Work Package 12 (ER 2)

Diapause and sexual behaviour in the Olive fly

General details

Oxitec Ltd. (UK)

Dr. Martha Koukidou

Christa Kistenpfennig

Other partners involved
University of Wuerzburg


University of Wuerzburg for 3 months to train in fly neuroanatomy/ICC; year 1
Actual Analytics for 1 month to train in software development; year 2
Bio-Fly for 2 months to train in field studies; year 2


  • We will identify and characterise the clock network in the Olive fly, anatomically and genetically
  • We will investigate reproductive diapause and mating behaviour in Olive fly


  • Train ESR7 and ESR12 in olive fly rearing and general biology
  • Identify key clock genes in Olive fly using conserved sequences in Drosophila
  • Compare genomic and expression profiles of the clock genes identified above among a) the RIDL olive fly strain, b) two laboratory strains with differential photoperiodic responses to mating and c) wild insects collected in collaboration with our associate partner, BioFly.
  • Study reproductive diapause of wild olive fly and evaluate the ability of the RIDL olive fly strain to obtain successful copulations with wild females during the reproductive diapause period under natural environmental conditions


Clock genes and phenotypes in Bactrocera
The olive fruit fly Bactrocera oleae is the single most important insect pest for the great majority of olive plantations in the world. The Sterile Insect Technique (SIT) is a highly effective, targeted, and environmentally friendly method of control, but the main obstacles connected with SIT and Bactrocera are: a) laborious and expensive mass rearing, b) absence of a sexing strain that allows for a male only release, c) reduced quality of mass reared flies (e.g. different mating time to wild insects under natural photoperiods).  Oxitec developed a system called RIDL® (Release of Insects carrying a Dominant Lethal gene or genetic system). Specifically, engineered dominant lethal genes replace the radiation-induced dominant lethal mutations of classical SIT. The lethality is conditional, so the strain can be propagated under permissive conditions, specifically the presence of tetracycline as a dietary ‘antidote’ which prevents expression of the lethal system. Furthermore, these lethal genes are designed to kill females selectively allowing integrated genetic sexing simply by rearing the flies intended for release under restrictive conditions. We have developed several conditional female-lethal strains of olive fly. These are based on a (tetracycline-repressible) ‘positive feedback’ design, made female-specific by incorporation of a sex-specific intron from Cctra, the Medfly homologue of the Drosophila sex determination gene transformer.  Thorough analysis of transgene penetrance, copy number and tetracycline repressibility resulted in strain OX3097D being selected for further development. Our proposal falls into four sections:

a) We will identify and characterise key clock genes in the olive fly, based on their counterparts in Drosophila and medfly and perform a comparative sequence analysis among OX3097D and wild strains, because sequence differences between clock genes such a period are known to alter mating times in Drosophila and Bactrocera cucurbitae (melon fly). Cloned genes will be used to generate antibodies (commercially) and these and heterospecific clock antibodies already available such as PDF, will be used to characterise the circadian neuronal network of olive fly strain OX3097D in comparison with wild strains, in collaboration with Helfrich-Forster.

b) We will investigate the effect of light and/or temperature in relation to behavioural traits such as circadian rhythms, rhythmic egg-laying and fecundity, longevity and male courtship in an attempt to entrain strain OX3097D for a more robust field performance. This project will be in collaboration with the Leicester group.  We will model our findings with Beersma for a more advanced field release of strain OX3097D.

c) In partnership with Actual Analytics we shall develop software for automated trapping systems in the field and mating monitoring systems using filters for fluorescence excitation and detection.

d) Following “entrainment” of the olive strain OX3097D based on natural photoperiod and circadian rhythms, we will undertake a much more detailed analysis of the “new” strain in its natural environment. We will investigate the mating performance of the strain in field cage trials using wild males and females from Israel. Furthermore, we will analyse a number of mass-rearing parameters including egg fecundity, adult longevity and egg to adult recovery according to standard tests as these are outlined by IAEA/USDA/FAO. Data will be compared to similar experiments conducted prior to the entrainment of the strain. This part of the project will be in collaboration with Bio-Fly in Israel.

Results and milestones

  • Project plan and personal development plan for training; month 8
  • Description of the key clock genes in olive fly; month 20
  • Complete mating behavioural analysis in olive fly; month 28

Synergies, Risks & Exploitation

The partners enhance the study with field studies, ICC, genetic analyses, and software development. Risks, minimal. Results will be incorporated in an Olive fly SIT application and Actual will commercialise the novel tracking method.

See more Work Packages in Research Area C