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Research Article
 

The Activity of Aspergillus terreus as Entomopathogenic Fungi on Different Stages of Hyalomma anatolicum anatolicum under Experimental Conditions



Elham Abdelbasit Suliman and Yassir Osman Mohammed
 
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ABSTRACT

Ticks are obligate blood sucking ectoparasites. They are regarded as a major constraint to improve cattle production in sub-Saharan Africa. They are vectors of many kinds of microorganisms than any other single arthropods. Fungi are capable to infect a wide range of living organisms including insects and acaroids. Numerous natural enemies of ticks including pathogens such as fungi were investigated. In the present study, the mould Aspergillus terreus was isolated from Hyalomma anatolicum anatolicum eggs. Spore suspension of A. terreus at different concentrations was tested against different developmental stages of H. a. anatolicum. Assessment of immature stages of H. a. anatolicum seven days post treatment showed that the unfed larval stage was more susceptible than other stages. Spore suspensions at different concentrations, induced no mortalities among unfed and fed females. It was also demonstrated that the treatment affected reproductive capacity of the both unfed and fed females by reducing egg-conversion factor and arresting oviposition. Hatchability of the treated eggs decreased with increasing the contact periods. The findings were assessed and implications on bio-control of ticks using A. terreus were discussed. Accordingly, A. terreus might play a substantial role in future IPM programmes for tick control.

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Elham Abdelbasit Suliman and Yassir Osman Mohammed, 2012. The Activity of Aspergillus terreus as Entomopathogenic Fungi on Different Stages of Hyalomma anatolicum anatolicum under Experimental Conditions. Journal of Entomology, 9: 343-351.

DOI: 10.3923/je.2012.343.351

URL: https://scialert.net/abstract/?doi=je.2012.343.351
 
Received: November 03, 2011; Accepted: April 11, 2012; Published: July 27, 2012



INTRODUCTION

Ticks are obligatory temporary blood sucking parasites that mainly feed on vertebrates. This constantly poses higher risks of diseases transmission by ticks. Ticks and tick-borne diseases are of extreme economic importance hence represent a major constraint for livestock industry. In practice, ticks are controlled mainly by acaricides via two common application approaches; on-host and off-host. In both, environmental hazards are considerably stimulating. Hence, biological control is becoming an increasingly attractive approach to tick management (Bittencourt, 2000) because of; increasing concerns about environmental safety; cost of chemical control and emergence of acaricide-resistant strain of ticks. Numerous natural enemies of ticks (Kaaya, 2003), including pathogens (Hoogstraal, 1977; Chandler et al., 2000), parasitoids (Mwangi et al., 1997) and predators (Mwangi et al., 1991) have been documented, though only a few species have been evaluated as potential tick bio-control agents.

The interest and concept for using fungi to control vectors of veterinary and medical importance was established in the 19th century as a result of their successful use in controlling agricultural pests (Ferron, 1981; Hall and Papierok, 1982). Recently, fungal enzymes such as chitinase have been developed as biopesticides against plant pests (Sharma et al., 2011). Entomopathogenic fungi were found to infect wide range of insects and parasitoids. Pathogenicity of Metarhizium anisopliae and Paecilomyces species were assessed against the subterranean burrower bug Cyrtomenus bergi Froeschner (Jaramillo and Borgemeister, 2006). Nielsen et al. (2004) studied the effect of Metarhizium anisopliae on survival and reproduction of the filth fly parasitoid, Spalangia cameroni. Infection of malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae revealed reduction of blood feeding and fecundity (Scholte et al., 2006). Furthermore, an oil-based conidia formulation of three isolated fungi was evaluated against fourth instars larvae of hairy caterpillar (Sahayaraj and Borgio, 2012).

Over the all species of entomopathogenic fungi have been reported only few species of fungi have been accounted to be associated with ticks in nature (Kaaya et al., 1996; Zhioua et al., 1997; Saquis et al., 2002), the most promising fungi appear to be Metarhizium anisopliae and Beauveria bassiana. M. anisopliae has been tested as a biological control agent against different insects and tick species (Nielsen et al., 2004; Frazzon et al., 2000; Bittencourt, 2000).

In the Sudan ticks and tick-borne diseases represent a key constriction for intensive livestock industry due to great economic losses induced. Prevalence of Hyalomma anatolicum anatolicum, the potential vector of Theileria annulata, in the most important region (central Sudan) for raising livestock consequently hinders the execution of policies aimed at improving livestock due to considerable losses caused by T. annulata infection (Osman, 1976; Latif, 1994; Salih et al., 2004). Conversely systemic tick control has not been applied in the country, yet acaricides have been in use. Reliance on acaricides together with their abuse has led to emergence of resistant-tick strains (Latif, 1984; Mohammed, 2003). Hence, new approach for tick control is needed, even so, to date bio-control agents against ticks of medically or veterinary important in the Sudan have not been tried.

Biological control is likely to play a substantial role in future of tick control. Because of fungi diversity taxa that show high potential as tick bio-control agents. Estrada Pena et al. (1990) addressed the pathogenicity of Aspergillus ochraceus on Rhipicephalus sanguineus adult females. The present study is carried out to isolate and identify the naturally occurring fungi associated with H. a. anatolicum in the Sudan, develop them as bio-control agents, establish their effectiveness on the survival and biotic potential of the target tick and devise production strategies to bring them to practical use. Thus, mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides (Bittencourt, 2000; Leger et al., 1996). Vertalec was found effective against lettuce Aphids (Fournier and Brodeur, 2000).

MATERIALS AND METHODS

Ticks collection: Ticks used in this study were randomly collected from different cattle breeds found in the central region of the Sudan including Khartoum, Gezira and North Kordofan States. About 600 engorged female ticks were collected, transferred to the Laboratory and identified according to Hoogstraal (1956). Only engorged female ticks of H. a. anatolicum of each State were selected and separately kept into sterilized test tubes. Then they were incubated at 27±2°C and 75-80% Relative Humidity (RH) in order to lay eggs.

Tick maintenance: Feeding of the flat phase of the different stages of H. a. anatolicum was done according to the method of Bailey (1960). Eggs for hatching and the engorged phases for moulting were incubated under optimum laboratory conditions of 27±2°C and 75-80% RH (Yassir et al., 1992).

Fungal isolation: Eggs yielded from each tick group were surface disinfected as described by Mwangi et al. (1995), consequently ground and cultured onto Sabouraud's dextrose agar medium (SDA, Oxoid) containing chloramphenicol 0.05 g L-1. Slant and plate culture were incubated at 27±2°C and observed daily for fungal growth for two weeks. Growth obtained was sub-cultured onto Malt extract Agar (MEA, Oxoid). The isolated fungus was identified as Aspergillus terreus according to Raper and Fennell (1973) and preserved for infectivity trials and further studies.

Experimental infection of ticks: Spore-suspension stock of Aspergillus terreus was prepared (Maniania, 1993; Kaaya and Hassan, 2000) by suspending the spores in 50 mL of distilled water with a small amount of the dispersing agent Triton x-100 into sterilized bottle. The concentration of the stock suspension was assessed using a counting-chamber. Then serial dilutions of A. terreus spore-suspension of varying concentrations were prepared and tested against different developmental stages of a laboratory colony of H. a. anatolicum by dipping method (Maniania, 1994; Mwangi et al., 1995). The treated ticks were observed daily.

Seven days post treatment the tick percentage mortality corresponding to spore concentrations used was determined. Data obtained were analyzed using a probit analysis programme (Steel and Torrie, 1986) and the probit values of LC50 for each developmental stages tested were assessed. Surface disinfection and re-culture of cadavers were carried out according to Kaaya et al. (1996) method to determine the cause of death.

Effect of the treatment on biotic potential of the treated fed and unfed H. a. anatolicum females were assessed and measured by percentage inhibition of oviposition or Egg Conversion Factor (ECF) as described by Drummond et al. (1973):

Image for - The Activity of Aspergillus terreus as Entomopathogenic Fungi on Different Stages of Hyalomma anatolicum anatolicum under Experimental Conditions

where, ER is estimated reproductive factor.

The inhibition hatchability of treated eggs with larval LC50 level of concentration at 6.7x105 spore mL-1 based on exposure period was assessed.

RESULTS

Effect of A. terreus spores on 7 day-old immature stages of H. a. anatolicum assessed 7 days post treatment: Table 1 shows the susceptibility levels of the immature stages of H. a. anatolicum to A. terreus spores. Based on LC50 value it was apparently evident that the unfed larval phase was more susceptible than the unfed and fed nymphal phases.

Table 1: Probit values of LC50 for immature stages of H. a. anatolicum treated with different concentrations of A. terreus spore-suspension
Image for - The Activity of Aspergillus terreus as Entomopathogenic Fungi on Different Stages of Hyalomma anatolicum anatolicum under Experimental Conditions

Table 2: Treatment efficiency of A. terreus spore-suspension on fed females of H. a. anatolicum under laboratory conditions
Image for - The Activity of Aspergillus terreus as Entomopathogenic Fungi on Different Stages of Hyalomma anatolicum anatolicum under Experimental Conditions
*Significant (p<0.05), ns: Not significant (p>0.05)

Table 3: Effect of A. terreus spore-suspension on reproductive capacity of unfed females of H. a. anatolicum under laboratory conditions
Image for - The Activity of Aspergillus terreus as Entomopathogenic Fungi on Different Stages of Hyalomma anatolicum anatolicum under Experimental Conditions
**High significant (p<0.01), ***Very high significant (p<0.001), X: Weight of eggs produced in grams, Y: Estimated percentage hatchability, Z: Weight of engorged female in grams, ER: Estimated reproductive factor, IO: Inhibition of oviposition

Table 4: Effect of fungal treatments on 12 h-old egg masses of H. a. anatolicum
Image for - The Activity of Aspergillus terreus as Entomopathogenic Fungi on Different Stages of Hyalomma anatolicum anatolicum under Experimental Conditions

Effect of A. terreus spore-suspension on oviposition of treated fed females of H. a. anatolicum: No moralities for female fed stage at the all levels of the concentration used were induced. Although, the treated and untreated control females succeeded to oviposit, yet, there were significant (p<0.05) reductions in hatchability of the eggs laid by ticks treated with highest concentrations of the spore-suspension. There was a strong positive correlation (r = 0.9981) between the concentrations used and resultant percentage inhibition of egg hatchability. The probit value of the concentration able to induce 50% (LC50) inhibition of egg hatchability was 5.3x108 spore mL-1. The results obtained are summarized in Table 2.

Effect of A. terreus spore-suspension on biological performance capacity of unfed females of H. a. anatolicum: The results are presented in Table 3. The treatment induced high significant reductions in engorgement weight gained (p<0.01) and the total egg-mass (p<0.001) laid by the treated flat female ticks. Consequently reduction induced in Egg Conversion Factor (ECF) for such ticks coincided with potency of spore concentration used. Moreover, the treatment has inhibited eggs hatchability, as a result, arrested oviposition of the treated female ticks.

Effect of A. terreus spores on H. a. anatolicum eggs: The result obtained indicated that the susceptibility of the treated eggs increased with increasing contact periods (Table 4). Consequently there was a strong positive linear correlation (r = 0.99) between exposure period and percentage inhibition of egg hatchability.

DISCUSSION

Ticks of Hyalomma anatolicum anatolicum examined for susceptibility to artificial infection with Aspergillus terreus in the present study is of common occurrence (Osman, 1976) in the central region of the Sudan. Where it has been approved to be the potential efficient vector of tropical theileriosis (Salih et al., 2007), which causes economic losses that hinder livestock up-grading programmes (Latif, 1994).

However, a number of H. a. anatolicum engorge females randomly collected from central region of the Sudan including Khartoum, Gezira and North Kordofan States were found naturally infected with A. terreus according to Raper and Fennell (1973). In nature, a higher percentage of adult ticks seem to be infected by fungi than their immature stages and engorged females seem to be most readily infected (Zhioua et al., 1999). The percentage of ticks infected by fungi in nature varies considerably according to tick stage and species, season and to ecological conditions at the sample sites (Kalsbeek et al., 1995; Mwangi et al., 1995).

In the present study, A. terreus spores to be tested against different stages of the target tick species were prepared as suspension formulation since in most laboratory tests the spores were suspended only in water with a small amount of dispersing agent (Kaaya and Hassan, 2000). Evidently, the formulation in which the spores are applied is critical to the level of control obtained, but very little has been published as yet on the subject.

The dipping method applied in this work has been used basically for assessing efficiency of fungal infection against various tick species and other arthropod pests (Maniania, 1994; Mwangi et al., 1995). Usually fungi take several days to kill ticks. For instance, the LT50 of the majority of the entomopathogenic fungi so for tested against unfed and fed stages of different tick species generally ranged from few days to few weeks (Hall and Papierok, 1982). For this reason in current work the tick percentage mortality corresponding to spore concentrations used was determined 7 days post treatment.

Both larvae and nymphs of H. a. anatolicum were affected by A. terreus spores treatment, which resulted in death. Death of ticks was attributed to penetration of their soft cuticle by the fungus which rapidly invades their internal organs and ultimately kills them (Kaaya and Essuman, 1995; Kaaya et al., 1996). Recently, adhesion of fungal conidia to cuticle of insect larvae and germination were demonstrated to facilitate penetration of the cuticle (Altre et al., 1999; Altre and Vandenberg, 2001). Data obtained were analyzed (Steel and Torrie, 1986) and the concentrations that caused 50% mortality of the various developmental stages of H. a. anatolicum were assessed. The values of LC50 obtained could be regarded as base line data for susceptibility of this tick species, as it is the first times to be tested against A. terreus infection. Similarly, in a study carried by (Gindin et al., 2001) under laboratory conditions Ixodid tick species showed unpredictable susceptibility level to entomopathogenic fungi (Gindin et al., 2001). Free-living larval, nymphal, and adult Ixodes scapularis showed high infection rate when treated with entomopathogenic fungi (Zhioua et al., 1999).

Based on the calculated value of LC50 for both unfed and fed phases of the immature stages of H. a. anatolicum, the unfed larval phase could be considered more susceptible to A. terreus infection. This finding is in agreement with Munshi et al. (2008) who recorded LC50 values in a range of 1.9x103-1.3x1011 on using Fusarium species as a bio-control agent against caterpillar larvae.

Variation in susceptibility levels observed for the examined immature stages was also reported by Kaaya and Okech (1990) and such phenomenon might be attributed to ticks physiological factor. Comparison between the susceptibility of the unfed stages of Rhipicephalus appendiculatus and Amblyomma variegatum or of H. excavatum and R. sanguineus demonstrated decreasing susceptibility to fungi in progression through the larval, nymph and adult stages (Kaaya, 2000; Samish, 2000) and unfed stages seem to become more resistant after engorgement (Reis et al., 2001). High mortality of immature stages might be due to their incubation condition as they had not been incubated individually a matter that enhances infection via tick to tick contact in the tube.

Moreover, it was observed that A. terreus fungal infection affected reproductive potential of the treated both fed and unfed females of H. a. anatolicum rather than inducing mortality. These findings are in agreement with those observed by Kaaya et al. (1996) who tested B. bassiana and M. anisopliae on R. appendiculatus and A. variegatum. The treatment altered the development processes of H. a. anatolicum by reducing the egg-mass laid and inhibiting oviposition and eggs hatchability (Gindin et al., 2001). This suggests that there is a relatively long-lasting sub-lethal action of the fungi. Germination of the spores might have produced metabolites that affected females’ reproductive ability. Oliver et al. (1991) suggested that a toxin produced by Rhizopus thailandensis, R. arrhizus and Curvularia lunata might affect reproductive efficiency of Rhipicephalus sanguineus. Further investigations in this point would be useful.

In this study, the ability of A. terreus to inhibit the treated egg hatchability increased with increase of the exposure period. This finding is in agreement with that reported by Mwangi et al. (1995). Tick eggs, in contrast to many insect eggs, are highly susceptible to fungi and up to 100% of the eggs exposed to fungi under laboratory conditions did not hatch (Kaaya, 2000).

Tick management is principally geared towards the prevention of development of the next generation (Bittencourt, 2000). This goal can be achieved either by destroying ticks or arresting their reproduction. Hence, A. terreus treatment verifies the goal of tick control strategy as it demonstrated mortality and arrested the reproduction of the treated ticks. Accordingly the fungi might be a successful candidate as a biological control agent and play a substantial role in future of tick control. Moreover, they can easily be produced in the laboratory (Soundarapandian and Chandra, 2007).

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