on the road to a mosquito sit programme: mass rearing ......on the road to a mosquito sit programme:...

Joint FAO/IAEA Food and Agriculture Programme

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

Fabrizio Balestrino, Mark Benedict, Clélia Oliva, Hanano Yamada, Sharon Soliban, Jérémie Gilles*

Insect Pest Control Laboratory

Food and Agriculture Organization of the United Nations International Atomic Energy Agency

R&D for Mosquito SIT in Northern State, Sudan


Ideal qualities of field sites for Ideal qualities of field sites for technology technology developmentdevelopment

• 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


Food and Agriculture Organization of the United Nations International Atomic Energy Agency Food and Agriculture Organization of the United Nations International Atomic Energy Agency


Ideal qualities of field sites for Ideal qualities of field sites for technology technology developmentdevelopment

• 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


The massThe mass--rearing technologyrearing technology

Create / Develop tools for a production of 1 million sterile males per day

ObjectivesObjectives

Produce sterile males of a good quality



5 mm ABS plasticDimension: 60 x 100 cm; max 3.5 cm hsurface area/larva = 2 cm2(3000 larvae per tray; 1 larva/ml using 3 l water)2,8 - 3 Kg weight

SLOPE ( 15° ) OVERFLOW SYSTEMRIDGE ( 76 cm x 3 cm )

SLITS > WATER LEVEL

POURING OUT LIP

Larval stages



SLOPE ( 15° )

OVERFLOW SYSTEMPLATE ( 15 cm x 4 cm )

Larval stagesFood and Agriculture Organization of the United Nations International Atomic Energy Agency

The massThe mass--rearing technologyrearing technologyLarval stages


STAINLESS STEEL RACKROLLING AND TILTING

STOCK CAPACITY : 50 TRAY STACKED 3 CM DISTANCE

POTENTIAL PRODUCTIVITY:150,000 – 200,000 L4-P

TECHNICAL CHARACTERISTICS:

HEIGHT : 190 cmSTRUCTURE WEIGHT: ~ 190 KgTRAYS WEIGHT : ~ 150 KgWATER CAPACITY: 150 l - 250 lTILTING RANGE: 0° - 20° (0 – 30 cm)




ENDLESS SCREW JACK FIXED ON THE MAIN STRUCTURE (BOTTOM) AND TO A MOVEBLE FRAME (TOP)



MOVABLE FRAMEWITH PIN SUPPORTING THE SHELVES (FRONT SIDE)


SHELVESPIVOT CONNECTIONS BETWEEN THE SHELVES AND THE FIX FRAME IN THE DRAINING SIDE







The massThe mass--rearing technologyrearing technologyLarvaLarva--pupapupa separatorseparator

End of process = mixture of larvae and pupaeOnly the pupae will go for irradiation

=Separation

How????????



VORTEX BREAKER

PUPAE

LARVAE

PUPAE + LARVAE CONCENTRATED

FLOW WATER (ICE WATER)

1 MILION L4/P TO PROCESS IN 2 HNO PUPAE IN LARVAL HARVEST

GENTLE VORTEX + ICE WATER (10°C)



�

�

�

� � �

��


THERMOMETER FLOW VALVE LARVAE/PUPAE INLET

WATER TANK

PUPAE OUTLETLARVAE OUTLET







LarvaLarva--pupapupa separatorseparatorThe massThe mass--rearing technologyrearing technology



√ 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 performanceMales 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

Diet elaboration, Anopheles arabiensisDiet elaboration, Anopheles arabiensis


The massThe mass--rearing technologyrearing technologyIdentification of the potential candidatesIdentification of the potential candidates

for the for the corpuscorpus of the dietof the diet

� Koi Fish Food� Tuna Meals� Squid Liver Powder� AP100 – Shrimp meal� Brewer’s Yeast� Bovine Liver Powder� EZ Larva� Protein Selco Plus� Spirulina



Survival � Pupae and adultDuration � Pupae and adult

Wing Length

AvailibilityPrice

Distribution

Concentration “Minimalist approach” (Gilles et al. 2010, JME) � Petri dishes with 32 mL of water

� Larval density 32 L1

� Food concentration640 µL of a subset of diet concentrations (0.5, 1, 2, 4 or 8% w/v)mg of diet / mL of water / day (0.05, 0.1, 0.2, 0.4, 0.8)


The massThe mass--rearing technologyrearing technology32 young larvae (L1)

32 mL of deionised water

640 µL of diet every 2nd dayRange of concentration 0.5% to 8% (w/v)



No developmentSpirulina

No developmentProtein Selco Plus

-?-(2419)

- - -(0.02)

+ +(9.17)2%EZ Larva

-+ ++ (2583)

- - -(0.30)

+ +(8.93)2%AP100 – Shrimp meal

+ + ++ +(2650)

- - -(0.08)

+ (10.00)2%Brewer’s Yeast

-+ ++ +(2627)

+ + +(0.67)

+ + +(8.33)2%Bovine Liver Powder

+ + ++ + + (2583)

+ (0.54)

+ (10.062)2%Squid Liver Powder

+ + ++ + + (2591)

+(0.61)

+(10.12)2%Tuna Meals

+ + - - -+(2584)

+ + +(0.78)

+ +(9.85)2%Koi Fish Food

PriceAvailabilityWing length(um)

Survival Rate until

the pupal stageDuration from

L1 to pupae (days)Best

concentrationDiet compound



No developmentSpirulina

No developmentProtein Selco Plus

-?-(2419)

- - -(0.02)

+ +(9.17)2%EZ Larva

-+ ++ (2583)

- - -(0.30)


+ + ++ +(2650)

- - -(0.08)


-+ ++ +(2627)

+ + +(0.67)


+ + ++ + + (2583)

+ (0.54)


+ + ++ + + (2591)

+(0.61)


+ + - - -+(2584)

+ + +(0.78)



Survival Rate untilthe pupal stage

Duration fromL1 to pupae (days)

Best concentrationDiet compound



No developmentNo developmentNo developmentSpirulinaSpirulinaSpirulina

No developmentNo developmentNo developmentProtein Protein Protein SelcoSelcoSelco PlusPlusPlus

-?-(2419)

- - -(0.02)

+ +(9.17)2%EZ Larva

-+ ++ (2583)

- - -(0.30)


+ + ++ +(2650)

- - -(0.08)


-+ ++ +(2627)

+ + +(0.67)


+ + ++ + + (2583)

+ (0.54)


+ + ++ + + (2591)

+(0.61)


+ + - - -+(2584)

+ + +(0.78)










---???---(2419)(2419)(2419)

--- --- ---(0.02)(0.02)(0.02)

+ ++ ++ +(9.17)(9.17)(9.17)2%2%2%EZ LarvaEZ LarvaEZ Larva

---+ ++ ++ ++ + + (2583)(2583)(2583)

--- --- ---(0.30)(0.30)(0.30)

+ ++ ++ +(8.93)(8.93)(8.93)2%2%2%AP100 AP100 AP100 ––– Shrimp mealShrimp mealShrimp meal

+ + + + ++ ++ ++ ++ ++ +(2650)(2650)(2650)

--- --- ---(0.08)(0.08)(0.08)

+ + + (10.00)(10.00)(10.00)2%2%2%BrewerBrewerBrewer’’’s Yeasts Yeasts Yeast

-+ ++ +(2627)

+ + +(0.67)


+ + ++ + + (2583)

+ (0.54)


+ + ++ + + (2591)

+(0.61)


+ + - - -+(2584)

+ + +(0.78)










---???---(2419)(2419)(2419)

--- --- ---(0.02)(0.02)(0.02)


---+ ++ ++ ++ + + (2583)(2583)(2583)

--- --- ---(0.30)(0.30)(0.30)


+ + + + ++ ++ ++ ++ ++ +(2650)(2650)(2650)

--- --- ---(0.08)(0.08)(0.08)


-+ ++ +(2627)

+ + +(0.67)


+ + ++ + + (2583)

+ (0.54)


+ + ++ + + (2591)

+ (0.61)

+ (10.12)2%Tuna Meals

+ + + + + + --- --- ---+++(2584)(2584)(2584)

+ + ++ + ++ + +(0.78)(0.78)(0.78)

+ ++ ++ +(9.85)(9.85)(9.85)2%2%2%KoiKoiKoi Fish FoodFish FoodFish Food









---???---(2419)(2419)(2419)

--- --- ---(0.02)(0.02)(0.02)


---+ ++ ++ ++ + + (2583)(2583)(2583)

--- --- ---(0.30)(0.30)(0.30)


+ + + + ++ ++ ++ ++ ++ +(2650)(2650)(2650)

--- --- ---(0.08)(0.08)(0.08)


-+ ++ +(2627)

+ + +(0.67)


+ + ++ + + (2583)

+ (0.54)


+ + ++ + + (2591)

+ (0.61)

+ (10.12)2%Tuna Meals

+ + + + + + --- --- ---+++(2584)(2584)(2584)

+ + ++ + ++ + +(0.78)(0.78)(0.78)

+ ++ ++ +(9.85)(9.85)(9.85)2%2%2%KoiKoiKoi Fish FoodFish FoodFish Food





MIXTURE DESIGNFood and Agriculture Organization of the United Nations International Atomic Energy Agency


Figure 1. Mean larval survival rate (CI 95%)

Food concentration

Surv

ival r

ate

0.2

0.4

0.6

0.8

0.5 1 2 4 8

Tuna Meal Bovine Liver Powder

0.5 1 2 4 8

Squid Liver Powder

Brewer Yeast0.5 1 2 4 8

0.2

0.4

0.6

0.8

Shrimp Meal AP100

aba a

b

a

a aa ab b

b

bcb

cbcc


The massThe mass--rearing technologyrearing technologyTable 1. Larval and pupal mortality of Anopheles arabiensis reared

under different diets and food concentrations

Diet N0.5 1 2 4 8 0.5 1 2 4 8

Bovine Liver Powder 96 0.83 0.48 0.33 0.54 0.63 0.88 0.68 0.55 0.55 0.42

Squid Liver Powder 96 0.84 0.72 0.46 0.74 0.99 0.67 0.52 0.31 0.24 1.00

Tuna Meal 96 0.99 0.77 0.40 0.41 0.84 1.00 0.50 0.26 0.26 0.27

Larval Mortality Pupal Mortality Food concentration (%) Food concentration (%)


Food concentration (%)

Durat

ion (d

ays)

6

8

10

12

14

16

18

0 1 2 3 4 5 6 7 8

Figure 2. Mean time to pupation (SE) for An. arabiensis reared under different diets and food concentration.



The massThe mass--rearing technologyrearing technologyMixture design

Squid Liver Powder (SLP)

Tuna Meals

(TM)

Bovine L

iver P

owder

(BLP)



SLP

TM

BLP

(1,0,0)

(0,1,0) (0,0,1)

(1/3,1/3,1/3)(1/2, 0,1/2)(1/2,1/2,0)

(0,1/2,1/2)



SLP

TM

BLP

(1,0,0)

(0,1,0) (0,0,1)

(2/3,1/6,1/6)

(0,1/2,1/2)

(1/6,2/3,1/6) (1/6,1/6,2/3)


0.0

1.0

0.0

1.0

0.0

1.0

6.6335 6.9219 7.2103 7.4988 7.7872 8.0757 8.3641 8.6526 8.9410 9.2295 9.5179 9.8064 10.0948 10.3833 10.6717

SLP

TM BLP

Mixture Contour Plot of PD days(component proportions)

0.0

1.0

0.0

1.0

0.0

1.0

8.20 8.45 8.70 8.95 9.20 9.45 9.70 9.95 10.20 10.45 10.70 10.95 11.20 11.45 11.70

SLP

TM BLP

Mixture Contour Plot of AD Femal(component proportions)




Feeding schedule

Pupation and emergencetimes significantlyincreased at 2%...ONLY FOR MALES

1 1.5 2

0.40.5

0.60.7

0.80.9

1.0

Food

Mean

Larva

l surv

ival ra

te (±

CI)



500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0 1 2 3 4 5 6 7

1%1.50%2%

Duration (days)

Larva

lleng

th(m

icron

)


The massThe mass--rearing technologyrearing technologyTrial on Aedes albopictus





1 % 1.5 % 2 %

0.5

0.6

0.7

0.8

0.9

1.0

Food

Mean

Larva

l surv

ival ra

te (±

CI)

1 % 1.5 % 2 %

0.5

0.6

0.7

0.8

0.9

1.0

Food

Mean

Pupa

l surv

ival ra

te (±

CI)

High survival rate !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

Trial on Aedes albopictus



1 % 1.5 % 2 %

24

68

Food

Mean

pre-a

dult s

tage d

uratio

n tim

e ± SE

M (in

days

)

1 % 1.5 % 2 %

12

34

56

7

Food

Mean

larva

l stag

e dura

tion t

ime (

± SEM

)

Need more food…develop faster….



Easy to rear…Large game / kind of food…

Tolerant to high food concentration…

Aedes albopictus has also its LIMITS





Aedes albopictus has also its LIMITS





Aedes albopictus has also its LIMITS8%


The massThe mass--rearing technologyrearing technologyFor the adults

Creation of a mass rearing cageDevelop a blood feeding system

Study of the behaviour and biology of the mosquito strains


MASS PRODUCTION CAGE (MPC)



RESTING AREA



SUGAR FEEDER RESTING AREA

OVIPOSITION SITEECLOSION BOX


MASS PRODUCTION CAGE (MPC) – MPC PLASTIC VERSION


BLOOD FEEDER PLATE (HEMOTEK DEVICE MODIFICATION)


BLOOD FEEDER PLATE (HEMOTEK DEVICE MODIFICATION)


CONTAINER FOR PUPAL IRRADIATION, TRANSPORT AND RELEASE

OUTSIDE FRAME

LOCK RING

INOX (PLASTIC) NET

HOLDER CYLINDER AND LID

SIEVE

5000 P / SIEVE200 REL. STAT.17 SIEVES / HC

12 HC


CONTAINER FOR PUPAL IRRADIATION, TRANSPORT AND RELEASE


M a les fl ight performance after i rradiation(October 0 9)

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25

Time after the first record (hrs)

Mean

heigh

t (± se

m) re

ache

d by m

ales (

cm)

No irradiation 70 Gy 120 Gy

Joint FAO/IAEA Food and Agriculture Programme

Soonbovine liver

powdersquid liver

powdertuna

meals

Code Peak mg/g DW

mg/g DW

mg/g DW

* 9 R 18:0 21,5 8,7 7,3* 10 18:1(n-9) 21,0 10,5 13,4* 11 18:1(n-7) 1,3 2,5 2,5* 12 18:2(n-6)-t 0,1 0,2 0,2

* 13 R 18:2(n-6)-c 6,6 1,6 2,6* 14 19:0 0,1 0,5 0,6* 15 18:3(n-6) 0,1 0,1 0,1 *** (ACTIVE) 16 19:1(n-9) 0,0 0,1 0,1* 17 18:3(n-3) 0,8 0,7 1,1 *( semi active) 18 18:4(n-3) 0,1 0,5 0,8* 19 20:0 0,3 0,4 0,4* 20 20:1(n-9) 0,5 1,3 1,1 21 20:1(n-7) 0,2 0,1

* 22 R I.S.* 23 21:0 0,1 0,1 0,0* 24 20:3(n-6) =DGLA 1,5 0,1 0,2 *** (ACTIVE)* 25 20:4(n-6) = AA 6,8 3,1 2,3 *** (ACTIVE)* 26 20:3(n-3) 0,3 0,3 0,2 *(semi active) 27 20:4(n-3) 0,3 0,4 0,5* 28 22:0 0,6 0,3 0,3

29 R 20:5(n-3) = EPA 1,0 5,6 5,4 *** (ACTIVE)* 30 22:1(n-9) 0,2 0,2 31 22:1(n-7) 0,1 0,1* 32 23:0 0,3 0,3 0,1 33 21:5(n-3) 0,2 0,2 **** 34 23:1(n-9) 0,1 0,1 0,1* 35 22:4(n-6) 0,8 0,5 0,3* 36 22:3(n-3) 37 22:5(n-6) 0,1 1,5 1,6 38 22:4(n-3) 0,1* 39 24:0 40 22:5(n-3) =DPA 4,0 1,7 1,8 *** (ACTIVE)* 41 24:1(n-9) 0,2 0,5 0,0

* 42 R 22:6(n-3) = DHA 0,7 19,9 22,1 *** (ACTIVE)

on the road to a mosquito sit programme: mass rearing ......on the road to a mosquito sit programme:...

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