Insulinlike androgenic hormones and monosex culture of prawns

Insulin-like androgenic hormones and monosex culture of prawns: The first implementation of RNA-interference RNA-i in aquaculture Amir Sagi Ben-Gurion University of the Negev, Israel

M. rosenbergii dimorphic growth pattern in favor of males Cumulative Weight (g/2 m 2) 1000 800 & 600 400 ~60% higher income Field study in India Nair et al. , 2006 200 0 45 60 75 88 102 115 130 142 152 2 cm * Includes only the marketable yields (≥ 30 g) Time (days) Modified from Sagi et al. , 1986 Aquacult.

Mr-IAG silencing induces full functional sex reversal 1 cm 3 mm ♂♀ n. c. of em al F 1 (control female progeny) Ne Co ♂ ♀ nt fe rol m al e e Female specific sex marker F 1 (neo-female progeny) n. c. P. C. F. S. Ventura et al. , Heredity, 2011

IAG silencing and prawn monosex biotechnologial cascade WZ X 50% WZ ZZ 50% ZZ RNAi IAG Neo ZZ X 100% Modified from Aflalo et al. , 2006 Aquacult ZZ ZZ

Monosex all-male culture The androgenic gland Sex differentiation Insulin-like Patent: RNA silencing of insulin-like gene in prawn and uses thereof. PCT/IL 2009/000127 (priority: February 4, 2008) WO 2009/098683, US 2012/073508. Biotechnology

How is RNAi working (non GMO) Mr-IAG Insulin-like androgenic hormone RNA Temporal ds. RNA effect 5’- A U CU U G C A U C C G C C U U G C A A U U A A AA UU GG CC AA UU GG A A A A 3’- 5’- U A UC A A C C U A C G U A UU UU AA CC GG UU AA C DNA A gene encoding the 3’Insulin like androgenic hormone A T G C A T A A T G C A T C C T G C A T A C G T A T T A C G T A C G T A T -5’ -3’

Clearance of exogenous ds. RNA in injected prawns 7 Time (Days) 7 days 3 3 days Controls PC NC T 0 T 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 14 14 days 17 18 19 20 21 22 23 24

RNAi temporarily reduces Mr-IAG expression Real time RT-PCR Relative Mr-IAG expression (%) 100 90 80 70 60 50 40 30 20 10 0 Control 3 21 Days after ds. Mr-IAG injection 28

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 Local strain BGU line 1 st Generation BGU line Neo female Selected Local strain All-male PLs to growers G 1 2 nd Generation BGU line Neo female G 2 Modified from Aflalo et al. , 2012 Selected Local strain All-male PLs to growers G 2

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.

Up to this point, the technology Temporal RNAi No hormone treatment No chemicals Non GMO Price Delivery 17. 8 B $ Environmental Solutions Bio-control Aquaculture production enhancement 5. 4 B $

Thank you Dr. 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.