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Honeybees Surviving Varroa Destructor Infestations in France - LE CONTE Yves
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Honey bees that surviving
Varroa destructor infestation in France
Yves Le Conte
INRA, UMR 406 Abeilles et Environnement, Laboratoire Biologie et Protection de l'abeille,
AVIGNON, France
Historic :Apparition of Varroa in France in 1982
Untreated colonies die after 2 or 3 years of infestation !!!
1994
Come back of feral colonies
Untreated colonies
more or less abandoned
seem to survive !
1998 => Characterization of the survival of those colonies
to Varroa destructor
Aims :
- validate the survival phenomenon of the bees
- test different hypothesis to explain it
I-Validation of the survival of the honey bee colonies
1. Investigation into beekeeping journals
2. Collection and set up different apiaries with candidate colonies
3. Criteria studied:
• Survival of the colonies
• Swarming
• Honey production
• In Avignon, population dynamics of the varroa mite
I- Validation of the survival of the honey bee colonies
Investigation to the beekeepers => set up of a different apiaries with 70
candidate colonies :
- Four apiaries in Avignon Two in la Sarthe, One in Orne,
I- Validation of the survival of the honey bee colonies
Queen paint marked
2 monthly visits of the colonies from
early spring to early winter
Check for diseases
No other manipulations
I- Validation of the survival of the honey bee colonies
Survival of the colonies from ‘la Sarthe’: 7.9 years
7.63 ± 0.3 years
(maxi: 15).
151413121110987654321
248
174
X
692
124
264
URSS96
10BC3C
URSS25
F
27
V1
G1
601
S44
F35
B
222
535
757
ESSTA
ES5
ES4
ES3
ES2
ESCH
ES1
TAG
TAM
TAB
200920082007200620052004200320022001200019991998199719961995N°Colonie
Surviving colonies
I- Validation of the survival of the honey bee colonies
12,5
10
12,5
14
16,8
9,7
0
2
4
6
8
10
12
14
16
18
%
98-99 99-00 00-01 01-02 02-03 03-04
Year
Mortality
N=72No evidence for mortality due to varroa infestation
0
20
40
60
80
100
2000 2001 2002 2003 2004 2005
Year
Sw
arm
ing
(%
)
VSB Control
59
67
52
61
45
58
3455 33
41
*
**
7469
Swarming
Honey production
N=72Cost due to varroa infestation
0
5
10
15
20
25
30
35
40
1999 2000 2001 2002 2003 2004 2005
Year
Ho
ney
pro
du
cti
on
(K
g)
VSB Control
72
76
71
65 5766
49
61
40
58
37
48
85
82
I- Validation of the survival of the honey bee colonies
No particular deseases observed inside de collected colonies
Comparaison of varroa population dynamics :
- Surviving colonies from Avignon- ‘Non surviving’ colonies from Canada- Daugther queen of surviving colonies
I- Validation of the survival of the honey bee colonies
Counting varroa fall twice a week
Same study made in Canada
by Pr. Gard Otis
Gérard de Vaublanc (INRA,
Avignon)
Differences of varroa
infestation
0
200
400
600
800
1000
1200
1400
1600
1800
S13 S15 S17 S19 S21 S23 S25 S27 S29 S31 S33 S35 S37 S39
Nu
mb
er
of v
arr
oa
week
Sensitive colonies (per week)
EBA 15
EBA 23
EBA 03
EBA 21
EBA 14
EBA 37
EBA 38
EBA 18
0
200
400
600
800
1000
1200
1400
1600
1800
S13 S15 S17 S19 S21 S23 S25 S27 S29 S31 S33 S35 S37 S39
Nu
mb
er o
f varr
oa
week
Surviving colonies (per week)
EBA 24
EBA 51
EBA 77
EBA 44
EBA 76
EBA 45
EBA 259
Varroas infestation of the colonies
De Vaublanc, G., Otis, G.W., Le Conte, Y.,
Crauser, D., Kelly, P. 2003 – Am. Bee J. 143
(4): 319.
Varroa fall in surviving colonies and canadian colonies (per month)
0
500
1000
1500
2000
2500
3000
3500
4000
April May June July August September October November
Va
rro
a n
um
be
r
Canadian
Surviving daughter
Mother surviving
colonies
• Honey Bee Colonies surviving to Varroa
Different Hypothesis :
The honey
bee
Virus
Beekeeping
methods
Environment
The Varroa
Co-evolution
Hypothesis tested:
Virulence of the varroa:
First resultat: no variability, it’s a clonal population!
Navajas, M., Le Conte, Y., Solignac, M., Cros-Arteil, S. et Cornuet, J-M. 2002.
Mol. Biol. Evol. 19: 2313-2317.
Solignac, M., Vautrin, D., Pizzo, A., Navajas M., Le Conte, Y., Cornuet, JM. 2003
Molecular Ecology Notes, 3 (4): 556-559.
Need tolls :
Genetic markers to differentiate
populations
Complet sequence of mtDNA,
17 microsatellites nuDNA
Does not support hypothesis of less virulent varroa populations
9 different virus search in different honey bee populations
Immuno-diffusion and elisa
- Cloudy Wing Virus : CWV
- Deformed Wing Virus : DWV
- Black Queen Cell Virus : BQCV
- Acute Paralysis Virus : APV
- Bee Virus Y : BVY and bee virus X : BVX
- Sacbrood Bee Virus : SBV
- Chronic Paralysis Virus : CPV
- Kashmir Bee Virus : KBV
The virusHypothesis tested:
Isabelle Mazet (INRA Avignon)
Brenda Ball (IACR- Plant and Invertebrate
Ecology Division, Rothamsted)
and Magali Ribière (AFSSA, Sophia Antipolis)
Results:
Significative differences beetwen surviving and control colonies
Control colonies have more APV et CPV
Almost all the colonies have DWV
The virusHypothesis tested:
No significant differences between surviving and
control colonies for APV
Nor for CPV
RS 15 AFSSA
control
0
20
40
60
80
100
0 3 6 9 12 15days after inoculation
Survival %
TNI ABPV d10-6 ABPV d10-8
CBPV d10-6 CBPV d10-8
RR 51
surviving colony
0
20
40
60
80
100
0 3 6 9 12 15day after inoculation
Survival (%)
TNI ABPV d10-6 ABPV d10-8
CBPV d10-6 CBPV d10-8
The virusHypothesis tested:
Injections of virus:
Resistance
mecanisms
Grooming
behavior
Varroa Hygienic
Behavior (SMR)
Capping
duration
Regulation of the
environment
(T°C et HR%)
swarming
Reduction of
Varroa
fertility
Regulation of Varroa population
development
individual response
populationnal response
honeybee - Varroa
Interaction
Resistance of the honey bee :
Rosenkranz P. (1999) Apidologie 30, 159-172.
Hypothesis tested:
Resistance of the bees to the mite:
Hypothesis tested : Grooming behavior
Ability of the bees to recognize
and destroy the varroa mite
Differences in detection of the mite beetween the differents type of colonies
Behavioral test =>
• Solid Phase Micro-Extraction (SPME)
• Capillary insert in the injecteur of the GC
Seringue SPME
FibreTube with 50 Varroa
30°C
• Solid Injection (IS)
Identified Substances with SPME and IS
• Micro-Extraction– Cuticular
hydrocarbons– 3 Acids (palmitic, oleic,
stearic)– 1 alcool
• Injection Solide– Cuticular
hydrocarbons– 8 acids– 3 esters (palmitate,
oléate, stéarate d’éthyle)
Martin C., Provost E., Roux M., Bruchou C., Crauser D., Clement J.L., Le Conte Y.
Physiological Entomology. 2001. 26: 4, 362-370.
Behavioral effects on the bees
Three of the compounds trigger a behavioral response
Surviving bees are responding more than control bees
EAG gave the same results
Behavioral contacts between surviving and control bees.
0
5
10
15
20
25
30
35
40
45
T S 0,1 1 10 100
Ethyl oleate dose
% p
erc
ep
tio
n o
f th
e t
ag
colonies S
colonies R
Behavioral effects on the bees
Confirm the better capacity of the surviving bees to recognize the mite
Resistance of the bees to the mite:
Hypothesis tested : Grooming behavior
Better ability of the resistant bees to recognize
and destroy the varroa mite
Differences in detection of the mite beetween the differents type of colonies
Behavioral test and electro-antennography =>
MARTIN C., SALVY, M., PROVOST E., BAGNÈRES A.G., ROUX M., CRAUSER D., CLÉMENT J.L., LE CONTE Y., 2002 –Physiological Entomology. 27: 175-188.
Method:
Hygienic test: frozen brood
Results:
Our surviving bees show that behavior,
but no more compared to sensitive one.
Resistance of the bees to the mite:
Hypothesis tested : Hygienic behavior
Method:
Varroa Hygienic Sensitive bees(Harbo JR, Harris JW. 2005. J. APIC. RES. 44:21-3)
Results:
Our surviving bees show that behavior
Resistance of the bees to the mite:
Hypothesis tested : Varroa Hygienic behavior
Resistance of the bees to the mite:
Hypothesis tested : Infertility of the varroa mite and honey bee survival.
From 2000 to 2005,
very variable results depending of the
season:
From 70% to 19% for surviving
colonies.
From 49% to 15% for canadian
colonies.
Gérard de Vaublanc (INRA, Avignon)
Last week:
50% for surviving colonies.
20 % for control colonies.
Barbara Locke
Honey bee Varroa tolerance :
Complexe phenomenon, involve many genes associated with :
metabolism, behavior, chemical communication, reproduction…
Pangenomic approachmethod for the identification of candidat genes
Using gene expression as a tool :
• to study molecular basis of host/parasite interactions andfunctional genomics
• for honey bees selection against Varroa destructor (Varroa tolerant bees could have a specific gene expression pattern that wecould use for selecting bees against the mite)
Maria NAVAJAS, INRA Montpellier, FranceYves LE CONTE, INRA Avignon, FranceGene ROBINSON, University of Illinois, USACharlie WHITFIELD, University of Illinois, USAJay EVANS, USDA, Beltsville, USA
Use gene expression to investigate
response of immature honey bees to Varroa
destructor
Effects of the parasitism and of the honey bee genotype
DNA chips
EST Chips
≈ 7000 cDNAs - ≈ 5000 genes ≈ 50% annoted
Oligo ChipsUniv. Illinois – USDA - NSF
Hypothetical pathways and models of honey bee responses to Varroa-parasitism. Arrows and dashes indicate positive and negative regulation, respectively; dotted lines are possible links between two factors.
NAVAJAS M., MIGEON A., ALAUX C., MARTIN-MAGNIETTE ML., ROBINSON GE., EVANS JD., CROS-ARTEIL S., CRAUSER D., LE CONTE Y. 2008. BMC Genomics 2008, 9:301doi:10.1186/1471-2164-9-301.
Immune system
Deformed
adult
Rab7
Deformed
Wing Virus
Atg18, pUf68
Pcmt , Nedd8
baz , dgl1, sgl ple , Atg18, Dlic2
BrainEmbryonic
development
Cognitive
impairment
Cellular and molecular
damages
Inflammatory
response
(A)
Immune system
Deformed
adult
Rab7
Deformed
Wing Virus
Atg18, pUf68
Pcmt , Nedd8
baz , dgl1, sgl ple , Atg18, Dlic2
BrainEmbryonic
development
Cognitive
impairment
Cellular and molecular
damages
Inflammatory
response
(A)
Effects of the parasitism:
31 genes were moving
Hypothetical pathways and models of honey bee response to the bee tolerant genotype. Gene names are up-regulated.
NAVAJAS M., MIGEON A., ALAUX C., MARTIN-MAGNIETTE ML., ROBINSON GE., EVANS JD., CROS-ARTEIL S., CRAUSER D., LE CONTE Y. 2008. BMC Genomics 2008, 9:301doi:10.1186/1471-2164-9-301.
(B)
Nervous system
Mushroom bodies
Responsiveness to stimuli
Olfaction
Immune system Resistance to toxins
Futsch , scrt , otk ,
Mhcl , gro , fng , Bchs
poe , GluCl α, para
smi21F , su(w a), poe ,
para , rogdi
Dscam , fwd
Dscam , otk Ahcy13, para
Candidate genes for a behavioural resistance
Others genes
Dhc64cNervous system
Mushroom bodies
Responsiveness to stimuli
Olfaction
Resistance to toxins
Bchs
poe, GluClα, para
smi21F, su(wa), poe,
para, rogdi
Ahcy13, para
Candidate genes for a behavioural resistance
Others genes
Dhc64c
Effects of the genotype:
99 genes were moving
2006 :Year of the sequencing of the honey bee genome
=> New ADN cheap including the hole genome
conception: Université d’Illinois + USDA + NSF
13440 oligos
Gene expression on the Varroa hygienic trait
USDA Baton Rouge, LouisianeJ. Harris and J. Harbo
Second approach: adult worker responses
Prospects:
Make replicates on other selected beesFind the genes involvedUse them as toll in honeybee selection
Results:
• olfaction involved
Conclusion
As for the case of Acarapis woodi in the past, we can hope that most of the bees will become resistant to Varroa destructor
• Some honey bee strains can survive to the Varroa mite in France
• It is a good start to develop IVM.
• Olfaction clearly involved in varroa tolerance
• Varroa hygienic behavior seems to be a good trait to select to get resistant bees (behavioral and molecular results on olfaction).
• Genome sequencing and gene expression techniques should bring new tools for honey bee selection in the future