Efficacy of Two Fungus-based Biopesticide Against the Honeybee Ectoparasitic Mite, Varroa destructor
Abdelaal A. Ahmed
Hany K. Abd-Elhady
The varroa mite, Varroa destructor (Anderson and Trueman)
(Acari: Varroidae), is known as the most serious ectoparasitic mite on honeybee,
Apis mellifera (Hymenoptera: Apidae) in the world. Based on the spores
of entomopathogenic fungi, two commercial preparations; Bioranza (Metarhizium
anisopliae) and Biovar (Beauveria bassiana) were evaluated through
application into the hives against varroa mite. Data showed significant differences
between treatments with Bioranza and Biovar, the results were significant after
7 and 14 days post-treatment. Mean a daily fallen mite individual was significantly
different between the hives before and after the applications of the two biopesticides
and wheat flour. Also, mites' mortality was, significantly, different between
the hives before and after treatments. There were significant differences between
treatments with the two biopesticides in workers body weight. Bioranza
and Biovar did not infect the honeybee in larval, prepupal, pupal and adult
stages. Scanning and transmission electron microscopy images showed spores and
hyphae penetration through stigma and wounds on varroa. The results suggest
that Bioranza and Biovar are potentially are effective biopesticides against
V. destructor in honeybee colonies.
January 09, 2013; Accepted: March 18, 2013;
Published: April 12, 2013
The parasitic mite V. destructor is currently a worldwide parasite on
the honeybee (Apis mellifera L.). It feeds on the hemolymph of immature
and adult bees (Harbo and Harris, 2001). This hemophagous
mite species reproduces within sealed brood, showing strong preference for drone
brood more than worker brood (Martin, 2001). The honeybee
is considered of great economic importance, not only for honey production but
also for crop pollination. A balanced host-parasite relationship is established
in the sense that the host fitness loss due to parasitism is limited because
mite reproduction occurs in drone brood only (Peng et
al., 1987; Tewarson et al., 1992). This
parasitic mite causes many biological effects like, weight loss, morphological
abnormalities and reduces the lifespan of infested individuals (De
Jong et al., 1982).
Chemical control methods and alternative methods including biotechnical and
genetic have been assayed against V. destructor (Fries
and Hansen, 1993, De Guzman and Delfmado-Baker, 1996,
Schmidt-Bailey et al. 1996; Rinderer
et al. 1997; El-Ghamdi and Hoopingarner, 2004).
Development of resistance in V. destructor mite populations to coumaphos
was studied by Elzen et al., (1998) and Elzen
and Westervelt (2002). Control of varroa mites with organic acids often
requires multiple applications to achieve a high degree of control and pose
a safety way to beekeepers (Veen et al. 1998).
Essential oils and their components have also been tested (Calderone
et al., 1997). In previous studies, the dust or strip applications
of M. anisopliae to honey bee hives provided satisfactory control of
varroa mite under field conditions (Kanga et al.,
2003). The fungi B. bassiana has been used as mycopesticide to control
of many pests belong to arthropod species (Goettel and Johnson,
1992). From honeybee colonies collected in France. Meikle
et al. (2006) discovered several isolates of B. bassiana from
varroa mites. Collecting fungal or bacteria isolates from the target pests themselves
is intended to increase the probability of finding the best suitable isolates
against these pests.
The present study aimed to determine the effect of two biopesticides, namely
Bioranza (M. anisopliae) and Biovar (B. bassiana) against V.
destructor, a honeybee ectoparasitic mite in Egypt. Compare between the
two commercial formulations and inert powder on the fallen mites and mean weights
of honeybee worker. Scanning and transmission electron microscopy images were,
also, used to show penetration of the spores and hyphae.
MATERIAL AND METHODS
Efficacy of the two fungus-based biopesticide and inert materials
Biopesticides: Two commercial formulations: Bioranza (10 % WP) of dry conidia
of fungus M. anisopliae and Biovar (10% WP) of dry conidia of fungus
B. bassiana. A weight of 0.1 gm of each formulation was suspended in 50
mL of water to be used per each colony.
Inert materials: Both of wheat and maize flour were used to make the
treatments more economical for beekeepers. Five grams of powder of maize or
wheat flour were used per each colony.
Experimental honeybee colonies: The present investigation was carried
out in special apiary during spring season of 2011. Fifteen colonies of hybrid
Carniolan honeybees, A. mellifera naturally heavily infested by varroa
mites were used.
Mite collection: To test the efficacy of selected commercially formulated
of entomopathogenic fungi against V. destructor, female mites were collected
from infested frames of sealed brood taken from honeybee colonies and additional
mites were collected from nurse and adult bees on the frames. The mites were
placed into glass scintillation vials (20 mL) containing two late instars honeybee
larvae as food source. Twenty to thirty mites were held in each glass vial and
used in the bioassays within an hour after they were collected.
Treatments: Four groups of colonies and control, all treated by spraying
with fine atomizer. The 1st treated with Bioranza, the 2nd with Biovar, the
3rd with wheat flour, the 4th was treated with maize flour and 5th is the control.
Mite mortality was recorded daily for 7 days and the experiments were repeated
three times. Also, all treatments were repeated with using wheat flour. Dead
mites were collected daily from the two biopesticides treatments and tested
in the following way to see if mortality was due to infection.
Establishing the hives: The hives used in these experiments were established
by dividing larger honeybee colonies that were severely infested with varroa
into 15 nucleus colonies to ensure uniform bee and mite populations. Used queens
were progeny of the same mother. The nucleus colonies were placed about 1.5
m apart from each other. Twelve days after they were established, hives were
ready for treatment. Honeybee workers were collected from each treatment and
Hive treatments: The experimental started from April to October (2011).
A new set of hives was established for each experimental run. To treat the colonies,
each hive was opened and each frame (both sides) was sprayed. Data on mite mortality
were recorded daily for 14 days.
Data collection: Before and after the fungal applications, the mite
infestation levels in the colonies were estimated using sticky-boards (De
Jong, 1990; Delaplane and Hood, 1997; Delaplane,
1998; Calderone and Turcotte, 1998). These were
coated on the upper surface with a clear adhesive material the bottom board
of the observation hive was removed and replaced with an 8-in mesh screen. The
screen was used to prevent the bees from removing the mites and from becoming
stuck to the cards. Mites that fell to the bottom of the hive passed through
the screen and were trapped on the sticky board. The sticky boards were placed
under each observation hive 1-7 days before the treatment in order to determine
pretreatment mite fall. The mites that fell onto the boards over a period of
24 h were counted and removed. The boards remained sticky for up to 7 days and
then they were replaced by a new set of sticky boards. The collected mites were
daily assessed for fungal infection by surface-sterilizing and incubating them
as described above for the laboratory assays. The number of mites was then recorded.
Electron microscopy examination: Mites were surface sterilized by dipping
them for 1 min in a sterilant-disinfectant and rinsing them with 95% ethanol.
The mites were then transferred with a camel-hair brush to Petri-dishes containing
wateragar and incubated at 25°C
for 4-7 days. Also, dead mites in the controls were also surface-sterilized
and incubated as described above. Mites were observed daily for the presence
of external fungal hyphae. The numbers of mites with external hyphae were counted;
these mites were removed from the Petri dishes. Only mites that showed fungal
growth were considered to have died by infection and photographed with electron
Statistical analysis: The data was analyzed using analysis of variance
(ANOVA) followed by the Student-Newman Keuls test to determine significance
between different groups. These tests were performed using a computer software
CoStat system for Windows, version 6.311, CoStat Program,
2006, Berkeley, CA, USA.
Efficacy of the two fungus-based biopesticide: Data in Table
1 showed significant differences between treatments with Bioranza and Biovar.
Results were significant after 7 and 14 days of application in the treatment
with Bioranza which was more effective than Biovar which differed in its effect
from 7 and 14 days.
||Mean number of fallen V. destructor mites on sticky
boards by using formulations of fungi and inert materials in hives during
|Means followed by the same letter (s) within each vertical
column are not significantly different
||Mean weights of honeybee workers before and after using formulations
of fungi and inert materials in hives during 2011
|Means followed by the same letter(s) within each vertical
column are not significantly different
Daily mite mortality was significantly different between the hives before and
after the applications of the fungus. Treating the hives with Bioranza resulted
in a significant increase in mites mortality. After treatments were initiated,
mite populations were found to be significantly smaller in treated hives than
in the control hives. Mites mortality varied significantly over time within
Table 2 showed significant differences between treatments
with Bioranza and Biovar in workers body weights. Results were significant after
treatments than before; the fungus Bioranza was the highly effective fungus,
followed by Biovar. Mean workers weight was not significantly different between
the hives before and after the applications of the fungus. Overall, V. destructori
was found to be a suitable host for the entomopathogenic fungi, Bioranza was
more virulent to varroa because it killed the host more quickly. Fungal applications
did seem to affect mite infestation levels in hives. It is possible that the
mites could become infected after they emerged from the brood cells if sufficient
quantities of conidia are present. Mite infestation level varied, significantly,
between treatments 7 and 14 days post-treatments.
ELECTRON MICROSCOPY EXAMINATION
Scanning electron microscopy (sem): The present scanning electron microscopy
(SEM) study describes the external development of B. bassiana and M.
anisopliae on the surface (cuticle) of the mite V. destructor. In
Fig. 1 to 5 illustrates the scanning electron
micrographs of the cuticle of V. destructor infected with M. anisopliae
and B. bassaina.
||Germ tubes emerged from a single conidia and extended over
the cuticle, Selected examples of scanning electron micrographs showing
the external development of B. bassiana and M. anisopliae
on the cuticle of the mite V. destructor
||Penetration the cuticle to find the more suitable exo-penetration
sites, Selected examples of scanning electron micrographs showing the external
development of B. bassiana and M. anisopliae on the cuticle
of the mite V. destructor.
|| Mycelium development on the host, Selected examples of scanning
electron micrographs showing the external development of B. bassiana
and M. anisopliae on the cuticle of the mite V. destructor.
Conidia of M. anisopliae were observed adhering to all parts of the
body of the mite and the fungus rapidly colonized the surface of the host's
cuticle. Germ tubes grew from conidia and extended over the cuticle (Fig.
1). The fungus was found to form germ-tubes and started to penetrate the
structures of the host within 24- 48 hrs after inoculation. Penetration started
in the epicuticle, then to the other regions of the cuticle and thereby inside
the mites body.
The epicuticle was penetrated without formation of an appressorium-like structure.
Later on, an appressorium was formed and full development of the germination
tube which started to find possible site of penetration. The present observations
demonstrate the following sequence of events in the infection of the mite V.
destructor as a host by M. ansopliae: (1) Attachment to the host
(2) Conidium germination and formation of germ tube (3) Mycelium development
on the host (Figs. 3 and 4) (4) Penetration
and growth of the pathogen on and in the host and (5) Sporulation on the surface
of the host's body (Fig. 5).
||Growth of the pathogen on and in the host, Selected examples
of scanning electron micrographs showing the external development of
B. bassiana and M. anisopliae on the cuticle of the mite V.
||Sporulation on the surface of the hoss body, Selected
examples of scanning electron micrographs showing the external development
of B. bassiana and M. anisopliae on the cuticle of the mite
Transmission electron microscopy (tem): Transmission Electron Microscopy
(TEM) studies were carried out to describe the internal development of M.
anisopliae and B. bassiana inside the body-cavity of the mite V.
destructor. TEM sections of the mite individuals infected with M. anisopliae
are shown in Fig. 6 to 9. It is well known
that the infection mainly takes place through the cuticle due to body contamination
with the conidia of the entomopathogenic fungus (Fig. 6).
Therefore, the first step of infection is to adhere to the cuticle to find the
more suitable exopenetration sites.
||The infection mainly takes place through the cuticle, Selected
examples of transmission electron micrographs showing the internal development
of M. anisopliae inside the body-cavity of the mite V. destructor
||The nucleus of a mother conidium starts to divide into two
nuclei, Selected examples of transmission electron micrographs showing the
internal development of M. anisopliae inside the body-cavity of the
mite V. destructor
||The nucleus of a mother conidium divide, forming two sister
conidia, Selected examples of transmission electron micrographs showing
the internal development of M. anisopliae inside the body-cavity
of the mite V. destructor
||Death of the host might be due to the effect on hemolymph
cells and hemolymph characters, Selected examples of transmission electron
micrographs showing the internal development of M. anisopliae inside
the body-cavity of the mite V. destructor
After the exopenetration, cylindrical conidia spread through the body fluid
and started to produce a toxin (s) that weakness the host's immune system and
supply the fungus conidia with the favorable conditions to start the division
process Fig. 7. An electron micrograph of a full structure
of M. anosopliae conidium showed its main components (cell wall, mitochondria,
vacuole and nucleus). The nucleus of a mother conidium starts to divide into
two nuclei, forming two sister conidia Fig. 8. The division
of the conidia of the fungus M. anisopliae inside the body-cavity of
the mite is a mitosis one with the haploid nuclei. Conidium division is apparently
required. The resulting increase in number of conidia makes it possible to excrete
more toxin(s) to suppress the hosts immunity system and complete the life-cycle
within the host which eventually lead to hosts mortality. Death of the
host might be due to the effect on hemolymph cells and hemolymph characters
(Fig. 9). Also, conidiagerminate in the hemolymph and penetrate
muscles, nervous system, malpighian tubules etc.
Efficacy of the two fungus-based biopesticide: The biopesticide Bioranza
which is dependent upon the entomopathogenic fungi, M. anisopliae was
the most efficacious fungal formulation tested because it caused the highest
reduction in mite counts in the hives. Mites are susceptible to entomopathogenic
fungi (Chandler et al., 2000). Among the pathogens
of varroa, only entomopathogenic fungi have the desired characteristics of a
control agent (Chandler et al., 2001). Observations
in this investigation indicated that it was critical to have fungal spores with
good germination, pathogenicity and virulence. In the fungal treatments, mite
infestations in the hives were significantly lower than those recorded in the
control at day 14 after treatment. Fungi of the genus Beauveria can be considered
as natural enemies of the mite since they have been found naturally-occurring
on varroa (Steenberg et al., 2010). Despite
the fact that they show specificity towards the mite, results of field tests
have been mixed, with some research groups reporting a measure of success and
other groups reporting no effect as biopesticides (Meikle
et al., 2012). This could simplify future registration procedures.
The overall differences between all treatments and the controls were statistically
significant at the end of the experiments. All treatments caused significant
change in mite infestations on adult bees over time and that the relationships
between the treated and control hives varied over time. It was determined that
fungal formulated spores provided successful control of mite populations in
established honeybee colonies at 10 g of conidia per hive applied two times
(day 0 and day 7). Microbial control of varroa mite with M. anisopliae
is feasible and could be a useful component of an integrated pest management
program for the honeybee industry. In addition, organic beekeepers, homeowner,
hobby beekeepers should benefit from a novel, user-friendly and chemical-free
strategy to manage the destructive pest of honeybees. Overall, this user-friendly
delivery method for the fungus, M. anisopliae could provide beekeepers
with effective, environmentally sound and sustainable control option for varroa
mite populations in honeybee colonies. B. bassiana appeared to be harmless
to honeybee workers (Martin, 2001; James
and Hayes 2006; James et al., 2006; Meikle
et al., 2006). The high correlation between mite mortality and fungal
infection is an indication that the fungus was the major mortality factor of
the varroa mitepopulation in the observation hive experiments. However, the
fungus proved of good persistence as the infecting mites were recorded 14 days
after the application. Also, the low infection rates found in the control hives
may be an indication that bees carrying fungus-treated mites drifted the fungus
between hives. The current and future target of the present research is to develop
a more efficient application technology to reduce the time required per application
and to make the treatments more economical for beekeepers. Biological control
could provide a key component in the development of a sustainable integrated
pest management strategy for V. destructor.
Electron microscopy examination: The aforementioned explanations agree
with Moino et al. (2002) who concluded that
the infection processes varies in different entomopathogenic fungi. He reported
that the initiation of B. bassiana conidial germination happened between
12 to 48 hrs after the inoculation of the subterranean termite Heteroterms
tenuis (Order: Isoptera). Altre et al. (1999)
reported that a higher germination percentage might help increase the probability
of infection before spores are removed from the cuticle surface. An early event
in germination of hydrophobic conidia is very probably favored by increasing
fungal affinity to insect-like cuticle components. B. bassiana produce
lipases, proteases and chitinases which can degrade insect cuticle (Charnley,
2003). These observations coincide with the commonly described sequence
of events characterizing other entomopathogenic fungal infections (Askary
and Yarmand, 2007). Therefore, the use of entomopathogenic fungi such as
B. bassiana, alone or associated to safe chemicals is an efficient and
environmentally favorable method for controlling the mite V. destructor
attacking honeybee. In this respect, Fargues et al.
(1994) stated that penetration, colonization and sporulation occurs faster
in the fungus M. anisopliae than in B. bassiana resulting in the
earlier death of hosts infected with the former fungus.
In conclusion, results obtained from this study demonstrate that Bioranza which
is dependent upon the entomopathogenic fungi, M. anisopliae was the most
efficacious fungal formulation tested against V. destructor in honeybee
colonies. Electron microscopy examination describes the external and the internal
development of spores of the two fungus-based biopesticide on and inside the
mite V. destructor.
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