1. biosecurity built on science
Understanding the microbiome of Queensland
fruit fly to improve sterile insect technique
1. Plant Biosecurity Cooperative Research Centre (PBCRC), Australia
2. Elizabeth Macarthur Agricultural Institute (EMAI), NSW Department of Primary Industries
3. Hawkesbury Institute for the Environment (HIE), Western Sydney University
Plant Biosecurity Cooperative Research Centre
D Woruba1,2,3, O Reynolds2, M Riegler3, J Morrow3 & T Chapman1,2

2. biosecurity built on science
Queensland fruit fly (Qfly), Bactrocera tryoni
 Australia’s most significant
horticultural insect pest
threatening crops valued at
$640 million
 Damages fruits and
vegetables and affects trade
 Broad-spectrum chemicals
are being phased out
 Current management includes monitoring, targeted chemical
application, cultural practices and Sterile Insect technique (SIT)

3. biosecurity built on science
Sterile Insect Technique
 SIT is “a method of pest control using area-wide inundative
releases of sterile insects to reduce reproduction in a field
population of the same species” FAO, 2014 (Insect birth control)
 The success of SIT depends on sterile males mating with wild
females
 Mass rearing and irradiation lead to reduced fitness and
performance of sterile males

4. biosecurity built on science
Probiotics
 Greek “pro” + “bios” = "for life.”
 “Live microorganisms which when administered in
adequate amounts confer a health benefit on the
host” FAO & WHO, 2006.

5. biosecurity built on science
Probiotics
Human products
Metchnikoff 1907
Animal products

6. biosecurity built on science
Beneficial bacteria in Insects
 Beneficial bacteria in insects have been well
documented
- E.g. Obligate endosymbiosis of aphids and the Buchnera
bacteria
- E.g. Gut bacteria linked to evolution of herbivory in ants
 Tephritid fruit flies gut bacteria studied since early
1900s

7. biosecurity built on science
Aim
 Characterization of complete gut microbiome of
Qfly
- Reared (lab) and wild Qfly populations
- Ecological distribution (location & host)
 Formulation of probiotic supplements to improve
Qfly SIT

8. biosecurity built on science
Literature Review
 Woruba et al (review manuscript) submitted
 Roles of bacteria in tephritids:
- Protein metabolism
- Carbohydrate metabolism
- Longevity
- Attraction and communication
- Mating and reproductive performance

9. biosecurity built on science
-80°C StorageGut isolation
Fitness tests
Probiotic
formulations Culture bacteria
Field Collection Molecular ID
Effective “sexy”
sterile male Qfly

10. biosecurity built on science
Reared Qfly Microbiome
 Pupae;
- EMAI (lucerne as diet bulking agent)
 Sterile
 Fertile
- HIE (carrot as diet bulking agent)
 Fertile
 Adults
- Tenerals (≈12 hrs post-eclosion)
- Mature (14 days post-eclosion).
 yeast and sugar (3:1)
 sugar only
Adult rearing

11. biosecurity built on science
0
10000
20000
30000
40000
50000
60000
♂ ♀ ♂ ♀ ♂ ♀ YS♂ YS♀ S♂ S♀ S♂ S♀ YS♂ YS♀ YS♂ YS♀ S♂ S♀
Average count of 16s rDNA reads
Reared Qfly Microbiome: Results
TENERALS
MATURE
EMAI EMAIHIE HIE
 Sterile
 Fertile
COUNTS

12. biosecurity built on science
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
♂ ♀ ♂ ♀ ♂ ♀ YS♂ YS♀ S♂ S♀ S♂ S♀ YS♂ YS♀ YS♂ YS♀ S♂ S♀
FertileFertileSterileSterileFertileFertileFertileFertileFertileFertileSterileSterileSterileSterileFertileFertileFertileFertile
Enterobacteriaceae
Acetobacteraceae
Lactobacillaceae
Xanthomonadaceae
Flavobacteriaceae
Legionellaceae
Moraxellaceae
Sphingomonadaceae
Hyphomicrobiaceae
Pseudomonadaceae
Others
EMAI EMAI
MATURE
HIE
Reared Qfly Microbiome: Results
TENERALS
Bacterial diversity of mass-reared teneral and mature Bactrocera tryoni
HIE

13. biosecurity built on science
Reared Qfly Microbiome: Conclusion
 Microbiome of mature Qfly is dominated by
Enterobacteriaceae
 Teneral flies have low bacterial density
 Bacterial population increases with fly maturation
(either through cell division or uptake from
environment)
 Ideal time point of supply of probiotic candidates is
in teneral stage

14. biosecurity built on science
Source: Dominiak & Daniels 2012
Mareeba
Atherton
Kuranda
Redlands
Mt. Coot-tha
Sunshine
Coast
Central Coast
Hawkesbury
Camden
Wild Qfly Microbiome
Qfly Sampling
 Wild
 Native and
Commercial
Habitats
 Host fruits
 Locations
 Lab reared
 Sterile and fertile

15. biosecurity built on science
 Use of 5 selective media for culturing bacterial
isolates
 Identification of isolates using molecular and
bichemical tools
Candidates identification and culturing
Culture bacteria

16. biosecurity built on science
 Identification of potential probiotic candidates and
develop formulations
 Test for:
 Improved stress resistance & survival
 Enhanced sexual mating performance (Flyagra)
Fitness tests
Fitness testsProbiotic formulations

17. biosecurity built on science
Thank you
The gutsy search for a sexy solution