Biocontrol Science and Technology

ISSN: 0958-3157 (Print) 1360-0478 (Online) Journal homepage: http://www.tandfonline.com/loi/cbst20

In vitro pathogenicity of different Metarhizium

anisopliae s.l. isolates in oil formulations against
Rhipicephalus microplus

Wendell M. S. Perinotto, Isabele C. Angelo, Patricia S. Golo, Mariana G.

Camargo, Simone Quinelato, Fillipe A. Sá, Caio J. B. Coutinho Rodrigues,
Allan F. Marciano, Caio M. O. Monteiro & Vânia R. E. P. Bittencourt

To cite this article: Wendell M. S. Perinotto, Isabele C. Angelo, Patricia S. Golo, Mariana G.
Camargo, Simone Quinelato, Fillipe A. Sá, Caio J. B. Coutinho Rodrigues, Allan F. Marciano, Caio
M. O. Monteiro & Vânia R. E. P. Bittencourt (2017): In vitro pathogenicity of different Metarhizium
anisopliae s.l. isolates in oil formulations against Rhipicephalus microplus , Biocontrol Science and
Technology, DOI: 10.1080/09583157.2017.1289151

To link to this article: http://dx.doi.org/10.1080/09583157.2017.1289151

Published online: 17 Feb 2017.

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BIOCONTROL SCIENCE AND TECHNOLOGY, 2017
http://dx.doi.org/10.1080/09583157.2017.1289151

RESEARCH ARTICLE

In vitro pathogenicity of different Metarhizium anisopliae s.l.

isolates in oil formulations against Rhipicephalus microplus
Wendell M. S. Perinottoa, Isabele C. Angelob, Patricia S. Goloc, Mariana G. Camargoc,
Simone Quinelatod, Fillipe A. Sáe, Caio J. B. Coutinho Rodriguesc, Allan F. Marcianoc,
Caio M. O. Monteirof and Vânia R. E. P. Bittencourtc
a
Centro de Ciências Agrárias, Ambientais e Biológicas, Universidade Federal do Recôncavo da Bahia, Cruz das
Almas, Brazil; bDepartamento de Saúde Pública, Instituto de Veterinária, Universidade Federal Rural do Rio de
Janeiro, Seropédica, Brazil; cDepartamento de Parasitologia Animal, Universidade Federal Rural do Rio de
Janeiro, Instituto de Veterinária, Seropédica, Brazil; dFundação Osvaldo Cruz, Coleção de Culturas de Fungos
Filamentosos, Pavilhão Rocha Lima, Rio de Janeiro, Brazil; eDepartamento de Bioquímica, Instituto de Ciências
Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; fDepartamento de Microbiologia,
Imunologia, Parasitologia e Patologia, Instituto de Patologia Tropical e Saúde Pública, Universidade Federal
de Goiás, Goiânia, Brazil

ABSTRACT ARTICLE HISTORY

The present study aimed to evaluate the in vitro pathogenicity of the Received 24 August 2016
Metarhizium anisopliae s.l. CG 112, CG 347, CG 32, CG 148 and CG 629 Returned 5 December 2016
isolates formulated in vegetable or mineral oil against Rhipicephalus Accepted 25 January 2017
microplus. The bioassays were performed with R. microplus engorged
KEYWORDS
females. The ticks were immersed for three minutes in oil-based Formulation; biological
formulations containing 10% oil (mineral or vegetable). The effects control; ticks
of the different fungal isolates were evaluated analyzing the females’
reproductive parameters (the total weight of the egg mass, hatching
percentage, egg production index and nutritional index). The
present study showed that all of the tested isolates and both oil
formulations (vegetable or mineral oil) changed the biological
parameters of the R. microplus females. However, the mineral oil
formulation was more effective than the vegetable oil formulation,
as the former showed a higher tick control percentage. It was
concluded that there was variation in the virulence among the
different M. anisopliae s.l. isolates and between the different types
of oil. Moreover, that either of the isolates GC 148 and GC 629
formulated in oil confers the good potential for controlling
R. microplus engorged females.

Highlights

. Evaluation of the in vitro oil formulations of Metarhizium anisopliae s.l. isolates against
Rhipicephalus microplus ticks.
. Bioassays were performed with engorged females.
. Mineral oil formulation was more efficient against R. microplus.
. The isolates GC 148 and GC 629 formulated in oil confers the good potential for con-
trolling R. microplus engorged females.

CONTACT Wendell M. S. Perinotto wendell@ufrb.edu.br CCAAB, UFRB, Rua Rui Barbosa, 710, Centro, CEP, Cruz das
Almas, BA 44380-000, Brazil
© 2017 Informa UK Limited, trading as Taylor & Francis Group
2 W. M. S. PERINOTTO ET AL.

1. Introduction
The cattle tick Rhipicephalus microplus cause economic losses to the Brazilian cattle indus-
try estimated at U$3.24 billion per year (Grisi et al., 2014). These economic losses are due
to the spoliative action on the host, predisposition to myiasis, leather depreciation, delay in
animal weight gain, decreased production, overspending with labor and acaricides and
transmission of pathogens that cause diseases such as anaplasmosis and babesiosis (Gui-
marães, Lima, & Ribeiro, 1998; Heuchert, de Gulli, De Athaide, Bose, & Friedhoff, 1999;
Horn & Arteche, 1985). The control of R. microplus is usually performed by the appli-
cation of insecticides on the cattle herd. However, frequent treatments with acaricides
and inadequate management practices have led to the development of resistant tick
populations.
The reduction of the tick population in the cattle herd can be achieved by improving the
efficacy of its control by selecting and correctly applying acaricides and using strategic
control systems associated with other management practices (Furlong, Martins, &
Prata, 2004). Microbial control using arthropod-pathogenic fungi has become a promising
tool for tick control. Arthropod-pathogenic fungi are biocontrol agents widely studied and
used for pest control worldwide (Faria & Wraight, 2007) due to their greater efficiency in
killing the host, great genetic variability that allows the selection of highly virulent isolates
to control a large number of pests and its relative safety for the environment and humans
(Alves, 1998; Thomas & Read, 2007; Zimmermann, 2007).
The virulence of these biocontrol agents for R. microplus has been demonstrated in
several in vitro studies (Angelo et al., 2012; Fernandes & Bittencourt, 2008; Fernandes, Bit-
tencourt, & Roberts, 2012; Perinotto, Angelo, et al., 2012; Perinotto, Terra, et al., 2012).
The species Metarhizium anisopliae s.l. and Beauveria bassiana s.l. are the most widely
used in arthropod control in general. However, the efficacy of arthropod-pathogenic
fungi depends on environmental factors such as humidity, temperature and solar radiation
(Huang & Feng, 2009; Ment et al., 2010; Peng & Xia, 2011).
When mineral or vegetable oil is used in association with arthropod-pathogenic fungi,
it increases the adhesion of the conidia to the surface of the arthropods, thereby protecting
the fungi from adverse environmental conditions (Alves, 1998). Therefore, the use of oil
formulations containing these entomopathogens may increase the conidia stability, extend
their persistence in the field protecting fungi against heat stress, desiccation, and particu-
larly ultraviolet irradiation (Barreto et al., 2016; Bateman, 1994; Hedimbi et al., 2008;
Jackson, Dunlap, & Jaronski, 2010), and increase their virulence to ticks (Ângelo et al.,
2010; Camargo et al., 2012).
The aim of the present study was to evaluate the in vitro pathogenicity of different M.
anisopliae s.l. isolates formulated in vegetable or mineral oil against R. microplus engorged
females.

2. Materials and methods

2.1. Obtaining R. microplus
Fully engorged females collected from cattle artificially infested with R. microplus with no
recent contact with chemical acaricides were used in the bioassays. This study is part of a
project that was previously submitted to the Ethics Committee of the Federal Rural
 BIOCONTROL SCIENCE AND TECHNOLOGY 3

University of Rio de Janeiro (Universidade Federal Rural do Rio de Janeiro – UFRRJ) and
was approved under protocol number 185/2011. The engorged females were aseptically
cleaned using 1% sodium hypochlorite for three minutes and then washed twice in
sterile distilled water prior to their exposure to the fungi.

2.2. Obtaining fungal isolates

Five M. anisopliae s.l. isolates (CG 32, Bahia, 1984; CG 112, Distrito Federal, 1988; CG 148,
Mato Grosso do Sul, 1982; CG 347, Goiás, 1991 and CG 629, Alagoas, 1997) donated by
the Brazilian Enterprise for Agricultural Research (Empresa Brasileira de Pesquisa Agrope-
cuária – EMBRAPA), Genetic Resources and Technology, located in Brasília-Federal Dis-
trict, Brazil, were used in the present study. The isolates were cultured on potato, dextrose
and agar (PDA) medium (Himedia, Mumbai, India) at 25°C ± 1°C and ≥80% relative
humidity for 15 days. Isolates were chosen based on previous studies (Perinotto et al.,
2014) that demonstrated variation in Metarhizium virulence against R. microplus
larvae and adult females. Based on the reported data, five fungal isolates were selected
for use in the present study: two highly virulent isolates (CG 148 and CG 629), two
with low virulence (CG 112 and CG 347) and an isolate that showed intermediary viru-
lence (CG 32).

2.3. Preparation of fungal suspensions and oil formulations

With the aid of a sterile scalpel blade, the conidia were scraped from the surface of the
culture medium and suspended in sterile distilled water and 0.01% Tween 80 solution
(Luz, Tigano, Silva, Cordeiro, & Aljanabi, 1998). The aqueous suspension was quantified
using a Neubauer chamber and a light microscope, according to Alves (1998). After the
quantification, the suspension concentration was adjusted to 1.0 × 108 conidia/mL with
a final volume of 20 mL of aqueous suspension of each isolate.
The oil formulations were made in either 10% vegetable oil (soybean) (Cargil, Brazil) or
10% mineral oil (Vetec Química Fina Ltda., Brazil),which were sterilized by autoclave
(121°C for 20 minutes). The following proportions were used to prepare the oil formu-
lations: 8.9 mL of aqueous conidial suspension + 1 mL of vegetable or mineral oil +
0.1 mL of Tween 80 (Vetec Química Fina Ltda., Brazil). The following groups were
used as controls: aqueous control (10 mL of sterile distilled water and 0.01% Tween 80 sol-
ution); 10% vegetable oil and/or mineral oil control (8.9 mL sterile distilled water + 1 mL
vegetable and/or mineral oil + 0.1 mL Tween 80).Thus, there were 15 total experimental
groups.

2.4. Viability of conidial suspensions

A 10 μL sample of each conidial suspension was placed in Petri dishes containing PDA
medium to assess the fungi viability. The Petri dishes were kept in a climate-controlled
chamber at 25 ± 1°C and ≥80% relative humidity and were evaluated 24 hours later.
The spore germination was calculated according to Alves (1998). When fungal isolates
germination percent was evaluated on PDA at 25°C ± 1°C and ≥80% relative humidity,
4 W. M. S. PERINOTTO ET AL.

24 hours after suspension preparation, conidia showed 100% germination, showing that
the fungi were suitable to be used.

2.5. Bioassays with R. microplus engorged females

For the bioassays the engorged females were separated into groups of ticks with homo-
geneous weight and then immersed in 1 mL of fungal suspension for three minutes.
Each group consisted of 10 females. After treatment with fungal suspension, the
females were fixed in a dorsal decubitus position in Petri dishes containing double-
sided adhesive tape to realize the oviposition and facilitate egg collection. Tick females
were kept in a climate-controlled chamber at 27°C and ≥80% relative humidity. When
the females started to lay their eggs, the eggs were collected, weighed and placed in
16 × 125 mm test tubes (Pyrex®, Corning®, NY) and kept in a climate-controlled
chamber (27°C and ≥80% relative humidity) for further evaluation of the larvae hatching
percentage.
Biological parameters used to evaluate the effects of the different M. anisopliae s.l. iso-
lates on the engorged females were the initial weight of the females, egg mass weight, larvae
hatching percentage and final weight of the females. Bioassays were repeated two times.
The average of each parameter was used to calculate the egg production index (EPI) and
the nutritional index (NI) using the equations from Bennet (1974). The percentage of
R. microplus control by M. anisopliae s.l. was obtained by the calculation of the estimated
reproduction according to Drummond, Gladney, Whetstone, and Ernst (1971).

2.6. Statistical analysis

For the analysis of the parametric data (the initial weight of the females, egg mass weight
and final weight of the females), analysis of variance was performed followed by Tukey’s
test for comparison between the means with a significance level of 5% (P ≤ .05). The non-
parametric data (the larvae hatching percentage, EPI and NI) were analysed by the
Kruskal–Wallis test followed by the Student–Newman–Keuls test to compare the mean
ordinations at a significance level of 5% (P ≤ .05) (Sampaio, 2002).

3. Results
All of the isolates tested in the present study, in both formulations (vegetable oil and
mineral oil) were able to change the biological parameters of the R. microplus females.
When evaluating the total egg mass weight of the females treated with vegetable oil for-
mulations, the CG 629 isolate was the most efficient, significantly reducing the egg mass
weight by 65% compared to the aqueous control. The total egg mass weight of the females
treated with the other isolates in the vegetable oil formulation did not differ statistically
from each other; however, the weights were significantly lower than those of the
control group. Regarding the larvae hatching percentage from the eggs laid by females
treated with the vegetable oil formulations, it was observed that the CG 148 and the
CG 629 isolates significantly reduced the larvae hatching by 47% on average. In turn,
the CG 32, CG 112 and CG 347 isolates reduced larvae hatching by an average of 27%.
 BIOCONTROL SCIENCE AND TECHNOLOGY 5

The CG 629 isolate was the treatment that most interfered with the EPI, significantly
reducing the ability of the females to oviposit (by 65%, on average) compared to the
aqueous control group. The evaluation of the NI showed that all of the isolates tested
in the present study significantly reduced the metabolism capacity of the R. microplus
females.
From the analysis of the EPI and the NI, the percentage of control was calculated and is
shown in Tables 1 and 2. Females exposed to 10% vegetable oil only (control group) had a
reduction in all of the reproductive parameters with a percentage of control of 10.73%, so
the vegetable oil alone had a toxic effect on the R. microplus females; however, these results
did not differ statistically from the aqueous control group.
The total weight of the egg mass of the females treated with the mineral oil formulations
was significantly reduced for all of the isolates evaluated in the present study compared to
the control groups. However, among all of the isolates tested, the CG 148 isolate most
affected this biological parameter, significantly reducing the weight of the egg mass by
88%.
There was no statistical difference in the larvae hatching percentage of eggs laid by the
females treated with M. anisopliae s.l. isolates formulated in mineral oil and eggs laid by
the females of the control groups. However, among all of the isolates tested in the bioas-
says, the CG 629 isolate had the greatest influence on this parameter, decreasing the per-
centage of larvae hatching by 62%.
Regarding the EPI, all of the isolates were able to significantly reduce this parameter
compared to the control groups.
In the assessment of the NI, all of the isolates tested in the present study significantly
reduced the metabolism capacity of the R. microplus females. However, the CG 148 isolate
induced the greatest decrease in the NI, reducing the ability of the females to use the
ingested blood for egg production by 85%.
From the analyses of the EPI and the NI, the percentage of control was calculated and is
shown in Table 3. The females in the 10% mineral oil control group experienced a

Table 1. Mean values ± standard deviation of egg mass weight (EMW); larval hatch percent (LHP); egg
production index (EPI); nutritional index (NI) and control percent (CP) of Rhipicephalus microplus
females treated with fungal vegetable oil formulation of five Metarhizium anisopliae s.l. isolates.
Treatment EMW LHP EPI NI
Aqueous control 131.2 ± 4.03ª 98.2 ± 0.40ª 54.12 ± 1.66ª 89.23 ± 4.91ª
Oil control 119.2 ± 4.17ab 96.5 ± 1.64ab 49.15 ± 1.67ab 81.25 ± 4.83ab
c c
CG 112 73.25 ± 15.18 71.7 ± 9.83 29.96 ± 6.16cd 59.07 ± 8.70bc
CG 347 76.25 ± 31.61c 76.7 ± 23.38bc 31.40 ± 13.12c 66.07 ± 15.61bc
bc c
CG 32 99.7 ± 7.28 70.0 ± 6.32 40.70 ± 3.89bc 71.07 ± 7.17bc
CG 148 79.9 ± 14.15c 44.2 ± 21.54d 32.89 ± 5.68c 54.57 ± 11.98c
CG 629 46.2 ± 15.65d 61.0 ± 31.93cd 19.08 ± 6.46d 51.91 ± 8.13c
Note: Mean values followed by the same letter in the same column do not differ statistically (P > .05).

Table 2. Control percent of Rhipicephalus microplus females treated with vegetable or mineral oil
formulations of five Metarhizium anisopliae s.l. isolates.
Oil control CG 347 CG 112 CG 32 CG 148 CG 629
Vegetable oil formulation 10.7 59.4 50.5 46.5 73.3 79.9
Mineral oil formulation 13.1 83.5 86.7 91.5 94.3 93.8
6 W. M. S. PERINOTTO ET AL.

Table 3. Mean values ± standard deviation of egg mass weight (EMW); larval hatch percent (LHP); egg
production index (EPI); nutritional index (NI) and control percent (CP) of Rhipicephalus microplus
females treated with fungal mineral oil formulation of five Metarhizium anisopliae s.l. isolates.
Treatment EMW LHP EPI NI
Aqueous control 131.2 ± 4.03a 98.2 ± 0.40a 54.12 ± 1.66a 89.23 ± 4.91a
Oil control 116.9 ± 11.59a 95.5 ± 1.22ab 48.31 ± 4.66a 79.19 ± 20.17ab
CG 112 30.45 ± 23.25b 70.8 ± 19.60bc 12.58 ± 9.58b 54.65 ± 16.89bc
CG 347 29.2 ± 12.62b 58.3 ± 25.63c 12.0 ± 5.13b 39.65 ± 8.91cd
CG 32 18.16 ± 8.05b 62.5 ± 33.72c 7.52 ± 3.32b 27.9 ± 7.18de
CG 148 16.1 ± 3.05b 45.8 ± 25.38c 6.62 ± 1.28b 13.88 ± 2.97e
CG 629 18.05 ± 17.31b 38.0 ± 13.04c 7.39 ± 7.04b 29.0 ± 23.48ce
Note: Mean values followed by the same letter in the same column do not differ statistically (P > .05).

reduction in all of their reproductive parameters compared to the aqueous control group
and had a percentage of control of 13.11%, thus showing that the mineral oil had a toxic
effect on R. microplus; however, the results of the mineral oil control group and the
aqueous group were not statistically different. In general, CG 148 and CG 629 isolates
were the most virulent, providing the highest percentage of R. microplus female control.

4. Discussion
The main barriers to the use of arthropod-pathogenic fungi in the field are environmental
factors, especially solar radiation, humidity and temperature (Benz, 1987; Ibrahim, Butt,
Beckett, & Clark, 1999; Ment et al., 2010). Therefore, the use of fungal conidia in formu-
lations is recommended (Alves, 1998). Vegetable and mineral oils have been used in fungal
formulations to protect the conidia against desiccation and provide a synergistic effect,
thereby improving the fungi efficiency against the target pest (Alves, 1998). According
to Prior, Jollands, and Patourel (1988), concentrations of 5% or more of oil improve
the adhesion of the fungi conidia to the arthropod cuticle due to the cutinophilic proper-
ties of the oil and the hydrophobic characteristics of the cuticle.
The present study, the objective of which was to verify the effect of different oil formu-
lations, demonstrated an increase in the pathogenic potential of M. anisopliae s.l. isolates
formulated in two types of oil (vegetable and mineral) at a 10% concentration. The best
results were observed when the fungi were in mineral oil formulations, for which the per-
centages of control of R. microplus females were close to 95% in three of the five isolates
tested. Even the two isolates that had been considered less virulent in the experiment when
used in an aqueous suspension had a high percentage of control (close to 85%) when they
were tested in mineral oil formulations. The control group containing 10% mineral oil
influenced the R. microplus reproductive parameters, reaching a 13.6% percentage of
control.
According to Abdel-Shafy and Soliman (2004), some oils are toxic to ticks, most likely
due to their components. Camargo et al. (2012) observed that there is variation in the
mineral oil toxicity between the tick stages; the larvae are more sensitive to the toxic
effect of the oil as the exposure time increases. The oil toxicity properties against the
ticks and the synergism with arthropod-pathogenic fungi have provided promising
results in the control of several tick species, such as Amblyomma variegatum (Maranga,
Kaaia, Mueke, & Hassanali, 2005), R. microplus (Ângelo et al., 2010; Camargo et al.,
2012; Leemon, Turner, & Jonsson, 2008) and Amblyomma cajennense (Lopes, Alves,
 BIOCONTROL SCIENCE AND TECHNOLOGY 7

Padulla, & Pérez, 2007). In addition to potentiating the fungi toxicity and enhancing their
virulence, mineral oils have the advantage of maintaining the conidia viability for a long
period at room temperature, even if the conidia have more than 10% water content,
because the mineral oil does not contain nutrient residues (Alves & Faria, 2010). Therefore,
mineral oils are an excellent adjuvant for fungal formulations (Camargo et al., 2014, 2016).
Another factor to be considered in oil formulations is the oil concentration because it
can affect the product cost, product application, formulation homogenization and its tox-
icity to microorganisms (Aguda, Rombach, & Shepard, 1986) and arthropods (Abdel-
Shafy & Soliman, 2004). Thus, some studies have evaluated the effects of different fungi
species on R. microplus control. These studies also evaluated different mineral oil concen-
trations. Ângelo et al. (2010) tested the fungus Lecanicillium lecanii in a formulation con-
taining 15% mineral oil, which showed a 97.6% percentage of control (Ângelo et al., 2010).
B. bassiana s.l. in formulations containing 10%, 15% and 20% of mineral oil showed
18.07%, 0.71% and 21.67% percentages of control, respectively (Camargo et al., 2012).
M. anisopliae s.l. at 10%, 15% and 20% of mineral oil resulted in 58.12%, 93.69% and
87.54% percentages of control, respectively (Camargo et al., 2012).
With regard to vegetable oil formulations, previous studies have shown an increase in
the treatment efficacy compared to aqueous suspensions (Kaaya & Hassan, 2000). In the
present study, there was also an increase in the pathogenic potential of the different isolates
in formulations using vegetable oil, but the toxic effects were lower than the mineral oil
effects. However, it is important to emphasize that the CG 148 and CG 629 isolates main-
tained a high percentage of control of close to 75% when formulated in vegetable oil. This
result is extremely relevant in the context of microbial control, in which we seek to reduce
the parasitic load to levels that do not generate economic losses to the farmers but still allow
the development of animals’ enzootic stability to the bovine tick-borne fever pathogens.
The results of the present study show that among the five isolates tested, CG 148 and
CG 629 isolates have a higher potential to control the cattle tick particularly when using a
mineral oil formulation. Despite this, it is important to consider the environmental tox-
icity of mineral oil as well as its flammable propriety (Alves & Faria, 2010). In the
study realized by Perinotto et al. (2014) using the same isolates to determine enzymatic
activities of proteases, lipases and chitinases in vitro produced by M. anisopliae s.l. was
observed that highest activities were achieved with the isolates CG 629 and CG 148.
This difference among isolates may be related to their virulence.
Thus, taking into account the efficacy of the formulations tested in the present study,
further studies using the same isolates in oil formulations in future trials under field con-
ditions are recommended to establish the practical applications of the use of M. anisopliae
s.l. in R. microplus biological control.

Acknowledgements
We also thank the Office to Improve University Research (CAPES) for providing the Ph.D. scholar-
ship. Vânia R. E. P. Bittencourt is a CNPq researcher (1 C).

Disclosure statement
No potential conflict of interest was reported by the authors.
8 W. M. S. PERINOTTO ET AL.

Funding
This research was supported by grants from the Rio de Janeiro State Research Foundation
(FAPERJ) and the National Council for Scientific and Technological Development (CNPq).

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