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
NColonie

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

2006

2007

2008

2009

TAB
TAM
TAG
ES1
ESCH
ES2
ES3
ES4
ES5
ESSTA
757
535
222
B
F35

7.63 0.3 years

(maxi: 15).

S44
601
G1
V1
27
F
URSS25
10BC3C
URSS96
264
124
692
X
174
248

10

11

12

13

14

15

Surviving colonies

I- Validation of the survival of the honey bee colonies

Mortality
16,8

18
16
14

14

12
%

12,5

12,5
10

9,7

10
8
6
4
2
0

98-99

99-00

00-01

01-02

02-03

03-04

Year

No evidence for mortality due to varroa infestation

N=72

Swarming

100

Swarming (%)

52

**

80
61

60

59
67

40

69

74

58
34

20

55

33

41

45

0
2000

2001

2002

2003

2004

2005

Year
VSB

Control

Honey production

Honey production (Kg)

40
35

71

30

58

85

25
76

20
15

40

72

66

82

65

48

61

57

37

10

49

5
0
1999

2000

2001

2002

2003

2004

2005

Year
VSB

Control

Cost due to varroa infestation

N=72

I- Validation of the survival of the honey bee colonies

No particular deseases observed inside de collected colonies

I- Validation of the survival of the honey bee colonies

Comparaison of varroa population dynamics :

- Surviving colonies from Avignon
- Non surviving colonies from Canada
- Daugther queen of surviving colonies
Counting varroa fall twice a week
Same study made in Canada
by Pr. Gard Otis
Grard de Vaublanc (INRA,
Avignon)

Varroas infestation of the colonies

Sensitive colonies (per week)
1800

EBA 15

Number of varroa

1600

EBA 23

1400

EBA 03

1200

EBA 21

1000
800

EBA 14

600

EBA 37

400

EBA 38

200
0
S13 S15 S17 S19 S21 S23 S25 S27 S29 S31 S33 S35 S37 S39

EBA 18

week

Differences of varroa
infestation

Surviving colonies (per week)

1800
Number of varroa

1600

EBA 24

1400

EBA 51

1200
1000

EBA 77

800

EBA 44

600

EBA 76

400

EBA 45

200

EBA 259

0
S13

S15

S17

S19

S21

S23

S25

S27

week

S29

S31

S33

S35

S37

S39

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)

4000

3500

Varroa number

3000

2500
Canadian
2000

Surviving daughter

1500

Mother surviving
colonies

1000

500

0
April

May

June

July

August

September

October

November

 Honey Bee Colonies surviving to Varroa

Different Hypothesis :
Beekeeping
methods
Environment

The honey
bee

Co-evolution
Virus

The Varroa

Hypothesis tested:
Virulence of the varroa:
Need tolls :
Genetic markers to differentiate
populations
Complet sequence of mtDNA,
17 microsatellites nuDNA

First resultat: no variability, its a clonal population!

Does not support hypothesis of less virulent varroa populations
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.

Hypothesis tested:

The virus

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

Isabelle Mazet (INRA Avignon)

Brenda Ball (IACR- Plant and Invertebrate
Ecology Division, Rothamsted)

and Magali Ribire (AFSSA, Sophia Antipolis)

Hypothesis tested:

The virus

Results:
Significative differences beetwen surviving and control colonies
Control colonies have more APV et CPV
Almost all the colonies have DWV

Hypothesis tested:

The virus

Injections of virus:

RS 15 AFSSA
control

Survival %
100

100

80

80

60

60

40

40

20

20

6
9
days after inoculation

TNI

ABPV d10-6

CBPV d10-6

CBPV d10-8

12
ABPV d10-8

RR 51
surviving colony

Survival (%)

15

6
9
day after inoculation

TNI

ABPV d10-6

CBPV d10-6

CBPV d10-8

No significant differences between surviving and

control colonies for APV
Nor for CPV

12
ABPV d10-8

15

Hypothesis tested: Resistance of the honey bee :

Varroa Hygienic

Capping
duration

Behavior (SMR)

Regulation of the
environment
(TC et HR%)
swarming

Grooming
behavior

Resistance
mecanisms

individual response

Reduction of
Varroa
fertility

populationnal response
honeybee - Varroa
Interaction

Regulation of Varroa population

development
Rosenkranz P. (1999) Apidologie 30, 159-172.

Resistance of the bees to the mite:

Hypothesis tested : Grooming behavior

Ability of the bees to recognize

and destroy the varroa mite

Behavioral test =>

Differences in detection of the mite beetween the differents type of colonies

 Solid Phase MicroExtraction (SPME)

Solid Injection (IS)

Seringue SPME

Capillary insert in the injecteur

of the GC
Fibre
Tube with 50 Varroa
30C

Identified Substances with SPME and IS

Micro-Extraction

Injection Solide

Cuticular
hydrocarbons
3 Acids (palmitic, oleic,
stearic)
1 alcool

Cuticular
hydrocarbons
8 acids
3 esters (palmitate,
olate, starate
dthyle)

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

Behavioral effects on the bees

Behavioral contacts between surviving and control bees.
45

% perception of the tag

40
35
30
25

colonies S
colonies R

20
15
10
5
0
T

0,1

10

100

Ethyl oleate dose

Surviving bees are responding more than control bees

EAG gave the same results
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

Behavioral test and electro-antennography =>

Differences in detection of the mite beetween the differents type of colonies

MARTIN C., SALVY, M., PROVOST E., BAGNRES A.G., ROUX M., CRAUSER D.,
CLMENT J.L., LE CONTE Y., 2002 Physiological Entomology. 27: 175-188.

Resistance of the bees to the mite:

Hypothesis tested : Hygienic behavior

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 : Varroa 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 : Infertility of the varroa mite and honey bee survival.
Last week:
From 2000 to 2005,
50% for surviving colonies.
very variable results depending of the
season:
From 70% to 19% for surviving
colonies.
From 49% to 15% for canadian
colonies.
Grard de Vaublanc (INRA, Avignon)

20 % for control colonies.

Barbara Locke

Honey bee Varroa tolerance :

Complexe phenomenon, involve many genes associated with :
metabolism, behavior, chemical communication,
reproduction

Pangenomic approach
method for the identification of candidat genes

Using gene expression as a tool :

to study molecular basis of host/parasite interactions and
functional genomics
for honey bees selection against Varroa destructor (Varroa
tolerant bees could have a specific gene expression pattern that we
could use for selecting bees against the mite)

Maria NAVAJAS, INRA Montpellier, France

Yves LE CONTE, INRA Avignon, France
Gene ROBINSON, University of Illinois, USA
Charlie WHITFIELD, University of Illinois, USA
Jay 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 Chips
Univ. Illinois USDA - NSF

(A)

Atg18, pUf68
Rab7
Immune system

Inflammatory
response

Deformed

Effects of the parasitism:

Wing Virus
Pcmt , Nedd8
Cellular and molecular

31 genes were moving

damages
baz , dgl1,

sgl

Embryonic

ple , Atg18,

Dlic2

Brain

development

Deformed
adult

Cognitive
impairment

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.

(B)

Candidate genes
for afor
behavioural
resistance
Candidate
genes
a behavioural
resistance
Futsch , scrt , otk ,
Bchs
Mhcl , gro , fng , Bchs
Nervous system

Dhc64c
Dhc64c

poe
, para
poe, GluCl
GluCl,
para
Responsiveness
to stimuli

Mushroom
bodies

smi21F
su(w a), poe
poe ,
smi21F , su(w
para
rogdi
para, rogdi

Effects of the genotype:

Dscam , fwd
Olfaction

99 genes were moving

Others genes
Others genes
Dscam , otk
Immune system

Ahcy13, para
Ahcy13, para
ResistanceResistance
to toxins to toxins

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.

2006 :
Year of the sequencing of the honey bee genome

=> New ADN cheap including the hole genome

conception: Universit dIllinois + USDA + NSF

13440 oligos

Second approach: adult worker responses

USDA Baton Rouge, Louisiane

J. Harris and J. Harbo

Gene expression on the Varroa hygienic trait

Results:
olfaction involved

Prospects:
Make replicates on other selected bees
Find the genes involved
Use them as toll in honeybee selection

Conclusion
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
As for the case of Acarapis woodi in the past, we can hope that most of
the bees will become resistant to Varroa destructor