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