The intestinal microbiota of tephritid fruit flies as a potential tool to improve

rearing and the SIT

Dept. of Entomology, Dept. of Microbiology and Plant Diseases, The Robert H. Smith Faculty of Agriculture, Food and Environment,

The Hebrew University of Jerusalem, Rehovot, Israel

Michael Ben-Yosef, Eyal Ben-Ami, Sagi Gavriel, Edouard Jurkevitchand Boaz Yuval

Outline

1. Tephritid - bacteria associations2. Bacteria and adult fruit fly fitness:

The olive fly as an example3. Bacteria and the Sterile Insect Technique:

The Medfly as a case study4. Final conclusions

Superfamily Tephritoidae Family Tephritidae

General saprophagy Plant saprophagy Phytophagy

Evolution of Tephritid – bacteria associations

Plant saprophagy:Self-rotting vegetation

Phytophagy:Fly mediated rot in living

plant tissuesDiaz-Fleischer et al. 2000

Olive fly-bacteria association

Petri 1910Capuzzo et al. 2005 Petri 1910

Adult flies feed opportunistically but mainly on honeydew

Effect of bacteria on olive fly fitness

Examining the effect of bacteria and diet on female fecundity

Mated wild females:

Sucrose Sucrose + non-essential amino-acids

Full diet (sucrose + Yeast

hydrolysate)

Ben-Yosef et al., Proc. R. Soc. B, 2010

+ / - Antibiotics

Effect of bacteria on olive fly fitness

Ben-Yosef et al., Proc. R. Soc. B, 2010

05101520

mean

no. o

f egg

s / fe

male P = 0.028

Honeydew

• Bacterial contribution to fecundity is diet dependent.

• Bacteria are needed only when females feed on an unbalanced amino-acid diet

intestinal bacteria contribute amino-acids or protein to the fly

Allows flies to subsist on low quality food such as honeydew

Other fruit flies may similarly depend on their intestinal bacteria

(Dacus, Ceratitis, Rhagoletis, Anastrepha)

Bacteria and adult fruit fly fitness: conclusions

• Egg disinfection • Diet Acidification • Use of antibiotics and preservatives

Disruption of symbiotic relationships during mass rearing

• Irradiation of pupae

The irradiation affects the gut microbiota

Reduced mating competence of sterile Medfly males

Irradiation effect on medfly gut microbiotaV8 (Day 1) Sterile V8 (Day 1) Sterile V8 (Day 5) Wild (Day 1)

1. Gut bacterial community profiling by Amplified rDNA restriction analysis (ARDRA)

Enterobacter sp.

Klebsiella sp.

EnterobacteriaceaePseudomonads

Enterobacter sp.

Klebsiella sp.

Enterobacter sp.

Klebsiella sp.

18.67%

4%

Ben-Ami et al. ISME Journal 2009

Irradiation effect on medfly gut microbiotaV8 (Day 1) Sterile V8 (Day 1) Sterile V8 (Day 5) Wild (Day 1)

EnterobacteriaceaePseudomonads

Enterobacter sp.

Klebsiella sp.4 %Klebsiella

sp.23%

Enterobacter sp.

Ben-Ami et al. ISME Journal 2009

1. Gut bacterial community profiling by Amplified rDNA restriction analysis (ARDRA)

2. Monitoring the establishment of Klebsiella oxytoca in the gut

Effect of “probiotic” diet on medfly gut microbiota

StrpRKlebsiella oxytoca

ChromoAgarMedium Strp Medium

Klebsiella treatmentSucrose only

Klebsiella + Enterobacter

Sterile V8 (Day 1)

0.00E+002.00E+074.00E+076.00E+078.00E+071.00E+081.20E+081.40E+081.60E+081.80E+082.00E+08

TO T3 T5 T7Days

Num

ber o

f col

onie

s

0 3 5 7

*

* *20

16

12

8

4

0

No. o

f colo

nies (

x107 )

Days

Klebsiella treatmentSucrose only

Pseudomonas spp.

0

4

8

12

16

0 3 5 7Days

No. o

f colo

nies (

x10 6

)

*

Ben-Ami et al. ISME Journal 2009

2. Monitoring the establishment of Klebsiella oxytoca in the gut

Effect of “probiotic” diet on medfly gut microbiota

StrpRKlebsiella oxytoca

ChromoAgarMedium Strp Medium

16SrRNA Sequencing:Klebsiella oxytoca

All StrpR colonies turned blue on ChromAgar

Sterile V8 (Day 1)

0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

1.00E+08

1.20E+08

1.40E+08

1.60E+08

1.80E+08

T0 T3 T5 T7Days

Num

ber o

f col

onie

s

0 3 5 7Days

16

12

8

4

0

No. o

f colo

nies (

x107 )

DaysBen-Ami et al. ISME Journal 2009

Effect of “probiotic” diet on male competitiveness

3. Examining copulatory success of sterile males fed with Klebsiella oxytoca

Live bacteria

Sterile V8 (Day 1)

Autoclaved bacteria

Sucrose only

Sucrose only

“Sade” strain“Sade” strain

X5 days feeding on:

Ben-Ami et al. ISME Journal 2009

020406080

100

Livebacteria

SadeStrain

(control)

Autoclavedbacteria

Sucroseonly

Laten

cy to

mate

(min)

*0.00.10.20.30.40.50.60.70.80.91.0

Failin

g0 20 40 60 80 100 120 140 160 180

Time to mate

• The intestinal microbiota contributes significantly to fruit fly fitness.

• Mass reared V8 flies differ from wild flies in their gut bacterial community structure.

• The sterilizing irradiation affects the bacterial community within the gut.

• Klebsiella oxytoca added to the post-irradiation diet significantly improve sterile male performance.

Final conclusions

Colonization

Production

Sterilization

Holding & emergence

Release

Supplementing pre-release diets with

bacteria

Supplementing adult diets with bacteria

Applying bacteria in larval rearing procedures

• Minimizing larval requirements of nutrients•Reducing microbial contamination

• Enhancing sterile male performance•Extending life through nutrient provisioning, minimizing infections

• Helping newly established colonies to adapt to artificial rearing conditions

Implementing bacteria in mass rearing of fruit flies

• Shlomit Shloush and Batya Kamenski• Bio-Fly facility: Inbar Shouster-Dagan• BARD and IAEA/FAO for financial support

Many thanks to