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