Comparison of tau-fluvalinate, acrinathrin, and amitraz effects on susceptible and resistant populations of Varroa destructor in a vial test

Abstract

The parasitic mite Varroa
destructor is a major pest of the western honeybee, Apis mellifera. The development of acaricide resistance in Varroa populations is a global issue. Discriminating concentrations of acaricides are widely used to detect pest resistance. Two methods, using either glass vials or paraffin capsules, are used to screen for Varroa resistance to various acaricides. We found the glass vial method to be useless for testing Varroa resistance to acaridices, so we developed a polypropylene vial bioassay. This method was tested on tau-fluvalinate-, acrinathrin-, and amitraz-resistant mite populations from three apiaries in Czechia. Acetone was used as a control and technical grade acaricide compounds diluted in acetone were applied to the polypropylene vials. The solutions were spread on the vial surface by rolling the vial, and were then evaporated. Freshly collected Varroa females were placed in the vials and the mortality of the exposed mites was measured after 24 h. The Varroa populations differed in mortality between the apiaries and the tested compounds. Mites from the Kyvalka site were resistant to acrinathrin, tau-fluvalinate, and amitraz, while mites from the Postrizin site were susceptible to all three acaricides. In Prelovice apiary, the mites were susceptible to acrinathrin and amitraz, but not to tau-fluvalinate. The calculated discriminating concentrations for tau-fluvalinate, acrinathrin, and amitraz were 0.66, 0.26 and 0.19 µg/mL, respectively. These results indicate that polyproplyne vial tests can be used to determine discriminating concentrations for the early detection of acaricide resistant Varroa. Finally, multiple-resistance in Kyvalka may indicate metabolic resistance.

Full text

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1
REVIEW PAPER
2
3
Comparison of tau-fluvalinate, acrinathrin, and amitraz
4
effects on susceptible and resistant populations of Varroa
5
destructor in a vial test
6
Martin Kamler
1,2
•
Marta Nesvorna
3
•
Jitka Stara
3
•
7
Tomas Erban
3
•
Jan Hubert
3
8
Received: 5 November 2015 / Accepted: 18 February 2016
9
Ó Springer International Publishing Switzerland 2016
10
Abstract The parasitic mite Varroa destructor is a major pest of the western honeybee,
11
Apis mellifera. The development of acaricide resistance in Varroa populations is a global
12
issue. Discriminating concentrations of acaricides are widely used to detect pest resistance.
13
Two methods, using either glass vials or paraffin capsules, are used to screen for Varroa
14
resistance to various acaricides. We found the glass vial method to be useless for testing
15
Varroa resistance to acaridices, so we developed a polypropylene vial bioassay. This
16
method was tested on tau-fluvalinate-, acrinathrin-, and amitraz-resistant mite populations
17
from three apiaries in Czechia. Acetone was used as a control and technical grade acaricide
18
compounds diluted in acetone were applied to the polypropylene vials. The solutions were
19
spread on the vial surface by rolling the vial, and were then evaporated. Freshly collected
20
Varroa females were placed in the vials and the mortality of the exposed mites was
21
measured after 24 h. The Varroa populations differed in mortality between the apiaries and
22
the tested compounds. Mites from the Kyvalka site were resistant to acrinathrin, tau-
23
fluvalinate, and amitraz, while mites from the Postrizin site were susceptible to all three
24
acaricides. In Prelovice apiary, the mites were susceptible to acrinathrin and amitraz, but
25
not to tau-fluvalinate. The calculated discriminating concentrations for tau-fluvalinate,
26
acrinathrin, and amitraz were 0.66, 0.26 and 0.19 lg/mL, respectively. These results
27
indicate that vial tests can be used to determine discriminating concentrations for the early
28
detection of acaricide resistant Varroa. Finally, multiple-resistance in Kyvalka may indi-
29
cate metabolic resistance.
A1 & Jan Hubert
A2 hubert@vurv.cz
A3
1
Department of Microbiology, Nutrition and Dietetics, Czech University of Life Sciences Prague,
A4 Kamycka 129, 165 21 Prague 6-Suchdol, Czechia
A5
2
Bee Research Institute at Dol, Maslovice-Dol 94, 252 66 Libcice nad Vltavou, Czechia
A6
3
Laboratory of Plant Active Substances in Crop Protection, Crop Research Institute, Drnovska
A7 507/73, 161 06 Prague 6-Ruzyne, Czechia
AQ1
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Exp Appl Acarol
DOI 10.1007/s10493-016-0023-8
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Keywords Varroa  Acaricide  Multiple-resistance  Discriminating concentrations 
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Apiculture
32
33 Introduction
34
The cosmopolitan parasitic mite, Varroa destructor Anderson and Trueman, is the most
35
devastating pest to the Western honeybee, Apis mellifera L. (Cornman et al.
2010). Bee
36
colony losses due to Varroa infestation primarily occur during winter (van Dooremalen
37
et al.
2012). Varroa mites reproduce in capped brood cells and feed on the hemolymph of
38
both immature and mature honeybees (Rosenkranz et al.
2010). Varroa infestation leads to
39
the reduction in body weight and nutrient contents of parasitized bees (Amdam et al.
2004;
40
van Dooremalen et al.
2013). In addition to causing reduced nutrient content, Varroa
41
transmits pathogenic viruses (Erban et al.
2015) and bacteria in honeybees (Hubert et al.
42
2015). Heavy Varroa infestation causes 100 % mortality within a few weeks in untreated
43
or poorly treated honeybee colonies (Kanga et al.
2010; Rosenkranz et al. 2010). Since the
44
first discovery of Varroa in (then) Czechoslovakia in 1978 (Peroutka et al.
2003), the mite
45
has become a major cause of recent honeybee losses in Czechia. The development of
46
resistance to acaricides disrupts the control of mite populations.
47
Different acaricidal compounds are used to control varroosis (Watkins
2011). However,
48
the chemical treatment produces residues in various bee products (Bogdanov et al.
1998;
49
Martel et al.
2007; Johnson et al. 2010). Requirements for successful acaricide treatment
50
are low toxicity to the non-target honeybees and low risk of contamination of bee products
51
(Santiago et al.
2000). There are only a few acaricides that can control Varroa (Milani
52
1999; Martin 2004; Maggi et al. 2011). However, resistance of Varroa to pyrethroids,
53
including fluvalinate, flumethrin, and acrinathrin, organophosphate coumaphos, and for-
54
mamide amitraz has been documented during the last three decades in Europe (Milani
55
1995; Colin et al. 1997; Trouiller 1998; Spreafico et al. 2001; Thompson et al. 2003), North
56
America (Elzen et al.
1999, 2000; Rodriguez-Dehaibes et al. 2005), and South America
57
(Maggi et al.
2009, 2011).
58
Of the listed compounds, organophosphates were banned and therefore not used by
59
beekeepers in Czechia. Despite the decreased effectiveness, fluvalinate, acrinathrin, and
60
amitraz are still commonly used to treat Varroa (Johnson et al.
2010) and are the most used
61
acaricides in Czechia. However, the treatment can be supplemented or changed with
62
formic acid and thymol to prevent the development of resistance to the synthetic var-
63
roacides (Brodschneider et al.
2014). Beekeepers have recently recorded decreased efficacy
64
of some commercially used varroacides, citing a large number of surviving mites in the
65
treated colonies as indicates the survey of Bee Research Institute at Dol. There is a need to
66
develop simple biotests that screen for Varroa resistance to acaricides.
67
Various researchers have conducted topical application bioassays on Varroa (Ritter and
68
Roth
1988). Methods involving Milani capsules with acaricides in paraffin, and glass vials
69
with the surface of the vials coated with pesticides, have been used (Milani and Della
70
Vedova
1996; Elzen et al. 1999; Thompson et al. 2002). In the Milani method, the aca-
71
ricide is dissolved in the paraffin covering the inner surface of the capsule, while in glass
72
method the acaricide is homogenously distributed on the inner glass surface. The glass vial
73
method (Elzen et al.
1999, 2000; Kanga et al. 2010 ) meets the recommendation of the
74
Insecticide Resistance Action Committee (IRAC;
http://www.irac-online.org/about/irac/)
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75
for detecting resistance in herbivorous insects. These two methods enable detection of
76
Varroa resistance. The methods were validated and diagnostic concentrations (discrimi-
77
nating doses) of the varroacides were estimated (Milani and Della Vedova
1996;
78
Thompson et al.
2002; Kanga et al. 2010). However, the methods are not comparable each
79
other due to the acaricide dispersion through paraffin and acaricide mono-layers on glass
80
vials. When we adopted glass for investigation of Varroa resistance to tau-fluvalinate, a
81
high natural mortality that complicated the testing was observed (Hubert et al.
2014). The
82
disadvantages of the glass vial can be overcome by application of acaricide on
83
polypropylene material. The present study aimed to develop tests and methods that have
84
low mite mortality in the control treatments and are comparable to the IRAC recom-
85
mendations for acaricide tau-fluvalinate, acrinathrin, and amitraz. The results provide also
86
information on the actual state of resistance in three apiaries in Czechia.
87 Materials and methods
88
Mites
89
Varroa females were collected from three apiaries: Kyvalka (N49°11
0
24
00
, E16°26
0
57
00
),
90
Postrizin (N50°13
0
59
00
, E14°23
0
12
00
) and Prelovice (N50°13
0
59
00
, E14°23
0
12
00
). The inves-
91
tigated apiaries had high level of Varroa infestation, i.e. the natural fall of mites was higher
92
than 50 mites per week. Varroa was collected from capped worker brood combs in
93
September 2014 and immediately placed in the treatment vials. Treatments of honeybee
94
colonies against Varroa mites were performed according to rules of the State veterinary
95
administration and veterinary law in Czechia (Law No. 166/99 Sb. in actual version). The
96
treatment is mandatory for all honeybee colonies each year (Anonymous
2013). Apiaries
97
Kyvalka and Prelovice were treated with the pyrethroid acrinathrin in the summer at least
98
2 years before sampling and with amitraz in the winter each year. The recorded efficacy of
99
acrinathrin in the Kyvalka apiary was lower than on previous apiaries based on beekeepers’
100
evidence. Experimental colonies at the Postrizin site were treated with formic acid in
101
summer seasons only and with amitraz in winter period for 3 years; no treatment was
102
applied in 2014 until Varroa sampling. There was no transfer of honeybee colonies
103
between these three apiaries in order to avoid mixing of the populations of Varroa. The
104
distances between apiaries were approximately 100 km; therefore, natural drift of Varroa
105
on the bees was unlikely.
106
Acaricidal compounds
107
The three acaricidal compounds used in the study were tau-fluvalinate, acrinathrin, and
108
amitraz (Sichuan Wangshi Animal Health Co., China). The compounds were diluted in
109
HPLC-grade acetone to the following concentrations: tau-fluvalinate: 44.4, 4.44, 0.444,
110
0.0444 and 0.00444 lg/mL; acrinathrin: 1.816, 0.1816, 0.01816, 0.001816 and
111
0.0001816 lg/mL; amitraz 10, 1, 0.1, 0.01 and 0.001 lg/mL. The control assay was the
112
pure acetone. The set of solutions was applied to 5-mL (inner surface: 20.1 cm
2
)
113
polypropylene vials (Cat No. I911241, P-LAB, Prague, Czechia) using micropipettes. The
114
vials were rolled to obtain homogenous distributions of the pesticide on the inner surface of
115
the vials. The vials were placed in darkness to allow the acetone to evaporate. The test vials
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116
were then capped and placed in a rack in a plastic box and refrigerated at 4 °C for no
117
longer than 3 weeks.
118
Bioassays
119
From 15 to 20 adult Varroa females were added to each polypropylene vial. Six replicates
120
per treatment, type of acaricide, and site were conducted. The test vials containing mites
121
were incubated in the dark, in desiccator cabinets, at 85 % relative humidity (RH), and at
122
28 °C for 24 h. At the end of the experiment, the vials were opened and the mites were
123
placed on 3-cm diameter pieces of filter paper. The mites that escaped from the circle were
124
recorded as surviving. The individuals inside the circle, including those in tremor, were
125
recorded as dead.
126
Data analyses
127
The mortality was analysed using probit regression. The dependent variable was mortality,
128
while the independent variables were concentration and apiary. The concentration of
129
acaricidal compounds was transformed logarithmically. LC
50
,LC
90
and LC
95
values, 95 %
130
confidence intervals, and the slopes and intercepts of the concentration–response curves
131
were determined (Tables
1, 2). Resistance ratios were determined by dividing the LC
50
132
value of the resistant population by the LC
50
value of the susceptible population (Table 2).
133
The data describing mortality of populations from Postrizin and Prelovice after acrinathrin
134
and amitraz treatment were pooled to estimate the lethal concentrations (Tables
1, 2). The
135
discriminating concentrations of the insecticides were determined as the concentrations
136
that caused 90 % mortality of the individuals in the sensitive population (LC
90
value). The
137
analyses were done in XLSTAT (Addinsoft, New York, NY, USA).
Table 1 Parameters for probit regression model description the relationship between mortality of Varroa
destructor mites and acaricide concentrations
Acaricidal
compound
Probit model
Population R
2
Slope 95 % CI Intercept 95 % CI
Tau-fluvalinate Postrizin 0.31 0.6783 0.5312 0.8255 1.3685 1.1688 1.5683
Prelovice 0.69 1.4241 1.1234 1.7249 0.8716 0.6384 1.1048
Kyvalka 0.83 2.4442 1.6614 3.2270 0.4182 0.0762 0.7601
Acrinathrin Postrizin/
Prelovice
0.34 0.6080 0.5153 0.7007 1.5798 1.3718 1.7879
Kyvalka 0.62 1.6444 1.1030 2.1858 1.3146 0.8348 1.7943
Amitraz Postrizin/
Prelovice
0.55 0.6080 0.5153 0.7007 1.5798 1.3718 1.7879
Kyvalka 0.73 1.8101 1.3228 2.2974 0.9468 0.5954 1.2983
The mites originate from three apiaries. For acrinathrin and amitraz no significant differences were found
between Postrizin and Prelovice, then the mortality data were pooled for these two apiaries
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138 Results
139
The natural mortality of Varroa females in vial bioassays varied between 6 and 13 %,
140
depending on the apiary and Varroa population. The probit model for tau-fluvalinate
141
(v
2
= 924; P \ 0.0001; R
2
= 0.56; N = 1721) showed a significant effect of transformed
142
concentrations of acaricide (v
2
= 482; P \ 0.0001) and apiary (v
2
= 72; P \ 0.0001)
143
(Table
1; Fig. 1a). The sensitivity of mites to tau-fluvalinate decreased in the following
144
order: Kyvalka, Prelovice, Postrizin (Fig.
1b). A comparison of LC
50
values for tau-flu-
145
valinate showed that the sensitive population (Postrizin) required an 85 times lower
146
effective concentration than did the resistant population (Kyvalka). The ratio of resistant to
147
susceptible concentrations increased to threefold for LC
90
and 1.3-fold for LC
95
. The
148
discriminating concentration of tau-fluvalinate was 0.66 lg/mL (Table
2).
149
The probit model for acrinathrin (v
2
= 713; P \ 0.0001; R
2
= 0.52; N = 1430)
150
showed a significant effect of transformed concentrations of acaricide (v
2
= 306;
151
P \ 0.0001) and apiary (v
2
= 120; P \ 0.0001) (Table 1; Fig. 1c). The sensitivity of
152
mites to acrinathrin was much lower in Kyvalka than in Postrizin and Prelovice (Fig.
1d).
153
The resistant population (Kyvalka) had 92-fold higher LC
50
for acrinathrin than did the
154
sensitive populations in Postrizin and Prelovice. The suggested discriminating concen-
155
tration of 0.26 lg/mL was estimated for acrinathrin (Table
2).
156
The probit model for amitraz (v
2
= 723; P \ 0.0001; R
2
= 0.63; N = 1120) showed a
157
significant effect of transformed concentrations (v
2
= 272; P \ 0.0001) and apiary
158
(v
2
= 81; P \ 0.0001) (Table 1; Fig. 1e). The sensitivity of mites to amitraz was similar
159
as to acrinatrin (Fig.
1f). LC
50
of the resistant population in Kyvalka was 31-fold higher
160
than that of the sensitive populations in Postrizin and Prelovice (Table
2). The estimated
161
discriminating concentration for amitraz was 0.19 lg/mL (Table
2).
Table 2 Fitted doses and determined discriminating concentrations of tau-fluvalinate, acrinathrin, and
amitraz to distinguish between sensitive and resistant population of Varroa destructor mites
Acaricidal
compound
Fitted
dose
Sensitive population
a
Resistant population
b
Resistance
ratio
Fitted val. 95 % CI Fitted val. 95 % CI
Tau-fluvalinate LC
50
0.00732 0.0031 0.0134 0.62539 0.4529 0.8488 85.4
LC
90
0.65972 0.3859 1.3717 2.18118 1.4788 4.2071 3.3
LC
95
2.30594 1.1442 6.3951 3.08692 1.9631 6.8722 1.3
disc. conc. 0.66
Acrinathrin LC
50
0.00149 0.0008 0.0024 0.13619 0.0858 0.2000 91.7
LC
90
0.25658 0.1468 0.5188 0.89267 0.5331 2.1851 3.5
LC
95
1.05195 0.5201 2.6326 1.49988 0.8100 4.6062 1.4
Disc. conc. 0.26
Amitraz LC
50
0.00802 0.0047 0.0123 0.25104 0.1646 0.3598 31.3
LC
90
0.18928 0.1170 0.3537 1.41668 0.9180 2.6927 7.5
LC
95
0.44984 0.2526 0.9885 2.27563 1.3763 4.9910 5.1
Disc. conc. 0.19
a
Sensitive Varroa populations are: Postrizin population for tau-fluvalinate and pooled data from Postrizin
and Prelovice populations for acrinathrin and amitraz
b
Resistant population is Kyvalka for all acaricides
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AB
CD
EF
Fig. 1 The effects of test concentrations of acaricidal compounds on the mortality of three in vitro Varroa
populations; the probit model for a tau-fluvalinate, c acrinathrin, and e amitraz; and fitted LC
50
concentration (columns) and 95 % confidence intervals (bars) for b tau-fluvalinate, d acrinathrin, and
f amitraz. The concentration of acaricidal compounds was transformed by decimal logarithms
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162 Discussion
163
In this study, we found populations of Varroa mites in Czechia that were resistant to the
164
commonly used acaricides, tau-fluvalinate, acrinathrin, and amitraz. The bioassay results
165
confirmed our previously reported results of sodium channel gene conferring tau-fluvali-
166
nate resistance of a Varroa population at the Kyvalka site (Hubert et al.
2014). The
167
recognized mechanism of the resistance was L–V single amino-acid substitution (Hubert
168
et al.
2014) which has been at the same time recognized in the UK (Gonzalez-Cabrera et al.
169
2013). However, the here recognized multiple-resistance makes future study of resistance
170
mechanism necessary.
171
Herein, we found a Kyvalka population resistant to acrinathrin, amitraz, and tau-flu-
172
valinate. Both acrinathrin and fluvalinate pyrethroids target the Varroa sodium channel.
173
The same mode of action is responsible for cross-resistance of other insect pests (Soder-
174
lund
2008). The mode of action of amitraz differs from pyrethroids in that amitraz, as a
175
formamidine pesticide, mimics octopamine and blocks the octopamine receptor (Casida
176
and Durkin
2013). The resistance to amitraz arises from modifications of the octopamine
177
receptor as described for amitraz-resistant cattle ticks (Chen et al.
2007). The mode of
178
resistance has not been studied in Varroa, but it has been studied in the tick Rhipicephalus
179
(syn. Boophilus) microplus, in which resistance is associated with the b-adrenergic octo-
180
pamine receptor gene (RmbAOR) (Corley et al.
2013). Selection with amitraz increased the
181
frequency of the RmbAOR mutation in ticks, thereby increasing the prevalence of amitraz-
182
resistance (Corley et al.
2013).
183
The resistance ratio for amitraz varied between 5 and 30, while for tau-fluvalinate and
184
acrinathrin, it varied between 1.3 and 92 (Table
2). According to the observation treatment
185
efficacy (estimation of mite fall after treatment in hives), fumigation by amitraz during the
186
broodless period is still highly effective (M. Kamler, unpublished data). Future investi-
187
gation of the RmbAOR might reveal whether a genetic mechanism of resistance exists.
188
The multiple-resistance, i.e. organophosphates (coumaphos and malathion) and pyre-
189
throids (fluvalinate and cypermethrin) of Varroa populations has been observed in the
190
absence of selection pressure (Kanga et al.
2010). Next, Varroa multiple-resistance to
191
fluvalinate and amitraz in Minnesota has been reported by Elzen et al. (
2000). The data
192
presented here most probably indicate a case of ‘multiple resistance’ and not ‘cross-
193
resistance’ although this can be addressed in a more specific (and detailed) study. One
194
possible scenario which could explain this fact is that the mites developed metabolic
195
resistance; that is, the detoxification enzymes are capable to detoxify different compounds
196
(Sammataro et al.
2005). We compared the toxicity of the various acaricides to sensitive
197
populations of mites. Acrinathrin was the most toxic, while tau-fluvalinate and amitraz
198
showed similar LC
50
efficacy. Amitraz LC
50
was 2.3-fold higher than the baseline of the
199
susceptible population, but for tau-fluvalinate, it was 327-fold higher than the baseline.
200
Pyrethroids were not used in experimental colonies at the Postrizin apiary for 2 years. This
201
can explain the high sensitivity of Varroa mites to tau-fluvalinate in our bioassay. Elzen
202
et al. (
2000) observed amitraz LC
50
of 16.35 lg, which was 20-fold higher than that
203
observed in the Kyvalka population, which is a resistant population. However, the
204
observed LC
50
for the tau-fluvalinate resistant population was much lower than was
205
reported for the resistant Varroa population in Poland (Bak et al. 2012). The difference
206
between our results and those from the Texas population is attributed to the higher sen-
207
sitivity of the Czech susceptible population (LC
50
0.001 and 0.1 lg) (Kanga et al. 2010).
208
The ascertained resistance ratio of 31 for amitraz within the Czech population is
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209
comparable to Argentina where the ratio was 35–39 (Maggi et al.
2009, 2011). The
210
difference between the effects of acaricide on susceptible Varroa populations can be due to
211
variable mite populations with putative differently developed detoxification mechanisms
212
(Maggi et al.
2011).
213
Screening of Varroa resistance to an acaricide just before its application is useful in
214
selection of appropriate treatment. In this study for detection of Varroa resistance we
215
recommended a test with polypropylene vials and found discriminating concentrations of
216
tau-fluvalinate, acrinathrin, and amitraz to be 0.66, 0.26 and 0.19 lg/mL/vial, respectively.
217
Acknowledgments The authors would like to thank the anonymous reviewers for their valuable comments
218
and suggestions that have improved the manuscript. We acknowledge the assistance from the beekeepers for
219
the collection of samples from honeybee colonies. The authors are obliged to Dalibor Titera and Jaroslav
220
Havlik for valuable comments on drafts of this manuscript and Martin Markovic for help. This study was
221
supported by The Ministry of Agriculture of the Czech Republic, project QJ1530148.
222
223
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