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In-vitro Evaluation of Entomopathogenic Fungus Metarhizium anisopliae against the Cattle Tick Rhipicephalus (B.) microplus (Acarina: Ixodidiae)

Greeshma Rao U. B. Babasaheb Wamanrao Narladkar
Vol 8(7), 167-175
DOI- http://dx.doi.org/10.5455/ijlr.20180109043235

The research was conducted to evaluate the pathogenicity of fungus Metarhizium anisopliae on the egg and adult stages of the cattle tick Rhipicephalus (B.) microplus. The fungal powder containing conidia procured in the year 2012 and manufactured in the year 2016 was used in the research to compare their efficacy against egg and adult stages of Rhipicephalus (B.) microplus ticks. The susceptibility of the tick to the species of entomopathogenic fungi was evaluated by treating engorged female ticks and eggs with fungi at concentrations 1g×108 conidia/litre, 2×108 conidia/litre 4g×108 conidia/litre, 5g×108 conidia/litre6g×108 conidia/litre. The treatment of ticks and egg was followed as per standard procedure. The both fungal treatments showed at par results in terms of mortality of adult ticks, reduction in egg laying capacity and hatchability of the treated eggs in comparison with the control and found to be concentration dependent. The hatching rates by the treated groups were inversely proportional to the concentration of conidia used. The results showed that the biological control of R. (B.) microplus by M. anisopliae infection might constitute an additional method to integrated tick management.


Keywords : Integrated Tick Management Metarhizium anisopliae Rhipicephalus (B.) microplus

Ticks are obligatory blood sucking arachnids that feed on vertebrates. While argasid ticks (soft ticks) feed for minutes or hours, the ixodid ticks (hard ticks) feed for days to weeks. The Rhipicephalus (B.) microplus is an obligatory haematophagus ectoparasite of terrestrial vertebrates with single host occurs in almost all tropical and subtropical countries and is a major vector for bovine babesiosis and anaplasmosis (Sharma et al., 2013) and also transmits various pathogenic microorganisms such as protozoa, rickettsiae, spirochaetes and viruses (Jongejan and Uilenberg 2004).

In general all tick species transmit  a  greater  variety of infectious organisms than any other group of arthropods and worldwide,  are  second  only  to mosquitoes  in  terms  of  their  public  health  and  veterinary  importance  (Levin, 2016). When  economic  significance   of  ticks  is  taken  into  account  it  proves  that   these  ticks  have  a  debilitating  action;  production  losses  by  acting  as  a  vector  to haemoprotozoan  diseases,  also  losses  to  the  tanning  industry  and  dairy  industry.  Tick control throughout the world is based mainly on the repeated use of chemical acaricides. Unlimited use of this tick controlling measures has resulted in problems related to environmental pollution, milk contamination and resistance development in the target species (Onofre et al., 2001). Thus scientists are concentrating on non-chemical alternatives, one of them is biological control. Biological method of tick control involves the use of entopathogenic fungi. Therefore  present  study  was  planned  with  the objective of evaluating the efficacy  of  fungal  bio-control  agent, Metarhizium  anisopliae against  egg  and   adult  stages   of   Rhipicephalus   microplus   ticks.

Materials and Methods

The research work was undertaken at Department of Veterinary Parasitology and College of Veterinary and Animal Sciences, (MAFSU) Parbhani, Maharashtra, India.

Fungal Species Studied in the Experiment-Metarhizium anisopliae

Procurement of the Fungal Powder

The fungal powder was procured from Biological control unit, Department of Agril Entomology, Mahatma Phule Krishi Vidyapeeth Rahuri Dist Ahemednagar (MS), India. The fungal powder procured five years back (2012) and manufactured in the year 2016 was used in the research to compare their efficacy against various developmental stages of Rhipicephalus (B.) microplus ticks.

Collection of Ticks

Requisite number of blood engorged female ticks was collected from cattle (cows/bullock) body using forceps at the junction of skin and ticks.

Identification

Ticks were identified under zoom stereoscopic microscope as Rhipicephalus (B.) microplus before being introduced in the experiment Walker et al. (2003).

Experimental Design

For all in vitro trials, working concentration were prepared as 1,2,4,5 and 6gm powder+ 5ml milk + 1 liter of water. Test procedure described by Bagherwal et al. (1994), Maske et al. (2000), Kaaya and Hassan (2000) and Narladkar et al. (2015) were followed with little modification.

For Adult Ticks

In each diluted concentration 10 female ticks were dipped for 1 minute, were dried on filter paper and then placed in test tube as single tick per tube, followed by closure of tube with muslin cloth and rubber band. The mortality of ticks was observed at 24 hrs till 96 hrs. The mortality data were tabulated and efficacy was worked out in terms of per cent mortality. If the ticks were not dead, they were judged for their egg laying capacity; observed for number of eggs laid and compared with control ticks which were only treated with water.

Action on Eggs of Treated Females

The treated female Rhipicephalus (Boophilus) microplus ticks not dead, were separately maintained for eggs collection. Eggs collected from such treated female ticks were counted in petri dishes in the batches of 100 numbers, were transferred in tubes which were closed with piece of muslin cloth tied with rubber band. These tubes were maintained in desiccators in which humidity levels were maintained @ 75 %. The eggs were observed for hatching, till the period hatching process of eggs in the control group were completed.

On Treated Eggs

The eggs laid by Rhipicephalus (B.) microplus ticks in the tubes separately maintained for egg collection, were drawn from tubes in petri dishes. By counting the eggs in 100 numbers were treated with different fungal concentration solution, dried on filter paper and then transferred in tubes. These tubes were maintained in desiccators in which humidity levels were maintained @ 75 %. The eggs were observed for hatching, till the period hatching process of eggs in the control group were completed.

Criteria’s for Assessment of Efficacy of Metarhizium anisopliae

  1. Mortality of adult ticks and eggs
  2. Reduction in egg laying capacity
  3. Hatchability of eggs from treated females
  4. Hatchability of treated eggs

Statistical Analysis

The data obtained from various parameters were analyzed by employing two factor Factorial Experiment using computer applications, WASP.

Results and Discussion

The average mortality, egg laying capacity, hatchability of eggs from treated female ticks and hatchability of the treated eggs count observed after the treatment with fungi showed variation in the mortality count at different concentrations and their average count at different concentrations are depicted in the Table 1,2,3,4 respectively.

On Mortality of Adult Ticks

If you look into numerical values, it appears that amongst 10 ticks treated for each fungus at different concentrations; maximum average mortality of 43% was recorded and minimum of 0 (no ticks dead); indicating and suggesting fungus has effect in causing mortality of adult ticks in-vitro, however mortality rate appears to be concentration dependent and with increasing concentration of fungal conidia mortality count was also increased (Table 1).

Table 1: Showing Mean mortality of Rhipicephalus (B) microplus female ticks after treatment with various fungi at different concentrations

S. No. Treatments 1g/l Mean±SE Range 2g/l Mean±SE Range 4g/l Mean±SE Range 5g/l Mean±SE Range 6g/l Mean±SE Range CD
I Metarhizium anisopliae Rahuri Old   p0.15a ±0.15

(0-1)

p0.29b ±0.18

(0-1)

p0.29b ±0.18

(0-1)

p0.29b ±0.18

(0-1)

p0.43c ±0.20

(0-1)

(5%) 0.12
II Metarhizium anisopliae Rahuri New p0.00a ±0.00

(0-0)

pq0.15b ±0.15

(0-1)

p0.29c ±0.18

(0-1)

p0.29c ±0.18

(0-1)

p0.43d ±0.20

(0-1)

III Control p0.00 ±0.00

(0-0)

q0.00 ±0.00

(0-0)

q0.00 ±0.00

(0-0)

q0.00 ±0.00

(0-0)

q0.00 ±0.00

(0-0)

   Critical Difference(CD) (5%)0.167 (1%)0.224

Superscripts a,b,c,d indicates significant differences between concentrations ( amongst the rows)

Superscripts p,q significant differences between different fungi (amongst the columns)

In the control group no mortality was recorded. Therefore as an adulticide all the tested fungal strains exerted much expected effect. The results obtained were in agreement to the author Monteiro et al., (1998) who recorded a mortality of 50%.

Egg Laying Capacity

The treatment with both fungi resulted in significant reduction in egg laying capacity of the female adult ticks compared to control group. If you look into numerical values, it appears that amongst 10 ticks treated for each fungus at different concentrations; maximum reduction in egg laying was recorded for new strain i.e. 23.57eggs while minimum reduction in egg laying was recorded with the treatment of old fungus i.e. 214.29 (Table 2). In contrast to these values, control ticks laid 1578.55 eggs. The result of eggs to an extent of 90% was noted by the author Lopez et al., 2009 on R. microplus ticks.

Hatchability of Eggs from Treated Females

The treatment with both fungi resulted in significant reduction in hatchability of the eggs, which were harvested from the treated female ticks (eggs were not directly exposed to the fungal treatment). Even 10% eggs could not be hatched as against hatchability of 97.7 in control tick eggs.  At the concentration of 6g/l, all the eight fungal treatments showed the effect to their maximum and hatching of eggs could be noted to the lowest as 0.71 to 2.29.These results were in contrast to the author Broglio-Micheletti  et al., (2012) who found no reduction in hatchability of B. microplus eggs.

Table 2: Showing mean egg laying capacity of Rhipicephalus (B) microplus female ticks after treatment with various fungi at different concentrations

S. No Treatments 1g/l Mean±SE Range 2g/l Mean±SERange 4g/l Mean±SERange 5g/l Mean±SERange 6g/l Mean±SERange CD
l Metarhizium anisopliae Rahuri Old p214.29± 76.94 (0-600) p125.71±53.99 (0-400) p118.57±69.36 (0-500) p102.85±53.75 (0-400) p70.000±55.807 (0-400) (5%)

80.218

II Metarhizium anisopliae Rahuri New q50.71±20.71 (0-150) p47.43±28.70

(0-200)

p34.85±11.21

(20-100)

p34.29±17.17 (0-100) p23.57±8.92

(0-60)

III Control r1578.55±261.32 (350-2315) q1578.55±261.32 (350-2315) q1578.55±261.32 (350-2315) q1578.55±261.32 (350-2315) q1578.55±261.32 (350-2315)
  Critical Difference(CD (5%) 107.621 ( 1%)141.448

Superscripts p,q,r indicates significant differences between different fungi (amongst the columns)

Table 3: Showing mean hatchability of eggs harvested from Rhipicephalus (B) microplus female ticks treated with various fungi at different concentrations

S. No Treatments 1g/l

Mean±SE

Range

2g/l

Mean±SE

Range

4g/l

Mean±SE

Range

5g/l

Mean±SE

Range

6g/l

Mean±SE

Range

CD

 

l Metarhizium anisopliae Rahuri Old p8.57a±1.78

0-12

p8.15a±1.84

2-15

p4.00b±0.81

0-6

p3.29bc±1.29

0-10

p2.29c±1.40

0-10

(5%)

1.290

 

II Metarhizium anisopliae Rahuri New q3.85a±2.30

0-15

q2.15b±1.36

0-10

q1.43bc±0.68

0-4

q0.85c±0.45

0-3

p0.71c±0.56

0-4

III Control r97.57±0.71

95-100

r97.57±0.71

95-100

r97.57±0.71

95-100

r97.57±0.71

95-100

q97.57±0.71

95-100

  Critical Difference(CD) (5%)1.748 (1%)2.295

Superscripts a,b,c indicates significant differences between concentrations (amongst the rows)

Superscripts p,q,r indicates significant differences between different fungi (amongst the columns)

Hatchability of Treated Eggs

The data from Table 5 indicates that, at all the concentrations may be for 1g/l, 2g/l, 4g/l, 5g/l or 6g/l for both fungi, the hatchability was significantly reduced with increasing concentration of conidia, as compared to control group eggs and it has achieved its lowest levels at 6g/l concentration. The hatchability of the control group was observed as 97.57. Similar types of results were recorded by Narladkar et al., 2015 who also found significant reduction in hatchability upon treatment with the fungus.

 

Table 4: Showing mean hatchability of the Rhipicephalus (B) microplus tick eggs after treatment with various fungi at different concentrations

Sl. No Treatments 1g/l

Mean±SE

Range

2g/l

Mean±SE

Range

4g/l

Mean±SE

Range

5g/l

Mean±SE

Range

6g/l

Mean±SE

Range

CD

 

l Metarhizium anisopliae Rahuri Old p40.60a±5.13

30-60

p35.83b±10.83

0-70

p32.00b±6.04

15-50

p11.80c±2.20

7-20

p5.60d±1.50

2-10

(5%)

4.521

 

II Metarhizium anisopliae Rahuri New q89.40a±2.21

82-96

q82.20b±2.08

77-89

q66.20c±3.33

59-77

q38.60d±4.29

28-52

p9.60e±1.72

5-15

III Control r97.57±0.86

95-100

r97.57±0.86

95-100

r97.57±0.86

95-100

r97.57±0.86

95-100

q97.57±0.86

95-100

 
  Critical Difference(CD) (5%) 6.067 (1%)7.974  

Superscripts a,b,c,d,e indicates significant differences between concentrations (amongst the rows)

Superscripts p,q,r  indicates significant differences between different fungi (amongst the columns).

 

Table 5: Overall Effect Rate (OER)

S. No. Fungus OER (At 5×108 conidia/litre) OER (At 1×108 conidia/litre)
11 MRO 100×(1-102.85/1578.57×11.8/96.8)= 99.20 100×(1-214.85/1578.57×40.6/96.8)= 94.29
22 MRN 100×(1-34.287/1578.57×38.60/96.8)=99.13 100×(1-50.714/1578.57×89.4/96.8)=70.35

Metarhizium anisopliae, a deuteromycete fungus, a general pathogen, that attacks diverse group of insects (Quesda-Moraga et al., 2004). This fungus invades the host arthropod body through the cuticle and subsequently reproduce in it; thus favouring horizontal transmission and may also have an effect on the reproduction of the organisms being attacked (Quesada-Moraga et al., 2004). Similarly Metarhizium anisopliae decreases the oviposition of the treated ticks and hatching could be interpreted as the vertical transmission in the future progeny of arthropods (Kaaya, 2000, Quesada-Moraga et al., 2004). Samish et al. (2001) observed that high pathogenicity of M. anisopliae to various developmental stages of R. sanguineus and R. appendiculatus. Similarly they also reported that M. anisopliae isolates caused a subsequent secondary infection in subsequently emerging stages and can also prevent egg laying by engorged R. sanguineus tick. Through these observations author further opined that fungal infection may be transmitted from an infected to a non infected host, both among tick at the same stage of development and also among ticks of different stage of development, indicating that vertical and horizontal transmission of M. anisopliae fungus. Similar type effect (vertical and horizontal) has been observed in the present study on R. (B) microplus ticks. Combining together all the studies, it is proved beyond doubt that, Metarhizium anisopliae is having horizontal and vertical modes of transmission in the tick host. Thus Metarhizium anisopliae fungus appears to be effective in controlling tick population in the same generation as well as in the next generation. According to Broglio-Micheletti et al. (2012), Metarhizium anisopliae gives the result and causes the mortality in less time with low concentration.

Present study results proved that fungus tested is found to be the promising biological control agents against an important tick pest of cattle. Hence present research has two important components, one as tick and other as fungi tested as BCAs. The use of entomopathogenic fungi can be adapted both to environmental conditions in the region in which they will be used and to ticks, which served as a substrate for reproduction, may be useful in the implementation of in vivo biological control programmes against ticks (Fernández et al., 2017). Potency and activity of the fungus is largely dependent on factors such as temperature and humidity. At a high temperature, low humidity fungus will not work.

In the present study Metarhizium anisopliae in 2 forms as old and new were evaluated. New means the fungal conidia which are recently harvested from the growing culture media and old means conidia harvested before 4 years from the growing culture media and stored in dark place away from the sunlight at room temperature. The idea behind testing new and old is to see

  1. Whether both old and new conidia has the similar potency
  2. To judge that for how much period conidia retains same potency after storage.

After study it was observed that, whether the fungus is new or old at par efficacy was recorded, though numerically slight differences were in evidence. Similarly after four years of storage, fungi hold the good potency. This species of entomopathogenic fungi established as BCA are having good amount of activity and can exert as cidal (killing) effect on adult ticks. Frazzon et al. (2000) developed the formula to scale the magnitude of effect of fungus on B. microplus ticks by calculating OER (Overall Effect Rate). By using the same formula the OER has been calculated. OER= 100× [1 – WEt/WEc –WLt/WLc], where WEt is the egg laying capacity of infected ticks; WEc the egg laying capacity of non-infected ticks (control); WLt the egg fertility of infected ticks; WLc the egg fertility of non-infected ticks. From the data of the Table 5, it can be assessed that both fungal treatment showed significant activity against the R. (B.) microplus ticks ranging between 70.35-99.20% OER, which was remarkable and proving this fungi as potential biological control agents.

Conclusion

The Metarhizium anisopliae fungus tested showed significant activity in terms of mortality of adult ticks, reducing egg laying capacity, less hatchability and less hatchability of treated eggs. The fungus when applied tick eggs caused the infection which resulted in reducing the hatchability of treated eggs and therefore if used at the sites where tick deposits the eggs, can prove as best biological control. The activity was optimum at 5g/l and hence it can be recommended for tick control programmes, subject to the evaluation under field conditions. When old stock of the fungus was evaluated, it had given at par activity with their respective newly harvested conidia, indicating that, if fungal powder is stored away from the sunlight at room temperature in dry conditions, it can hold activity for more than 4 years. Thus present study concludes that Metarhizium anisopliae is better choice as ovicidal as compared to tickicidal in the tick control programmes. Efficacy of fungal treatments was found to be concentration dependent. Hence it is concluded that minimum concentration of 5×108 conidia/litre can be recommended to achieve. desired effect and can be incorporated in the integrated pest management approach.

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