Insulin-like androgenic hormones and monosex culture of prawns:

The first implementation of RNA-interference in aquaculture

Amir SagiBen-Gurion University of the Negev, Israel

2cm45 60 75 88 102 115 130 142 152

0

200

400

600

800

1000

Time (days)

Cu

mu

lati

ve W

eigh

t (g

/2m

2)

Modified from Sagi et al., 1986 Aquacult.

M. rosenbergii dimorphic growth pattern in favor of males

* Includes only the marketable yields (≥30g)

~60% higher incomeField study in IndiaNair et al., 2006

&

♂♀ Neo fe

mal

e

F1 (neo-female progeny) n.c.

F.S.

P.C.

Contr

ol

fem

ale

♂♀ F1 (control female progeny) n.c.

1 cm

3 mm

Ventura et al., Heredity, 2011

Mr-IAG silencing induces full functional sex reversal

Female specific sex marker

ZZWZ X

Neo ZZ

RNAi IAG

ZZ X

50% WZ

50%ZZ

100% ZZ

IAG silencing and prawn monosex biotechnologial cascade

Modified from Aflalo et al., 2006 Aquacult

The androgenic gland

Sex differentiation

Insulin-like

Biotechnology

Monosex all-male culture

Patent: RNA silencing of insulin-like gene in prawn and uses thereof. PCT/IL2009/000127 (priority: February 4, 2008) WO2009/098683, US2012/073508.

CA AA AA A AU CCC C GGG U U U UU U U3-’ -5’

AA CC GU GA

A A A A A A A A A A A A ACA AAAAA A AU C CC C C C GGG GU U U U U U UA CU U5-’ -3’

A T G C A A A A A A A A AT T T T T T T T TC C C C C CG G G G G5-’ -3’

A A A A A A A A AAT T T T T T T T T TC C C C C C CG G G G G G3-’ -5’DNA

RNA

How is RNAi working (non GMO)

Temporal dsRNA effect

Mr-IAGInsulin-like

androgenic hormone

A AUCCAU

AU C CG UU

AC G UC G

A AC CGU U

A AA C GU U U

AAA CGU U

AA GUU U

CA AC GU U

G

A gene encoding theInsulin like androgenic hormone

3 days

1 4 5 6 7 82 3T0 T0 T1 T1

Controls

PC NC

7 days

9 10 11 12 13 1514 16 17 18 19 20 21 2322 24

14 days

Clearance of exogenous dsRNA in injected prawns

Time (Days)3 7 14

RNAi temporarily reducesMr-IAG expression

Real time RT-PCR

Control 3 21 280

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60

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Days after dsMr-IAG injection

Rel

ativ

e M

r-IA

G e

xpre

ssio

n (%

)

Neo females (sex reversed males) reproductive performance

Females Neo females

Body weight (g) 32.03 ± 0.28 41.31 ± 0.22

BSI (%) 13.3 ± 1.63 12.7 ± 1.28

Eggs/g BW 859 ± 120 845 ± 67

Selective breeding scheme combined with all-male selective production

BGU line

1st Generation

Local strain

G1 G1

All-male PLs to growers

G2G2

BGU lineNeo

female

SelectedLocal strain

2nd Generation

All-male PLs to growers

BGU lineNeo

female

SelectedLocal strain

Modified from Aflalo et al., 2012

Recommendations• Protect from decay by crossing BGU with

Local lines (do not reuse crosses, no backflow of genes).

• Insert a selectively bred line into the portfolio (improved lines in Vietnam, India, or other sources).

• Pathogen free declaration of the BGU line.

Temporal RNAiNo hormone treatmentNo chemicalsNon GMO

Aquaculture production

enhancement

Up to this point, the technology

17.8 B $ 5.4 B $

EnvironmentalSolutions

Bio-control

PriceDelivery

Thank youDr. Tomer Ventura Ohad Rosen

Dr. Simy Weill Dr. Ziv Roth

Dr. Eli D. Aflalo Yaara Lazar

Dr. Rivka Manor Omri Sharabi

Dr. Lilah Glazer

Funding sources: BARD TIRAN Shipping Ltd.