Scanning Electron Microscopy (SEM) analysis and biological control of Ixodes ricinus using entomopathogenic fungi

INTRODUCTION

Ticks are economically the most important pests of cattle and other domestic animals in tropical and subtropical countries. They are the vectors of a numerous pathogenic microorganisms including Protozoans (Babesiosis, Theileriosis), Rickettsiae (Anaplasmosis, Ehrlichiosis), Viral disease (e.g., Kyasanur Forest Disease reported from Karnataka State of India; Crimean-Congo Hemorrhagic Fever reported from Pakistan), Bacteria (e.g., Pasteurella, Brucella, Listeria, Staphylococcus) and also Spirochaetes (Jongejan & Uilenberg 2004).

Ixodes ricinus can be found on a wide variety of hosts, particularly mammals and birds but also reptiles (Gray and Khal 2001). The adult ticks feed mainly on large mammals such as cattle, sheep and deer, the larvae feed on small mammals (especially rodents), birds and reptiles, and the nymphs parasitize small- and medium-sized vertebrates. I. ricinus occurs in cool, relatively humid, shrubby or wooded areas. In addition to deciduous and mixed forests, it can be found in more open areas when the vegetation is dense and rainfall is abundant. This tick is endemic in most of Europe (with the exception of the Mediterranean region, which has a warm, dry climate). It also occurs as far south as the Caspian Sea and northern Iran, as well as in northern Africa and play an important role in transmission a number of pathogens Including Babesia divergens (Babesiosis), Babesia bovis in cattles, louping ill virus, tick born encephalitis virus, Borrelia burgdorferi (Lyme disease) and Anaplasma phagocytophila (Little 2008).

Although, economic losses due to ticks are mainly due to the diseases which they transmit (Garcia 2003), financial losses associated with nagging irritation and depreciation of the value of skins and hides (up to 20-30%) are also significant (Biswas 2003). In severely tick infested young cattle, sometimes ticks have been found in the oral cavity as well as in the stomach. They reach here as a result of constant licking induced by irritation. Since many years ago investigators have documented numerous potential tick biocontrol agents, including pathogens, parasitoids and predators of ticks (Kaaya 2003).

Application of chemical acaricides such as organophosphorus compounds (Malathion, Comaphous) and the carbamate carbaryl is the most common method for controlling tick populations (Rodriguez-Vivas et al. 2006), but they may be hazardous for the environment. Drawbacks to this strategy include environmental contamination (Pell et al. 2001), impacts on non-target organisms (Schulze et al. 2001), human health hazards due to chemical residues in food products (Ostfeld et al. 2006) and the development of resistance in ticks (Graf et al. 2004). These disadvantages have stimulated the search for alternative methods to control ticks.

Biological pesticides are natural, more environm-
entally friendly, potentially less expensive, and more effective than chemical pesticides, also problems with resistance are less likely to occur (Whipps & Lumsden 2001). Among biocontrol agents, entomopathogenic fungi received major attention in recent years (Briggs et al. 2006, Abolins et al. 2007, Tavassoli et al. 2008). One of the most pathogenic fungal species examined for pathogenicity against ticks under laboratory and field conditions is M. anisopliae (Ostfeld et al. 2006). It showed high pathogenic activity against the ixodid ticks Amblyomma maculatum and Amblyomma americanum (Kirkland et al. 2004), I. scapularis (Hornbostel et al. 2005), Rhipicephalus appendiculatus and Amblyomma variegatum (Kaaya and Hassan 2000) and Boophilus microplus (Alonso-Diaz et al. 2007). In Iran, some indigenous strains of M. anisopliae, Beauveria bassiana and Lecanicillium psalliotae have been isolated with promising results to control different life stages of Rhipicephalus (Boophilus) annulatus under laboratory conditions (Pirali-Kheirabadi et al. 2007). However, only few studies have been reported about the control of Ixodidae ticks by M. anisopliae or other entomopathogenic fungi (Zabalgogeazcoa et al. 2008).The aim of this study was to introduce the safe and alternative way to control ticks, and also to find the natural and virulent strains of Iranian entomopathogenic fungi as promising candidate for tick biological control.

MATERIALS AND METHODS

Tick rearing

Adult I. ricinus was collected from naturally infested cattle in Mazandaran province, north of Iran. Ticks were transferred within 3–4 h and maintained in the laboratory at 26 °C and 70% relative humidity (RH) in test tubes for further study.

Fungal strains

Four entomopathogenic fungi including M. anisopliae strains Iran 437 C, DEMI 001, Iran 715 C and DEMI 002 used in this experiment were obtained from the fungal culture collection of Iranian Research Institute of Plant Protection, Department of Botany, Tehran, Iran (Table 1.).

Table 1. Metarhizum anisopliae strains used in the study

Preparation of conidia suspension

The fungi were cultured on PDA (Potato Dextrose Agar; Merck, Germany) in Petri dishes for 2 weeks at 25 °C. Conidia were harvested by washing the plates with an aqueous solution of 0.005% sterile Tween 80. The conidial suspension was filtered through four layers of sterilized muslin to remove the fungal mycelia. Conidia numbers were determined using a Neubauer slide under the microscope and the concentration of conidia was adjusted to 2.4 × 10⁷ conidia/ml. These suspensions were used as the source of fungal propagules.

Treatment of different developmental stages of I. risinus with conidial suspension

The virulence (Pathogenicity) of each fungal strain (2.4 × 10⁷ conidia/ml) was tested by immersing ten engorged I. ricinus in conidial suspensions for 3–5 seconds per replication according to the method of Gindin et al. 2001. The experiment was carried out in three replicates and in total, 180 ticks were treated with the strains concentrations. A negative control group of ten ticks was immersed in the same volume of sterilized distilled water and three replicates were made for the controls and experimental groups. Like negative control a positive control group containing an acaricidal compound, Cypermethrin 15 ml/1 sterilized distilled water (Cypermethrin^®, Aria Shimi, Zahedan, Iran) was also considered. Then ticks were transferred to Petri dishes containing moist filter papers and incubated for 20 days at 26 °C and 70% RH as described by Gindin et al. 2001. Mortality rate were recorded in 4, 8, 12, 16, and 20 days post treatment with fungal suspension and chemical acaricides.

Scanning Electron Microscopy (SEM)

For SEM analysis, ticks were fixed overnight at
4 ºC with 2% (v/v) glutaraldehyde, 2% (v/v) paraformaldehyde in 0.1 M sodium cacodylate buffer at pH 7.2. Post fixation was carried out in 1% (w/v) osmium tetroxide in the same buffer. The specimens were rinsed in buffer, dehydrated in series of 30–100% acetone solutions, dried at critical point in CO₂ (CPD 030 BALTEC), and coated with gold in a sputter-coater (SCD 050 BALTEC). The material was examined with Jeol JSM 5800 scanning electron microscope (SEM) at the Pasteur Institute of Iran, Tehran, Iran.

Statistical analysis

The data were expressed as the Mean ± SEM. Groups were compared using one-way ANOVA for repeated measurements. Student t-test was used for post hoc analysis. The software Sigmaplot version 12 was used for data analysis. A value of (P < 0.05) was considered significant.

RESULTS

Mortality rate of ticks exposed to fungal suspensions for four revealed thatthere were significant differences between M. anisopliae IRAN 437 C and other three fungal strains and also negative and positive controls (Cypermethrin). Positive control killed all the ticks in first four days and there were significant differences between positive control and treated groups as well. (P < 0.05) (Table 2). In four next days (eight days  post treatment), the highest mortality rate was belonged to the strain Iran 437 C with 30% and minimum of mortality rate was 13.3 % in treatments by strain DEMI 002. There were significant differences between strain M. anisopliae Iran 437 C and M. anisopliae DEMI 002 and IRAN 715 C in killing activity against I. ricinus (Table 2). Maximum of 63.3% and minimum of 40% mortality was demonstrated by using M. anisopliae strains Iran 437 C and M. anisopliae strain DEMI 002 twelve days post inoculation, respectively and there was significant differences between M. anisopliae strain Iran 437 C and other fungal groups in this regard (P<0.05).

About 86.6% and 63.3% of ticks mortality was recorded treatment with M. anisopliae strains Iran 437 C and M. anisopliae strain   DEMI 002 in Sixteenthdays post treatment. Significant differences were observed between M. anisopliae strain Iran 437 C and M. anisopliae DEMI 002  and M. anisopliae Iran 715 C and between M. anisopliae DEMI 001 with  M. anisopliae DEMI 002  (P<0.05) (Table 2). After twentieth day treatment, mortality rate of I. ricinus caused by M. anisopliae Iran 437 C, DEMI 001, Iran 715 C and DEMI 002 reached to 100 %, 93.3%, 90% and 83.3%, respectively. So the mortality rate increased with pass the time. In positive control groups (Cypermethrin), ticks were being killed early and faster than other groups and no mortality occurred in negative control groups even after twentieth days.

Metarhizium anisopliae IRAN 437 C was found to be the most virulent strain to adult stage of I. ricinus, followed by M. anisopliae DEMI 001 and M. anisopliae Iran 715 C. M. anisopliae strains IRAN 437 C and DEMI 001 had the most conidial growth on the killed tick’s cuticles, respectively (Table 3). Figure 1 shows the mycelia growth of M. anisopliae on the cuticle of killed ticks.

Table 2. Mean of number of killed ticks by Metarhizum anisopliae in different times. Different lower cases (a-e) show the significant difference between rows of every columns (Values of p < 0.05 were considered significant).

Table 3. Mean of mycelium growth on killed ticks in different times. Different lower cases (a-e) show the significant difference between rows of every columns (Values of p < 0.05 were considered significant).

Fig. 1. Direct view of fungal mycelia growth on the cuticle of killed ticks. a. Metarhizium anisopliae DEMI 002; b. Metarhizium anisopliae DEMI 001; c. Metarhizium anisopliae IRAN 715 C; d. Metarhizium anisopliae Iran 437 C.

Fig. 2. Scanning electron microscopy (SEM) analysis of ticks contaminated with Metarhizium anisopliae. a–b. Growth of fungal mycelia on the cuticle of killed ticks which shows signs of fungal grow in dorsal and ventral surface of Ixodes ricinus, respectively (magnification ×25); c–d. Fungal grow with magnification × 100 (c) and × 500 (d); e. Fungus produces a thin amorphous mucilage layer and it firmly adheres the conidia and germ tubes to the tick integument; f. Each conidia usually produces only one germ tube that penetrates into the tick cuticle.