On the road to a mosquito SIT programme: Mass rearing tools and quality control

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On the road to a mosquito SIT programme: Mass rearing tools and quality control R&D for Mosquito SIT in Northern State, Sudan Fabrizio Balestrino, Mark

qualities of field sites for technology development isolation from immigration narrow geographic distribution appropriate size (small enough to manage,

1 On the road to a mosquito SIT programme: Mass rearing tools and quality control R&D for Mosquito SIT in Northern State, Sudan Fabrizio Balestrino, Mark Benedict, Clélia Oliva, Hanano Yamada, Sharon Soliban, Jérémie Gilles* Insect Pest Control Laboratory Joint FAO/IAEA Food and Agriculture Programme Ideal qualities of field sites for technology development isolation from immigration narrow geographic distribution appropriate size (small enough to manage, large enough to be convincing detailed entomologic and epidemiologic information one vector good transportation & communication low density malaria transmission

Ideal qualities of field sites for technology

geographic distribution appropriate size (small enough

entomologic and epidemiologic information one vector

2 Ideal qualities of field sites for technology development isolation from immigration narrow geographic distribution appropriate size (small enough to manage, large enough to be convincing detailed entomologic and epidemiologic information one vector good transportation & communication low density malaria transmission Dongola Kasinger

Objectives Larval stages Create / Develop tools for a production of 1

3 cm ) SLITS > WATER LEVEL Produce sterile males of a good quality 5 mm ABS

5 cm h surface area/larva = 2 cm 2 (3000 larvae per tray; 1 larva/ml using

3 Objectives Larval stages Create / Develop tools for a production of 1 million sterile males per day SLOPE ( 15 ) OVERFLOW SYSTEM RIDGE ( 76 cm x 3 cm ) SLITS > WATER LEVEL Produce sterile males of a good quality 5 mm ABS plastic Dimension: 60 x 100 cm; max 3.5 cm h surface area/larva = 2 cm 2 (3000 larvae per tray; 1 larva/ml using 3 l water) 2,8 3 Kg weight POURING OUT LIP OVERFLOW SYSTEM PLATE ( 15 cm x 4 cm ) Larval stages SLOPE ( 15 ) Larval stages

STAINLESS STEEL RACK ROLLING AND TILTING STOCK

PRODUCTIVITY: 150,000 200,000 L4P TECHNICAL

~ 190 Kg TRAYS WEIGHT : ~ 150 Kg WATER CAPACITY:

SCREW JACK FIXED ON THE MAIN STRUCTURE (BOTTOM) AND

SUPPORTING THE SHELVES (FRONT SIDE) SHELVES PIVOT

4 STAINLESS STEEL RACK ROLLING AND TILTING STOCK CAPACITY : 50 TRAY STACKED 3 CM DISTANCE POTENTIAL PRODUCTIVITY: 150, ,000 L4P TECHNICAL CHARACTERISTICS: HEIGHT : 190 cm STRUCTURE WEIGHT: ~ 190 Kg TRAYS WEIGHT : ~ 150 Kg WATER CAPACITY: 150 l 250 l TILTING RANGE: 0 20 (0 30 cm) ENDLESS SCREW JACK FIXED ON THE MAIN STRUCTURE (BOTTOM) AND TO A MOVEBLE FRAME (TOP) MOVABLE FRAME WITH PIN SUPPORTING THE SHELVES (FRONT SIDE) SHELVES PIVOT CONNECTIONS BETWEEN THE SHELVES AND THE FIX FRAME IN THE DRAINING SIDE

5 Larvapupa separator End of process = mixture of larvae and pupae Only the pupae will go for irradiation = Separation How???????? PUPAE LARVAE CONCENTRATED FLOW WATER (ICE WATER) Larvapupa separator 1 MILION L4/P TO PROCESS IN 2 H NO PUPAE IN LARVAL HARVEST GENTLE VORTEX ICE WATER (10 C) PUPAE VORTEX BREAKER LARVAE

6 Larvapupa separator WATER TANK Larvapupa separator LARVAE OUTLET THERMOMETER FLOW VALVE LARVAE/PUPAE INLET PUPAE OUTLET ❷ ❺ ❽ ❸ ❾ ❶ ❹ ❻ ❼ ❿ Larvapupa separator Larvapupa separator

Larvapupa separator Diet elaboration, Anopheles arabiensis Much of the potential adult performance is determined by the diet provided in the larval stage

the sterile insect technique must be capable of mating competitively Several powdered larval diets for their capacity to support Anopheles mosquito culture

proportions of each using a mixture design Koi Fish Food Tuna Meals Squid Liver Powder AP100 Shrimp meal Identification of the potential candidates for the

7 Larvapupa separator Diet elaboration, Anopheles arabiensis Much of the potential adult performance is determined by the diet provided in the larval stage Select diets which not only allow colony keeping, but also provide necessary components for adult performance including longevity and flight performance Males being used for release e.g. the sterile insect technique must be capable of mating competitively Several powdered larval diets for their capacity to support Anopheles mosquito culture in trays Cost and global availability Stepwise selection procedure in which the principle components were selected, followed by determination of the optimal proportions of each using a mixture design Koi Fish Food Tuna Meals Squid Liver Powder AP100 Shrimp meal Identification of the potential candidates for the corpus of the diet Concentration Survival Pupae and adult Duration Pupae and adult Minimalist approach (Gilles et al. 2010, JME) Petri dishes with 32 ml of water Larval density 32 L1 Brewer s Yeast Wing Length Food concentration Bovine Liver Powder EZ Larva Availibility 640 µl of a subset of diet concentrations (0.5, 1, 2, 4 or 8% w/v) Protein Selco Plus Spirulina Price Distribution mg of diet / ml of water / day (0.05, 0.1, 0.2, 0.4, 0.8)

32 young larvae (L1) 32 ml of deionised water Diet compound Koi Fish Food Tuna Meals Best concentration Duration from L1 to pupae (days) (9.85) (10.12) Survival Rate until the pupal stage (0.78) (0.

30) EZ Larva (9.17) (0.02) (2419)? 640 µl of diet every 2nd day Range of concentration 0.

8 32 young larvae (L1) 32 ml of deionised water Diet compound Koi Fish Food Tuna Meals Best concentration Duration from L1 to pupae (days) (9.85) (10.12) Survival Rate until the pupal stage (0.78) (0.61) Wing length (um) (2584) (2591) Availability Price Squid Liver Powder (10.062) (0.54) Bovine Liver Powder (8.33) (0.67) (2627) Brewer s Yeast (10.00) (0.08) (2650) AP100 Shrimp meal (8.93) (0.30) EZ Larva (9.17) (0.02) (2419)? 640 µl of diet every 2nd day Range of concentration 0.5% to 8% (w/v) Protein Selco Plus Spirulina Diet compound Koi Fish Food Tuna Meals Squid Liver Powder Bovine Liver Powder Brewer s Yeast AP100 Shrimp meal EZ Larva Best concentration Duration from L1 to pupae (days) (9.85) (10.12) (10.062) (8.33) (10.00) (8.93) (9.17) Survival Rate until the pupal stage (0.78) (0.61) (0.54) (0.67) (0.08) (0.30) (0.02) Wing length (um) (2584) (2591) (2627) (2650) (2419) Availability? Price Diet compound Koi Fish Food Tuna Meals Squid Liver Powder Bovine Liver Powder Brewer s Yeast AP100 Shrimp meal EZ Larva Best concentration Duration from L1 to pupae (days) (9.85) (10.12) (10.062) (8.33) (10.00) (8.93) (9.17) Survival Rate until the pupal stage (0.78) (0.61) (0.54) (0.67) (0.08) (0.30) (0.02) Wing length (um) (2584) (2591) (2627) (2650) (2419) Availability? Price Protein Selco Plus Protein Selco Plus Spirulina Spirulina

9 Diet compound Koi Fish Food Tuna Meals Squid Liver Powder Bovine Liver Powder Brewer s s Yeast AP100 Shrimp meal EZ Larva Best concentration Duration from L1 to pupae (days) (9.85) (10.12) (10.062) (8.33) (10.00) (8.93) (9.17) Survival Rate until the pupal stage (0.78) (0.61) (0.54) (0.67) (0.08) (0.30) (0.02) Wing length (um) (2584) (2591) (2627) (2650) (2419) Availability? Price Diet compound Koi Fish Food Tuna Meals Squid Liver Powder Bovine Liver Powder Brewer s s Yeast AP100 Shrimp meal EZ Larva Best concentration Duration from L1 to pupae (days) (9.85) (10.12) (10.06) (8.33) (10.00) (8.93) (9.17) Survival Rate until the pupal stage (0.78) (0.61) (0.54) (0.67) (0.08) (0.30) (0.02) Wing length (um) (2584) (2591) (2627) (2650) (2419) Availability? Price Protein Selco Plus Protein Selco Plus Spirulina Spirulina Brewer Yeast Shrimp Meal AP100 Diet compound Best concentration Duration from L1 to pupae (days) Survival Rate until the pupal stage Wing length (um) Availability Price 0.8 Koi Fish Food Tuna Meals Squid Liver Powder Bovine Liver Powder Brewer s s Yeast AP100 Shrimp meal EZ Larva Protein Selco Plus Spirulina (9.85) (10.12) (10.06) (8.33) (10.00) (8.93) (0.78) (0.61) (0.54) (0.67) (0.08) (0.30) (2584) (2591) (2627) (2650) (9.17) (0.02) (2419) MIXTURE DESIGN? Figure 1. Mean larval survival rate (CI 95%) Survival rate a Tuna Meal c c b b a a ab b Bovine Liver Powder c bc b b a Squid Liver Powder b a a a Food concentration

10 Table 1. Larval and pupal mortality of Anopheles arabiensis reared under different diets and food concentrations Larval Mortality Pupal Mortality Diet N Food concentration (%) Food concentration (%) Bovine Liver Powder Squid Liver Powder Tuna Meal Duration (days) Food concentration (%) Figure 2. Mean time to pupation (SE) for An. arabiensis reared under different diets and food concentration. Mixture design Squid Liver Powder (SLP) Mixture design SLP (1,0,0) (1/2,1/2,0) (1/3,1/3,1/3) (1/2, 0,1/2) Tuna Meals (TM) Bovine Liver Powder (BLP) TM (0,1,0) (0,1/2,1/2) (0,0,1) BLP

11 Mixture design Mixture design SLP (1,0,0) Mixture Contour Plot of PD days (component proportions) Mixture Contour Plot of AD Femal (component proportions) TM (0,1,0) (2/3,1/6,1/6) (1/6,2/3,1/6) (1/6,1/6,2/3) (0,1/2,1/2) (0,0,1) BLP 1.0 TM 0.0 SLP BLP TM 0.0 SLP BLP Mean Larval survival rate (± CI) Feeding schedule Pupation and emergence times significantly increased at...only FOR MALES Larval length (micron) % % Food Duration (days)

Trial on Aedes albopictus Trial on Aedes albopictus Trial on Aedes albopictus

0 1 % 1.5 % 2 % Food Mean Pupal survival rate (± CI) 0.5 0.6 0.7 0.8 0.9 1.

5 % 2 % Food Mean preadult stage duration time ± SEM (in days) 2 4 6 8 1 % 1.

12 Trial on Aedes albopictus Trial on Aedes albopictus Trial on Aedes albopictus Trial on Aedes albopictus Mean Larval survival rate (± CI) % 1.5 % 2 % Food Mean Pupal survival rate (± CI) % 1.5 % 2 % Food Mean larval stage duration time (± SEM) % 1.5 % 2 % Food Mean preadult stage duration time ± SEM (in days) % 1.5 % 2 % Food High survival rate!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!! Need more food develop faster.

13 Trial on Aedes albopictus Easy to rear Large game / kind of food Tolerant to high food concentration Aedes albopictus has also its LIMITS Trial on Aedes albopictus Easy to rear Large game / kind of food Tolerant to high food concentration Aedes albopictus has also its LIMITS Trial on Aedes albopictus Easy to rear Large game / kind of food 8% Tolerant to high food concentration Aedes albopictus has also its LIMITS For the adults Creation of a mass rearing cage Develop a blood feeding system Study of the behaviour and biology of the mosquito strains

14 MASS PRODUCTION CAGE (MPC) MASS PRODUCTION CAGE (MPC) RESTING AREA MASS PRODUCTION CAGE (MPC) MASS PRODUCTION CAGE (MPC) MPC PLASTIC VERSION SUGAR FEEDER RESTING AREA ECLOSION BOX OVIPOSITION SITE

15 BLOOD FEEDER PLATE (HEMOTEK DEVICE MODIFICATION) BLOOD FEEDER PLATE (HEMOTEK DEVICE MODIFICATION) CONTAINER FOR PUPAL IRRADIATION, TRANSPORT AND RELEASE CONTAINER FOR PUPAL IRRADIATION, TRANSPORT AND RELEASE HOLDER CYLINDER AND LID 5000 P / SIEVE 200 REL. STAT. 17 SIEVES / HC 12 HC INOX (PLASTIC) NET LOCK RING OUTSIDE FRAME Food and Agriculture Organization SIEVEof the United Nations

Soon Males flight performance after irradiation (October 09) bovine liver powder squid liver powder tuna meals Mean height (± sem) reached by males (cm) No irradiation 70 Gy 120 Gy 160 140 120 100 80

0,2 * 13 R 18:2(n6)c 6,6 1,6 2,6 * 14 19:0 0,1 0,5 0,6 * 15 18:3(n6) 0,1 0,1 0,1 *** (ACTIVE) 16 19:1(n9) 0,0 0,1 0,1 * 17 18:3(n3) 0,8 0,7 1,1 *( semi active) 18 18:4(n3) 0,1 0,5 0,8 * 19 20:0 0,3

16 Soon Males flight performance after irradiation (October 09) bovine liver powder squid liver powder tuna meals Mean height (± sem) reached by males (cm) No irradiation 70 Gy 120 Gy Time after the first record (hrs) Code Peak mg/ mg/ mg/ g DW g DW g DW * 9 R 18:0 21,5 8,7 7,3 * 10 18:1(n9) 21,0 10,5 13,4 * 11 18:1(n7) 1,3 2,5 2,5 * 12 18:2(n6)t 0,1 0,2 0,2 * 13 R 18:2(n6)c 6,6 1,6 2,6 * 14 19:0 0,1 0,5 0,6 * 15 18:3(n6) 0,1 0,1 0,1 *** (ACTIVE) 16 19:1(n9) 0,0 0,1 0,1 * 17 18:3(n3) 0,8 0,7 1,1 *( semi active) 18 18:4(n3) 0,1 0,5 0,8 * 19 20:0 0,3 0,4 0,4 * 20 20:1(n9) 0,5 1,3 1, :1(n7) 0,2 0,1 * 22 R I.S. * 23 21:0 0,1 0,1 0,0 * 24 20:3(n6) =DGLA 1,5 0,1 0,2 *** (ACTIVE) * 25 20:4(n6) = AA 6,8 3,1 2,3 *** (ACTIVE) * 26 20:3(n3) 0,3 0,3 0,2 *(semi active) 27 20:4(n3) 0,3 0,4 0,5 * 28 22:0 0,6 0,3 0,3 29 R 20:5(n3) = EPA 1,0 5,6 5,4 *** (ACTIVE) * 30 22:1(n9) 0,2 0, :1(n7) 0,1 0,1 * 32 23:0 0,3 0,3 0, :5(n3) 0,2 0,2 *** * 34 23:1(n9) 0,1 0,1 0,1 * 35 22:4(n6) 0,8 0,5 0,3 Joint FAO/IAEA Food and Agriculture Programme * 36 22:3(n3) 37 22:5(n6) 0,1 1,5 1, :4(n3) 0,1 * 39 24: :5(n3) =DPA 4,0 1,7 1,8 *** (ACTIVE) * 41 24:1(n9) 0,2 0,5 0,0 * 42 R 22:6(n3) = DHA 0,7 19,9 22,1 *** (ACTIVE)

Sterile Insect Technique (SIT): Perspectives for the management of Aedes mosquitoes in the region of the Americas

Sterile Insect Technique (SIT): Perspectives for the management of Aedes mosquitoes in the region of the Americas Rui Cardoso Pereira Insect Pest Control Sub-programme, Joint FAO/IAEA Programme of Nuclear