MAGANG ACTIVATE BATCH 2

Modul C.3: Pengenalan Nyamuk

dan Proses sampel

Modul C.4: Pengantar

Monitoring: Alat, Sampling, dan
Data

Jumat, 15 Juli 2022

Indah Nurhayati dan Sigit Setyawan
• TUJUAN: PENGANTAR: 1. Rearing
Rearing, Pengenalan Nyamuk, Monitoring,
– Memberikan pemahaman kepada peserta tentang tujuan dan alur
pembelajaran dalam rearing
dan Pemrosesan Sample Monitoring
– Memberikan pemahaman kepada peserta tentang sejarah Wolbachia
di lab Wolbachia
– Memberikan pemahaman kepada peserta tentang alur utama rearing
– Memberikan pemahaman kepada peserta tentang bagian-bagian
ruang laboratorium dan fungsinya
– Memberikan pemahaman kepada peserta tentang backcrossing,
outcrossing, closed population
– Memberikan pemahaman kepada peserta tentang quality assurance
dalam rearing
– Memberikan pemahaman kepada peserta tentang aktivitas di rearing,
jadwal dan pembagian peran stafnya
WMP Yogyakarta Team_Entomology Unit
(Sigit Setyawan & Indah Nurhayati)
28 Juni 2022
• MRU adalah bagian unit entomologi yang bertugas
untuk memproduksi nyamuk Aedes aegypti ber-
Wolbachia di laboratorium untuk kemudian
dilepasliarkan kembali ke alam
Denah Insektari
 SIKLUS HIDUP
NYAMUK AEDES AEGYPTI
terestrial 1-2 hari
akuatik

40-60 hari

1. Telur

2. Larva
4. Imago

• Rata-rata: 600 telur terestrial

• Jarak terbang = 200m
akuatik 5-10 hari
• Menggigit: untuk
produksi telur 2-3 hari 3. Pupa

https://www.cdc.gov/dengue/entomologyecology/m_lifecycle.html
 SEJARAH KOLONI NYAMUK AE. AEGYPTI
BER-WOLBACHIA
• Secara alami, nyamuk Ae. aegypti tidak mengandung
Wolbachia
• Metode memasukkan Wolbachia ke dalam nyamuk Ae.
aegypti:

. .. . • tekniknya sangat sulit,

• waktu yang lama,
• tingkat keberhasilannya
sangat rendah (1:100.000),

baru berhasil dilakukan

terhadap Ae. aegypti Australia
setelah bertahun-tahun
prosesnya,
1. Mikroinjeksi telur 2. Persilangan populasi
• BACKCROSSING • OPEN and CLOSED
with Bantul wild POPULATION “1O2C”
colony having Maintain the • 170 cages/3 weeks
highest insecticides adaptive colony
resistant • 3-4 millions eggs/week
• High Wolbachia
frequency (>96%)
Develop the adaptive • CLOSED POPULATION • Adaptive: good fitness
Wolbachia Ae. aegypti to be
• 24 cages and high insecticides
released in Bantul
• High quality lab resistant
colony
✔ High frequency
Wolbachia Produce numbers of
✔ High insecticides eggs enough for scale
resistant up release in Bantul
✔ Good fitness
BACKCROSSING

Guidelines: Backcrossing and outcrossing a Wolbachia colony

October 2019 Copyright © 2019 Monash University ABN 12 377 614 012
OUTCROSSING

Guidelines: Backcrossing and outcrossing a Wolbachia colony

October 2019 Copyright © 2019 Monash University ABN 12 377 614 012
 Ross et al., (2017)
Copyright © 2017 Journal of Visualized Experiments
CLOSED POPULATION

dari koloni yang sama

GENERAL ACTIVITIES
OF REARING
Pre-adult Maintenance

1. Hatching atau penetasan

telur
✔ Telur 100% Wolbachia
✔ Umur <3 bulan
✔ Larutan yeast
Pre-adult Maintenance

2. Split
✔ 24 jam setelah
penetasan
✔ Ember rearing 3,4 L
✔ Air: 1,2 L
✔ Densitas: 500
larva/ember
✔ Pakan: ¼ tsp pelet
ikan
Pre-adult Maintenance

3. Pemeliharaan larva-pupa
✔ Pakan: ¼ tsp/hari *
✔ Cek ketersediaan pakan
dan kondisi air
 Pre-adult Maintenance
4. Caging (pengandangan)
✔ Jumlah kandang vs kebutuhan produksi*
✔ 1,200 nyamuk per kandang*
✔ 3-4 gonotrophic cycles*
Adult Maintenance

1. Pemberian makan nyamuk

berupa larutan gula
● Larutan gula digunakan
sebagai sumber energi
bagi nyamuk
● Ganti 2x per minggu
Adult Maintenance

2. Pemberian pakan darah

(blood feeding)
✔ Darah manusia
dibutuhkan untuk
bertelur
✔ Satu minggu 1x
✔ Human blood feeding
✔ QA Dengue,
Chikungunya, and Zika
 Adult Maintenance
3. Eggs collection
✔ Panen 1x per minggu
⮚ Ovicup in: Senin, ovicup out: Rabu
Adult Maintenance
4. Eggs conditioning
✔ Dikeringanginkan 1 hari
 Adult Maintenance
5. Eggs storing
✔ Disimpan dalam ruang bersuhu 25 +/- 2 C

Four day old Ae. aegypti eggs under different conditions. (A) Intact eggs on sandpaper strips maintained at a high (> 80%)
humidity but without any visible moisture. Hatch rates should be above 90% for wild-type Ae. aegypti if maintained correctly. (B) Eggs that
hatch before being submerged in water (precocious hatching) are distinguished by a detached egg cap and visible larva. This indicates that the
sandpaper strip was kept too moist. (C) Eggs that are dried too harshly may collapse, and are clearly visible by their concave appearance. If the
sandpaper becomes stiff this also indicates that the eggs may be too dry. (Ross et al., 2017)

Copyright © 2017 Journal of Visualized Experiments

 Scale Up Release in Sleman and Bantul
“Production of Wolbachia Aedes aegypti”

KPI: Quality and quantity of

mosquito eggs production (3-4
MILLIONS/WEEK)

WMP Yogyakarta Team_Entomology Laboratory

 Some changes at MR…
CRCT SLEMAN BANTUL

Target release point/week 3,000 13,000 13,000

Target no. of eggs 3,000,000 -

700,000 3,000,000
produced/week 4,000,000
Strip Strip Strip
What is the end product
(150-200 eggs) (150-200 eggs) (200-250 eggs)
30x30x30 cm3 30x30x30 cm3 30x30x30 cm3
How big is each cage
BugDorm *custom *custom
How many cages need 30 100-135 150-170
How many adults in each
600 800-1200 1200
cage
How many females in each
300-350 400-480 500
cage

How may eggs are currently 24,000-28,800

18,000-21,000 30,000?
produced for each cage (fecundity 60)
 Some changes at MR…
CRCT SLEMAN BANTUL

How are you blood Human blood feeding

HBF HBF
feeding (HBF)
What % of the cages
are producing eggs 100% 100% 100%
(each week)
How many larvae per
400 600 500
rearing buckets
How many cages does 1,3 : 1 (sieved)
one bucket stock? 2,5 : 1 (sieved) 2:1 (sieved : un-sieved = 2,5 : 1 (UN-SIEVED)
(bucket : cage) 1:1)* *Updated Jun 21
3.4x4.5 m2 2(4.8*3.4) m2 2(4.8*3.4) m2
How big is the rearing (pre-adult room) (pre-adult room) (pre-adult room)
space (m2) 2.8x2.8 m2 2(3*2.8) m2 2(3*2.8) m2 + 2(3*1.5)
(adult room) (adult room) m2 (adult room)
How many FTE (Full
5+1 5+1 6+1
Time Equivalent) are
(divided into 2 groups) (divided into 2 groups) (divided into 2 groups)
assigned
ERC_MASS REARING – Yogyakarta, Indonesia (Scale Up Bantul)

To release mosquitoes each week daily:

⮚ Mass rearing in 4 parallel cohorts that start 3 weeks apart (hatch per 3 weeks: 1 open, 2 closed “1O2C”)
⮚ Activities overlap each 3 weeks
⮚ Rear for 3 gonotrophic cycles
⮚ Set up 150-170 cages? With ±1250 pupae/cage
⮚ QA colony:
1. Wolbachia% in each OPEN COLONY offspring
2. Dengue, Chikungunya, and Zika infection in each blood feeder of GC1.
 100% Wolb
WMELYOG Backcross1 WT
G31 BANTUL

7 cages
Generation
100% Wolb F1 CROSS
Closed Population (24 cages)
W F2CP
WMELYOG
100% Wolb F2CP
Open Population (59 cages)
WMELYOG F3
A1 F3 B1
closed 100% Wolb closed

F4CP
A2 C1 D1 B2
1O2C closed closed closed closed

F5CP
A3 C2 D2 B3
open open open open
open
… %Wolb? … %Wolb? … %Wolb? … %Wolb? F6

Updated: 03 Jun 22
ERC_MASS REARING – Yogyakarta, Indonesia (Scale Up Bantul)
Week 1 Week 2 Week 3 Week 4

Cohort A, Cohort B,
Gen 1 Gen 1
3 GC
MASS Hatch 60-90% to Collect
Blood-feed ERC
REARING eggs CP pupation cage eggs

Hatch Sort males, Egg

wildtype add at 10% OC colony
eggs stock

Cohort A,
Gen 2

QUALITY Insecticide Sample adults Sample blood-fed

ASSURANCE Resistance Testing mosquitoes (from
Screen samples for
each bloodfeeder)
Wolbachia frequency
Preserve adults
BC1&OC offspring only!
Screen samples for Dengue,
If Wolbachia frequency is Chikungunya, and Zika
<96%, stop rearing & cancel GC1 ONLY!
Confirm that mass-rearing process is following release. If dengue is detected in any
reaching targets and that mosquitoes Restart with another cohort. volunteers, do not use eggs from
are good quality. that cage.
Continue rearing with other eggs.
DAY WEEK 1 WEEK 2 WEEK 3 WEEK 4 WEEK 5 WEEK 6

Thu hatch bulk pupae, blood feeding blood feeding, blood feeding, store eggs,
set cage, change sugar, change sugar, kill cages
sugar pad in store eggs store eggs

Fri split, feed QA sample

Sat -

Sun -

Mon feed, change sugar, change sugar, OC in

prepare OC in OC in
cages
Tue feed,
prepare
cages
Wed check pupae, change sugar OC out OC out OC out,
feed? kill cages
 Mon Tue Wed Thu Fri Sat Sun
Change sugar ✔ ✔ - -
Blood-feed ✔ ✔ - -

Harvest eggs OC in OC out - -

Keep eggs damp ✔ ✔ - -

Slow-dry eggs ✔ ✔ - -
✔ ✔
Cut-pack & store eggs Mon&Tue Wed&Thu ✔ ✔ - -
releases releases
Updated: 10 Jun 22
Release Preparation

1. Cut egg strips

✔ Senin-Selasa
✔ Strip: 200-250
telur*
✔ 13,000 strip telur
per minggu*
Release Preparation

2. Pack egg strips

✔ Divide egg strips into release
team correspond to the ODK
system
✔ QA egg release batch
rates/week (LAB):
⮚ Randomly selected 3 strips
⮚ Count eggs on each strip
⮚ Count hatched larvae
QUALITY ASSURANCE

WOLBACHIA
RATE COLONY
MAINTENANCE

QA BUCKET
(RANDOMIZE) DENV, CHIK,
AND ZIKA
(EMERGENCE RATE)

RELEASE
MATERIAL EGG BATCH
HATCH RATE
 Modul G: Monitoring
Pengenalan alat, alur dan perekaman data
 Wolbachia Monitoring

Definisi & Tujuan:

Suatu rangkaian aktivitas

entomology dan data
manajemen yang
bertujuan untuk
mendapatkan data
entomologi yang berupa
persentase nyamuk Aedes
aegypti ber Wolbachia di
wilayah pelepasan (release
area).

Tujuan lainnya adalah

sebagai dasar untuk
menentukan adjustment
yang dapat dilakukan
berkaitan dengan proses
pelepasan nyamuk Aedes
aegypti ber Wolbachia.
 Pengenalan Alat & Cara Penggunaan

a. Biogents Sentinel Trap (BG Trap) (Gambar 1)

b. Ovitrap (Gambar 2)
c. Sweepnet (Gambar 3)
d. Aspirator (Gambar 4)
e. Prokopack (Gambar 5)
 a. Biogents Sentinel Trap (BG Trap)
•Deskripsi alat: Berbentuk tabung
dilengkapi dengan charger, kipas,
cover, akumulator, serta kantong
sampel.
•Tujuan: Alat untuk menangkap
nyamuk dewasa
•Cara kerja alat:
1. Menggunakan aliran listrik dan
backup akumulator untuk
menghidupkan alat
2. Diletakkan didalam rumah, dan
bekerja 24 jam nonstop
3. Dilengkapi dengan warna kontras
untuk menarik nyamuk datang ke
pada trap

Gambar 1. BGTrap
 b. Ovitrap
•Deskripsi alat: Terdiri dari
ember kecil yang dilengkapi
dengan kain flanel, kawat
strimin dan atraktan berupa
pelet kelinci
•Tujuan: Alat untuk menangkap
telur nyamuk Aedes aegypti
•Cara kerja alat:
1.Dipasang didalam rumah/ di
sekitar rumah
2.Diisi dengan air bersih dan
dimasukkan 1 butir pelet kelinci
sebagai atraktan
3.Koleksi telur 1 minggu setelah
dilakukan pemasangan Gambar 2. Ovitrap
 c. Sweepnet
•Deskripsi alat: Berbentuk
jaring dilengkapi dengan
gagang / pegangan
•Tujuan: Alat untuk
menangkap nyamuk dewasa
•Cara kerja alat:
1.Dirangkai gagang dan jaring,
lalu dilakukan proses sampling
dengan cara mengibaskan
jaring secara aktif untuk
menangkap nyamuk dewasa
2.Dilakukan didalam rumah
untuk target sampling Aedes
aegypti Gambar 3. Sweep Net
 d. Aspirator
•Deskripsi alat: Terdiri dari kipas
tangan yang dilengkapi dengan
cup sample, dan baterai
•Tujuan: Alat untuk menangkap
nyamuk dewasa
•Cara kerja alat:
1.Dikolaborasikan dengan
sweepnet untuk menangkap
nyamuk dewasa
2.Dengan membuka cup sample
terlebih dahulu, kemudian alat
dihidupkan dan dilakukan
penyedotan nyamuk yang
sebelumnya telah ditangkap
menggunakan sweepnet
Gambar 4. Aspirator
(modifikasi)
 e. Prokopack
•Deskripsi alat: Terdiri dari
kipas tabung dan dilengkapi
dengan cup sample, serta
akumulator
•Tujuan: Alat untuk menangkap
nyamuk dewasa
•Cara kerja alat:
1.Bekerja seperti vacum
dengan daya dari akumulator
2.Proses penangkapan dengan
cara aktif mencari nyamuk
didalam ruangan Gambar 5. Prokopack
aspirator
3.Proses penangkapan dengan
cara mengarahkan alat kepada
nyamuk yang akan ditangkap
Alur dan Waktu Pelaksanaan
 Alur persiapan logistik & koordinasi
proses monitoring
Persiapan Alat dan Bahan:
Alat yang digunakan saat ini : Seperangkat Sweepnet & Aspirator
Bahan yang dipersiapkan: Form data responden, barcode sample,
device dilengkapi dengan ODK, maps berbasis digital (avenza maps)
b. Alur koordinasi
•Koordinasi dengan PP (Pemilik Program) & PW (Pemilik Wilayah)
terkait agenda monitoring (pendataan & sampling)
•Koordinasi dengan kader & dukuh berkaitan pendataan responden
•Komunikasi dengan responden terkait waktu pelaksanaan sampling
•Proses pelaksanaan sampling (diampu oleh tim WMP)
•Integrasi proses lanjutan: pemrosesan sample (Lab), screening
Wolbachia (DU) dan Mapping hasil monitoring (DMU)
Mapping Target monitoring
Mapping / Dashboard Monitoring
 Monitoring Result vs Adjustment Release

Monitoring
Time %Wolb (plan) %Wolb (Result) Adjustment for Release
Period

1 servis ke 3 >= 35% ……….. ………..

………..
2 servis ke 7 >= 55% ………..

3 servis ke 9 >= 60% ……….. ………..

4 servis ke 11 >= 60% ……….. ………..

…. Pemrosesan Sample Monitoring
Wolbachia
SURVEILLANCE Monitor dengue activity – active fever surveillance

MAPPING & DATA Results

Operational data and mapping QA summaries
MANAGMENT
and maps

FIELD Release egg Wolbachia Collect samples QA

ENTOMOLOGY mosquitoes and service traps
QA

Identify and preserve

trap samples
QA QA
ENTOMOLOGY Mass-rear
Maintain high quality
LABORATORY mosquitoes
local Wolbachia colony
for release
QA QA

QA QA
DIAGNOSTICS Screen colony Screen colony Screen field samples
LABORATORY for dengue for Wolbachia QA
for Wolbachia
JENIS-JENIS NYAMUK UMUM DITEMUKAN
Ae. aegypti:
• Dewasa: 2 garis putih di bagian dada, sisik putih di tungkai.
• Aktif siang hari, dalam rumah.
• Vektor utama Dengue, Chikungunya, Zika.
• Suka darah manusia.
• Pradewasa: telur terpisah, menempel di wadah.

Ae. albopictus:
• Dewasa: 1 garis putih di tengah dada, tungkai tanpa sisik putih.
• Aktif siang hari, luar rumah.
• Vektor Dengue, Chikungunya, Zika (bukan utama).
• Pradewasa: telur terpisah, menempel di wadah.

Cx. quinquefasciatus:
• Dewasa: warna coklat hingga keemasan tanpa corak khusus di bagian
tubuhnya.
• Aktif malam hari.
• Vektor filariasis
• Pradewasa: telur mengelompok membentuk rakit, di permukaan air
• Dilakukan identifikasi
morfologis terhadap sampel
nyamuk dari lapangan
• Setiap sampel Ae. aegypti
ditempatkan dalam tube
khusus berisi etanol 80% dan
diberi barcode
• Dilakukan perekaman data ke
dalam Core System WMP
• Sampel dikirim ke
laboratorium diagnostik untuk
diuji kandungan Wolbachia-
nya
REFERENSI

• Ross, P.A., Axford, J.K., Richardson, K.M., Endersby-Harshman, N.M.,

Hoffmann, A.A. Maintaining Aedes aegypti Mosquitoes Infected with
Wolbachia. J. Vis. Exp. (126), e56124, doi:10.3791/56124 (2017).
• Guidelines: Backcrossing and outcrossing a Wolbachia colony; WMP:
Copyright © 2019 Monash University ABN 12 377 614 012
• Internal Training: Mosquito ID EDPY 20140204 by PHJ
• 2014.10.21_edp_medkom_gambaran-kegiatan-edp-yogya_by-enggal
• https://www.cdc.gov/dengue/entomologyecology/m_lifecycle.html
Sekian & Terimakasih
wmpyogyakarta.org
wmp-yogya@worldmosquito.org

Pusat Kedokteran Tropis FK UGM, Gedung PAU,

Jl Teknika Utara, Barek, Yogyakarta 55281

Phone: 0822 20000 385

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