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Genetic approaches to population suppression in Anopheles: from bench to the field
Tania Dottorini
New biotechnological tools
Università degli studi di Perugia
Transgenic insect technologyfor vector control
Population replacementstrategies
(also in combination with drivee.g. MEDEA)
RIDLFacilitating classic SIT
(Sterile Insect Technique)
Genetic SterilitySystems
Genetic Drive Systems(e.g. Homing Endonucleases,
Transposons)
Sex Ratio Distorters Transformation withWolbachia, Viruses...
SIT requires large numbers of sexually active but sterile insects to be released over large areas.
In the case of mosquito programs, male-only populations must be released as females would contribute to disease transmission.
SIT programs in mosquitoes have faced a series of hurdles:
loss of male fitness
lack of a suitable genetic sexing mechanism to separate male from female individuals
reinvasion of the treated areas by immigration of mated females
The Sterile Insect Technique
Transgenic technologies to improve SIT
MarkersAllow dispersal analysis and fitness studies
SexingFacilitate the production and release of male-only
SterilizationReduce fitness costs due to irradiation
β2EGFP marker predicts sex at the larval stage
β2EGFP expression offers the possibility to accurately identify and separate male and female mosquitoes at the larval stage
• 4th instar larvae were scored either positive or negative for green fluorescence.
• Larvae were allowed to mature to adulthood, and their sex determined.
Development of Anopheles sexing lines
Sorting Embryos with Sex-Specific GFP-Expression
Sorting DrosophilaImaginal Discs
A new version will be produced with the capability to separate 100,000 larvae per day
Technology is available for automatically sorting fluorescent mosquito larvae
Automated mosquito flow sorting
β2EGFP spermatozoa can be easily detected in female spermathecae
Spermathecae were dissected from wild-type females mated to males from different transgenic lines.
Transgenic insect technologyfor vector control
Population replacementstrategies
(also in combination with drivee.g. MEDEA)
RIDLFacilitating classic SIT
(Sterile Insect Technique)
Genetic SterilitySystems
Genetic Drive Systems(e.g. Homing Endonucleases,
Transposons)
Sex Ratio Distorters Transformation withWolbachia, Viruses..
Homing endonucleases
Engineering sex ratio distortion by destroying the X chromosome using highly specific nucleases
I‐PpoI recognition sites within rDNA genes located on the A.gambiae X chromosomes
(300‐400 repeats)
Development of mosquitoes expressing I-PpoI/EGFP fusion protein under the β2tubulin promoter
Line 1: "Anaximander"2L at position 49029419AAAACGCACCTTAA-PB-TTAAACACAATGAC
Line 2: "Otto von Schwarzenberg"3R at position 11872203AGAAAATCATTTAA-PB-TTAACACTCACATA
Heterozygous transgenic β2Ppo males are completely sterile
Testes of β2Ppo males develop normally
The EGFP expression pattern reflects the temporal activation of the β2 tubulin promoter and the spatial organization of spermatozoa differentiation process
Genomic rDNA is cleaved in the germline of transgenic males
Southern blot on genomic DNAfrom wt and β2Ppo testis
β2Ppo testis
Embryo development is arrested at an early stage
b2Ppo males x wt females wt Males x b2Ppo females
Laid eggs are fertilized by β2Ppo spermatozoa
1 2 3
1. S7 primerset (Chromosome 3L)2. Y specific primer set3. Both primer sets
PCR on total genomic DNA from sterile embryos
S7Y
Singe embryo genotyping of sterile and wt embryos
Multiplex PCR with primers designed to detect:
‐ β2Ppo transgene or‐Y chromosome specific sequenceand S7 control gene
Y chromosome transmission ratio distortion
(P < 0.0001)
(P = 0.1824)
All sperm cells of b2Ppo males are EGFP positive
Early Spermatogenesis
Spermatogonium (Stem cell derivative)
MITOSIS I
Spermatogonia(undergo 4 mitotic
divisions)
MEIOTIC DIVISION I
32 SecondarySpermatocytes
130 hrs
16 Primary Spermatocytes
(undergo 30-fold increase in volume)
90 hrsMITOSIS II-IV
β1
β3
Cytoplasmic bridgesMEIOTIC DIVISION II 140 hrs
64 Spermatids
160 hrsβ2
A. gambiae lines β2Ppo1 and β2Ppo2 meet a number of requirements for the implementation of transgenic SIT:
i) a visible marker for monitoring male dispersal and competitiveness
ii) a validated sexing system that can be effectively automated
iii) complete and dominant genetic male sterility
iv) laboratory cage experiments performed indicate that β2Ppo males can compete withWT males in mating experiments
Imperial College LondonSilwood Park Campus /NERC Centre for Population Biology:Prof. Austin BurtProf. Charles GodfreyDr. Anne Deredec Dr. Penny Hancock
South Kensington Campus:Prof. Andrea CrisantiDr. Nikolai WindbichlerMr. Phillipos Papathanos Daniel NaujoksAlex Pinder
CNR, Milan, ItalyProf Gianluca deBellis
University of CambridgeProf. Michael Ashburner Dr. Steve RusselllMr Yuk Sang Chan Dr. David Huen
University of Washington andFred Hutchinson Cancer Research Center, Seattle
Prof. David Baker Prof. Ray Monnat Prof. Barry Stoddardd
Transgenic insect technologyfor vector control
Population replacementstrategies
(also in combination with drivee.g. MEDEA)
RIDLFacilitating classic SIT
(Sterile Insect Technique)
Genetic SterilitySystems
Genetic Drive Systems(e.g. Homing Endonucleases,
Transposons)
Sex Ratio Distorters Transformation withWolbachia, Viruses..
Transgenic insect technologyfor vector control
Population replacementstrategies
(also in combination with drivee.g. MEDEA)
RIDLFacilitating classic SIT
(Sterile Insect Technique)
Genetic SterilitySystems
Genetic Drive Systems(e.g. Homing Endonucleases,
Transposons)
Sex Ratio Distorters Transformation withWolbachia, Viruses..
Homing-mediated genetic drive
HEG mediated homing in Anopheles gambiae
Transgenic Donor or a Recipient lines Anopheles gambiae have been created
The Donor line (GFP‐) contains a I‐SceI HEG casette driven by the testis specific beta2 tubulin promoter interrupting the reading frame of a GFP sequence
The Recipient line (GFP+) contains a I‐SceI recognition site within a functional GFP sequence
The constructs (and transgenic lines) or otherwise identical apart from a NotI molecular marker site unique to the Recipient construct
Donor line
GFP Homing Assay
GFP Homing Assay
Genetic crosses :Progeny of mosquitoes carrying HEG+ Donor and Recipient:
Females: 50% GFP-, 50% GFP+Males: >85% GFP-, <15% GFP+
Conclusion: HEG is active and homing in male mosquitoes
GFP + GFP -
In vivo dynamics of HEG based drive system
GFP- HEG+ mosquitoes seeded in a population of GFP+ HEG- recipien
Conclusions:• GFP target allele inactivated over time• HEG construct frequency increases in population -> genetic
drive• Estimated homing rate: up to 70%
Which are the optimal target genes for HEGs in Anopheles
• Sex determination
• Behaviour
• Longevity