with molecular size ≈ 5 KDa was not scored after 4 weeks in both mobile and immobile ticks.
Four new protein bands with molecular sizes ≈ 22, 24, 27 and 42 KDa) appeared in treated ticks specially after 4 weeks. Most bands were highly expressed in mobile ticks compared to immobile ones.
from the present work, we suggested that the elevated expression of proteins may represent stress proteins to overcome the effects of the fungus in treated individuals.
7.3.2. Esterases:
In the present study, esterase profile of untreated ticks revealed four bands (EST1 to EST4). The activity of EST1 was inhibited in immobile individuals 2 and 4 weeks after treatment. High levels of EST2 activity were observed particularly in mobile individuals after 4 weeks of treatment and lost its activity in all other treated ones along the examined periods. Moreover like EST1, EST3 lost its activity in immobile individuals 2 and 4 weeks after treatment. However, its activity increased only in mobile individuals during the 2nd and 4th weeks after treatment. The activity of EST4 showed high levels of activity in mobile and immobile individuals 2 and 4 weeks after treatment.
The present results revealed that new three esterase bands were recognized in mobile individuals after 4 weeks of treatment; one band between EST1 and EST2 and two faster ones following EST4.
from the present work we suggest that the loss of esterase bands in immobile individuals, increase levels of bands in mobile individuals with longer post-treatment intervals and recognition of new bands also in mobile ones indicate their possible association with resistance.
7.3.3. RAPD and ISSR:
In the present study, five RAPD primers and five ISSR primers were used to examine the probable genotoxic effects of B. bassiana in A. persicus. OPB-11 primer showed a prominent control band with a size of ≈ 540 bp in mobile treated ticks indicating its possible relation to resistance against the fungus. After using OPC-04 primer, a band with molecular size of ≈ 370 bp disappeared from immobile treated ticks after 2 and 4 weeks. This band might be a sensitive region of the tick’s genome. A new band was scored in mobile treated ticks (≈ 1300 bp) using HB-12 primer that may be related to resistance.
Also, OPA-02 and A-49 primers gave the highest percentage of polymorphism in RAPD and ISSR analyses, respectively. The polymorphisms ranged between 3.3% and 52.5% using RAPD primers, while they ranged between 23.5% and 77.3% using ISSR primers.
The present investigation indicated that the genotoxicity of B. bassiana increased and the genomic stability decreased with increasing the intervals post treatment (from 1st to 2nd to 4th week after treatment).
The changes in DNA bands observed in the present study after the use of RAPD and ISSR assays proved that they are reliable tools that could detect genotoxic effects induced by the fungus in ticks.