Identification of Pheromone Binding Protein Gene of Yellow Rice Stem Borer Scirpophaga Incertulas (Walker) (Lepidoptera: Crambidae)

 
 

IDENTIFICATION OF PHEROMONE BINDING PROTEIN
GENE OF YELLOW RICE STEM BORER
Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae)
 
 
 
JAZIROTUL FITRIYATI
 
 
 
 

 
 
 
 

GRADUATE SCHOOL

BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2011

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DECLARATION
I declare that this thesis entitled “Identification of Pheromone Binding Protein
Gene of Yellow Rice Stem Borer Scirpophaga Incertulas (Walker)
(Lepidoptera: Crambidae)” was entirely completed by myself with resourceful
help from the Department of Biology, Bogor Agricultural University. Information
and quotes which were sourced from journals and books have been acknowledged
and mentioned where in the thesis they appear. All complete references are given

at the end of the paper.

Bogor, August 2011

Jazirotul Fitriyati
Reg Number: G352090151

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ABSTRACT
JAZIROTUL FITRIYATI. G352090151. Identification of Pheromone Binding

Protein Gene of Yellow Rice Stem Borer Scirpophaga Incertulas (Walker)
(Lepidoptera: Crambidae) (Supervised by RIKA RAFFIUDIN and I MADE
SAMUDRA)

The yellow rice stem borers (YRSB) moth, Scirpophaga incertulas
(Walker) (Lepidoptera: Crambidae), is known as the most major rice stem borer in
tropical Asia. Pesticides are not effective to control the population of these insects
due to almost entire larvae phase and pupae are in the rice stem. Hence, other
control technique is needed such as based on the mating behaviour. Pheromone
binding protein (PBP) in male S. incertulas antennae plays a role in the
recognition of sex pheromone produced by the female, therefore influenced in
their mating behavior. The aim of this study was to identify PBP gene of S.
incertulas. For the purpose to collect imagoes as DNA source, S. incertulas were
reared. Touchdown PCR and touchdown-nested PCR were the main techniques
conducted to identify genomic of PBP gene from S. incertulas and revealed 700
and 600 bp amplicons, respectively. Those amplicons strongly expected as PBP
gene. Sequence analysis of S. incertulas from touchdown-nested amplicon
identified 575 bp which was consisted of 169 bp of exon 3 and 406 bp of intron 2.
This study revealed putative amino acid sequences of exon 3 from S. incertulas
has one conserved cysteine while other Lepidopterans PBP have three conserved
cysteine. In phylogenetic analysis, the putative amino acid sequences obtained,
showed a phylogenetic signal i.e. by clustering with PBPs from other Crambidae
moths. The result of this study is important as a basic data for PBP expression

analysis in female or male S. incertulas as the initial step to develope new insect
biocontrol.
Keywords: moth, sex pheromone, mating behavior, touchdown-nested PCR,
conserved cysteine

 
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ABSTRAK
JAZIROTUL FITRIYATI. G352090151. Identifikasi Gen Pheromone Binding

protein pada Penggerek Batang Padi Kuning Scirpophaga incertulas
(Walker) (Lepidoptera: Crambidae). (Dibimbing oleh RIKA RAFFIUDIN and
I MADE SAMUDRA)

Ngengat Penggerek Batang Padi Kuning (PBPK) Scirpophaga incertulas
(Walker) (Lepidoptera: Crambidae), diketahui sebagai hama penggerek batang
padi utama di kawasan Asia tropis. Penggunaan pestisida tidak efektif untuk
mengendalikan populasi serangga ini karena hampir seluruh fase larva dan pupa
serangga ini berada di dalam batang. Oleh karena itu, teknik pengendalian lain
diperlukan dalam pengendalian PBPK, seperti teknik pengendalian berdasarkan
perilaku kawin. Pheromone binding protein (PBP) pada antena S. incertulas
jantan mempunyai peranan dalam mengenali feromon sex yang dihasilkan oleh
betina sehingga mempengaruhi perilaku kawin mereka. Tujuan dari penelitian ini
adalah untuk mengidentifikasi gen PBP pada S. incertulas. Dilakukan
pengembangbiakkan S. incertulas untuk mendapatkan imago sebagai sumber
DNA. Touchdown PCR dan touchdown-nested PCR merupakan teknik amplifikasi
utama yang dilakukan untuk mengidentifikasi data genom dari gen PBP pada S.
incertulas dan menghasilkan dua amplikon berukuran 700 dan 600 pb berturutturut. Kedua amplikon tersebut diduga kuat sebagai target dari gen PBP. Analisis
sekuen dari S. incertulas dari amplikon hasil touchdown-nested dihasilkan sekuen
berukuran 575 pb yang terdiri atas bagian ekson 3 (169 pb) dan intron 2 (406
pb). Penelitian ini menemukan sekuen asam amino putatif bagian ekson 3 PBP
dari S. incertulas hanya memiliki satu sistein konservatif sedangkan PBP dari
Lepidoptera lain memiliki tiga sistein konservatif. Dalam analisis filogenetik,
sekuen asam amino putatif yang dihasilkan menunjukkan adannya sinyal

filogenetik dengan mengelompok bersama PBP dari ngengat Famili Crambidae.
Hasil penelitian ini sangat penting untuk digunakan sebagai data dasar untuk
mempelajari analisis ekspresi PBP pada S. incertulas jantan dan betina sebagai
langkah awal mengembangkan teknik baru pengendalian serangga hama.
Keywords: ngengat, feromon sex, perilaku kawin, touchdown-nested PCR, sistein
konservatif

 
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SUMMARY

JAZIROTUL FITRIYATI. G352090151. Identification of Pheromone Binding

Protein Gene of Yellow Rice Stem Borer Scirpophaga Incertulas (Walker)
(Lepidoptera: Crambidae) (Supervised by RIKA RAFFIUDIN and I MADE

SAMUDRA)

The yellow rice stem borers (YRSB) moth, Scirpophaga incertulas
(Walker) (Lepidoptera: Crambidae), is known as the most major rice stem borer
pest in tropical Asia. In China, India, and Southeast Asia, annual losses to yellow
rice stem borers average 5 to 10%, but losses in individual fields may reach 5060%. In Karawang, Indonesia, S. incertulas is dominant pest among other rice
stem borers, and it caused 20,5% losses.
S. incertulas is monophagous in rice, its larva bore into the rice stem and
complete the larva and pupa stages within the rice stem. During development,
larva feed voraciously causing tremendous yield losses. Larva of S. incertulas are
difficult to be controlled with insecticides because after hatching, the larva are
exposed only for a few hours before they enter the rice stem. In addition, the use
of insecticides can contaminate rice field and diminish the other non-target insect
such as pollinator and predator.
Currently, Sustainable biocontrol such as mating disruption has
implemented to control the population of S. Incertulas moth. Artificial sex
pheromone trap as a famous example of mating disruption that using the analogue
of sex pheromone which is produce by female moth to attract the male come to
the trap. This method can lower the copulation behavior hence the pest population
will decrease. The idea of artificial sex pheromone trap is influenced by natural

mating behaviour of moth that using sex pheromone as communication signal.
Female moth produce an airbone signal consisting of a single chemical
compounds or a particular blend of compounds called sex pheromone. Moreover,
male moth has pheromone binding protein (PBP) that selectively binds the sex
pheromone release from the female moth for a signal to find the female position to
mate.
PBP play a role in the recognition of sex pheromone in male moth
antennae. By binding selectively to different components of pheromone blends,
therefore, PBP can be a selective filter to bind the sex pheromone. Identification
of PBP gene in S. incertulas is needed as an initial step to developing new insect
biocontrol. Based on other species of Lepidoptera, PBP gene have two introns and
three coding regions (exons); however, none was reported from S. incertulas.
Male and female imagoes of S. incertulas from Bogor were collected on
August 30, 2010, whereas imagoes collection from Karawang was done on
September 27-28, 2010. Male and female imagoes of S. incertulas were reared
two periods for the purpose to collect imagoes as DNA source, first during
September 6th - October 26th 2010 and the second during October 4th –
November 15th 2010 . The first rearing used male and female parental imagoes of
S. incertulas that were collected from Bogor and the second used those from
Karawang. both were conducted in greenhouses. Subsequently, Total DNA of S.

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incertulas was extracted using a Phenol/Chloroform method and amplification of
PBP gene in S. incertulas was generated using eight degenerate primers. Five
polymerase chain reactions (PCR) methods were used to amplify this gene and to
improve the quality of generated PCR product, i.e. (i) Standard PCR, (ii)
polyacrilamide gel cutting – re-PCR, (iii) nested PCR, (iv) touchdown PCR, and
(v) touchdown-nested PCR. DNA sequencing and Phylogenetic analysis were
done to identify PBP gene of S. incertulas.
Fifty seven S. incertulas imagoes emerged from the first rearing (parental
collection from Bogor), seven male imagoes and 50 female imagoes. Whereas
second rearing (parental collection from Karawang) emerged more number of
imagoes than the first rearing, i.e. 88 imagoes consisted of 47 males and 41
females. Rain exposure was predicted as key factor that increased larval mortality
rates during larva inoculation, hence emerged less imagoes than the second one.
This is due to the first rearing was conducted in greenhouse covered with net roof;
hence all first rearing process was exposed by rain. The second rearing was
conducted in greenhouse with fiber roof, hence unexposed with rain. Life cycle

span of S. incertulas from the first rearing was longer (48-56 days) than second
rearing (44-51 days).
Although degenerate primers were designed in conserved region in
Lepidopteran PBP genes, multi amplicons using standard PCR was produced.
Polyacrilamide gel cutting-rePCR method revealed a single fragment from multi
amplicons; however, those did not show the PBP features based on BLASTN
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis hence, the cut amplicon was not
the PBP target. Nested PCR still could not reduce non target fragments
represented by multi amplicons having same pattern as amplicon with the same
primers in standard PCR. This might be due to amplicon from standard PCR as
DNA template in nested PCR, has several unselected fragments and caused
misleading amplification when it was used in nested PCR as a DNA template. The
last two PCR techniques touchdown PCR and touchdown-nested PCR gave one
dominant fragment strongly expected as PBP target with 700 bp and 600 bp sizes,
respectively.
There were six sequences were obtained from six amplicons by using three
PCR modification techniques: (i) polyacrilamide gel cutting – rePCR method (two
sequences: 217 and 235 bp), (ii) nested PCR (three sequences: 265, 317, One
sequence from 700 bp fragment was unreadable sequence), (iii) touchdown-nested
PCR (one sequence: 575 bp). There was no open reading frame (ORF) aswell as

found in four sequences from polyacrilamide gel cutting – rePCR amplicons and
nested PCR amplicons. Result of four sequences from amplicons in point (i) and
(ii) did not show the PBP features based on BLASTN analysis. However, 575 bp
sequence from touchdown-nested PCR approach was identified having 169 bo
ORF (named Since_PBPexpected) for 56 amino acids and 406 bp intron region.
That open reading frame was predicted as exon 3 of PBP expected sequence from
S. incertulas based on sequencing primer was designed to flank exon 3 region in
O. furnacalis PBP sequence and the intron was predicted as intron 2. The 3’end of
the intron had typical AG sequence. BLASTP (http://blast.ncbi.nlm.nih.gov/Blast.cgi) result of 56 amino acids sequence of Since_PBPexpected showed that
it has homology structure with General Odoran Binding Protein (GOBP) gene of
honey bee Apis mellifera. GOBPs and PBPs, both is member of the large family

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gene, Odorant Binding Proteins (OBPs). The characteristic of PBP amino acid
sequene are presence of six conserved cysteine residues, and so does the GOBP;
therefore, BLASTP result obtained homology with GOBP. Might be due to A.
mellifera is the second most complete genome data of insect; therefore,
characterization of novel genomic data in insect will always hit to A. mellifera.
Alignment of the third exon putative amino acid sequencess PBPs among
species showed there were three conserved cysteine residues, however,
Since_PBPexpected amino acid only showed one concerved cysteine.
Since_PBPexpected can be classified into PBP group by phylogenetic analysis
and confirmed have a closest relationship with other Crambidae’s PBP (Ostrinia
furnacalis and Ostrinia nubilalis). It has nearest distance with PBP of O.
furnacalis (distance = 3.366) and PBP of O. nubilalis (distance = 3,366). Three
disulfide bridges and hydrophobic pocket in PBP structure are formed by six
conserved cysteine. Out of six conserved cysteine in PBP, three are located in
exon 3 hence this region is more conserved than the other exons in PBP.
Accordingly, even only one conserved cysteine was found in third exon of
Since_PBPexpected, this sequence still gave phylogenetic signal by clustering
with other PBPs of Crambidae.

 
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RINGKASAN

JAZIROTUL FITRIYATI. G352090151. Identifikasi Gen Pheromone Binding

protein pada Penggerek Batang Padi Kuning Scirpophaga incertulas
(Walker) (Lepidoptera: Crambidae). (Dibimbing oleh RIKA RAFFIUDIN dan
I MADE SAMUDRA)

Ngengat penggerek batang padi kuning (PBPK), Scirpophaga incertulas

(Walker) (Lepidoptera: Crambidae), diketahui sebagai hama penggerek batang
padi yang utama di kawasan Asia tropis. Di Cina, India, dan Asia Tenggara,
persentase kehilangan hasil panen karena PBPK rata-rata 5-10% per tahun, namun
kehilangan hasil panen karena PBPK pada sawah individual dapat mencapai 5060%. Di Karawang, Indonesia, S. incertulas merupakan hama penggerek batang
padi yang paling dominan dibandingkan dengan hama penggerek lainnya, dan
kerugian yang ditimbulkan akibat hama ini mencapai 20,5%.
S. incertulas merupakan serangga monofag pada padi, larva dari S.
incertulas akan menggerek ke dalam batang padi dan menyelesaikan fase larva
dan pupa di dalam batang padi. Selama masa perkembangan, larva memakan
batang padi dengan sangat rakus sehingga dapat menimbulkan kehilangan
produksi gabah yang signifikan. Larva dari S. incertulas sulit dikendalikan
menggunakan insektisida karena segera setelah menetas, larva hanya akan
terekspose beberapa jam di luar lalu akan segera memasuki batang. Selain itu,
penggunaan insektisida secara berlebihan dapat menimbulkan terjadinya
kontaminasi pada sawah dan dapat membunuh serangga-serangga lain yang
menguntungkan seperti predator atau musuh alaminya.
Saat ini, penggunaan teknik pengendalian hama yang berkelanjutan seperti
Penggangguan proses kawin telah dilakukan untuk mengendalikan populasi
ngengat S. Incertulas. Perangkap feromon sex buatan merupaka salah satu metode
penggangu proses kawin yang paling dikenal. Perangkap ini menggunakan analog
dari feromon sex yang dihasilkan oleh ngengat betina untuk menarik ngengat
jantan untuk mendatangi perangkap. Metode pengendalian ini dapat menurunkan
perilaku kawin sehingga dapat menurunkan populasi hama generasi berikutnya.
Gagasan pembuatan perangkap ini di dasari dari perilaku kawin ngengat secara
alami di alam yang menggunakan feromon sex sebagai sinyal komunikasi.
Ngengat betina menghasilkan sinyal mudah menguap yang terdiri dari senyawa
kimia tunggal atau campuran dari senyawa tertentu yang disebut dengan feromon
sex. Sedangkan pada ngengat jantan memiliki pheromone binding protein (PBP)
yang akan mengikat secara selektif feromon sex yang dihasilkan ngengat betina
sebagai sinyal untuk menemukan posisi betina lalu melakukan kopulasi (kawin).
PBP yang terdapat pada antena ngengat jantan memainkan peranan untuk
mengenali feromon sex yang dihasilkan oleh ngengat betina. Kemampuan PBP
untuk mengikat secara selektif komponen berbeda dalam campuran feromon sex
sehingga PBP dapat digunakan sebagai penyeleksi untuk mengikat feromon sex
yang tepat. Identifikasi gen PBP pada S. incertulas diperlukan sebagai langkah
awal untuk mengembangkan metode baru pengendalian hama. Berdasarkan

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beberapa spesies dalam Lepidoptera, gen PBP memiliki dua intron dan tiga exon,
namun belum terdapat data tentang gen PBP pada S. incertulas.
Imago S. incertulas jantan dan betina dikembangbiakkan sebanyak dua
kali dengan tujuan untuk mendapatkan imago sebagai sumber DNA.
Pengembangbiakkan S. incertulas yang pertama dilaksanakan pada tanggal 6
September – 26 Oktober 2010 dan pengembangbiakkan yang kedua dilaksanakan
pada tanggal 4 Oktober – 15 November 2010. Pengembangbiakkan pertama
menggunakan imago indukan yang berasal dari Bogor sedangkan yang kedua
menggunakan imago indukan yang berasal dari Karawang. Proses
pengembangbiakkan S. incertulas dilaksanakan dalam rumah kaca. Selanjutnya
DNA total dari S. incertulas diekstraksi menggunakan metode fenol/kloroform
dan Amplifikasi gen PBP pada S. incertulas dilakukan dengan menggunakan
primer degenerate. Sebanyak lima metode polymerase chain reaction (PCR)
dilakukan untuk meningkatkan kualitas produk PCR (amplikon) yang dihasilkan,
yaitu: (i) standard PCR , (ii) polyacrilamide gel cutting-rePCR, (iii) nested PCR,
(iv) touchdown PCR, (v) touchdown-nested PCR.
Sebanyak 57 imago dihasilkan dari proses pengembangbiakkan yang
pertama dengan rincian yaitu tujuh imago jantan dan 50 imago betina. Sedangkan
hasil pengembanbiakkan S. incertulas yang kedua didapatkan 88 imago dengan
rincian 47 jantan dan 41 betina. Paparan hujan diduga sebagai factor kunci yang
meningkatkan laju mortalitas larva pada saat inokulasi larva, sehingga pada
pengembangbiakkan yang pertama dihasilkan jumlah imago yang lebih sedikit
dibandingkan dengan yang kedua. Hal ini disebabkan karena pengembangbiakkan
tahap pertama dilakukan pada rumah kaca yang atapnya terbuat dari jaring
sehingga air hujan dapat masuk ke dalam. Pengembangbiakkan S. incertulas yang
kedua dilakukan pada rumah kaca yang atapnya terbuat dari fiber sehingga air
hujan tidak dapat masuk ke dalam rumah kaca. Lama siklus hidup S. incertulas
dari pengembangbiakkan tahap pertama lebih lama (48-56 hari) dibandingkan
pengembangbiakkan tahap kedua (44-51 hari).
Posisi beberapa primer degenerate dalam penelitian ini sudah didesain
berada pada bagian paling konservatif di gen PBP dari anggota Lepidoptera
namun amplikon dengan banyak fragmen tetap dihasilkan oleh metode standard
PCR. Metode polyacrilamide gel cutting-rePCR telah berhasil menghasilkan
fragment tunggal dari amplikon dengan banyak fragmen. Hasil sekuen dari
amplikon tersebut tidak menunjukkan adanya karakteristik dari gen PBP
berdasarkan hasil aligment sekuen tersebut dengan data sekuen PBP lain pada
Genbank menggunakan BLASTN (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Hal ini
dapat disebabkan oleh fragmen yang dipotong bukan merupakan amplikon target
dari gen PBP. Metode nested PCR yang dilakukan seharusnya mengurangi
fragmen-fragmen non-target, namun pada penelitian ini terlihat bahwa metode
nested PCR kurang efektif mengurangi kemunculan fragmen-fragmen non-target.
Hal ini dapat terlihat dari dihasilkannya tiga fragmen yang memiliki pola sama
dengan amplikon pada standard PCR dengan menggunakan pasangan primer
yang sama. Hal ini dapat disebabkan karena cetakan DNA yang berasal dari
amplikon standard PCR belum terseleksi, sehingga saat dilakukan PCR ulang
menggunakan primer internal (nested) menyebabkan terjadinya misleading
amplifikasi. Dari kelima metode PCR yang digunakan dalam penelitian ini, dua
metode PCR dapat memberikan amplikon dengan satu fragmen dominan yang

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diduga kuat sebagai amplikon target gen PBP yaitu touchdown PCR (700 pb) dan
touchdown-nested PCR (600 pb).
Terdapat enam sekuen yang dihasilkan dari enam amplikon yan
menggunakan tiga metode PCR yang berbeda: (i) metode polyacrilamide gel
cutting-rePCR (dua sekuen: 217 pb (primer F2.1-R3.1) dan 235 bp (primers F2.2R3.1), (ii) nested PCR (tiga sekuen: 265 pb, 317 pb, dan satu sekuen dari
amplikon berukuran 700 pb tidak dapat terbaca), (iii) touchdown-nested PCR
(satu sekuen: 575 bp). Pada 4 hasil sekuen dari amplikon metode polyacrilamide
gel cutting-rePCR dan nested PCR tidak ditemukan adanya Open Reading Frame
(ORF). Hasil BLASTN dari keempat sekuen tersebut juga tidak menunjukkan
adanya karakteristik gen PBP pada sekuen tersebut. Amplikon dari metode
touchdown-nested PCR dapat menghasilkan sekuen sepanjang 575 pb dari S.
incertulas (dinamakan Since_PBPexpected) yang terdiri dari ORF sepanjang 169
pb (56 asam amino) dan bagian intron sepanjang 406 pb. Bagian ORF sekuen
tersebut diduga sebagai bagian dari ekson 3 dari gen PBP, sedangkan bagian
intron yang teridentifikasi diduga sebagai bagian intron 2 dari gen PBP pada S.
incertulas. Ujung 3’ pada bagian intron dari sekuen ini memiliki struktur AG yang
menunjukkan
batas
antara
intron
dan
ekson.
Hasil
BLASTP
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) dari 56 asam amino hasil translasi dari
ORF pada sekuen ini menujukkan bahwa sekuen asam amino tersebut memiliki
homologi dengan gen General Odorant Binding Protein (GOBP) pada Apis
mellifera. GOBP dan OBP merupakan anggota dari family gen Odorant Binding
Protein (OBP). Karakteristik adanya enam asam amino sistein konservatif yang
terdapat pada PBP juga terdapat pada GOBP. Karakteristik yang dimiliki bersama
oleh PBP dan GOBP ini diduga sebagai penyebab hasil BLASTP dari sekuen
asam amino Since_PBPexpected memiliki homologi dengan GOBP. Selain itu, A.
mellifera merupakan serangga dengan data genom terlengkap kedua di Genbank,
hal ini yang menyebabkan karakterisasi data genom baru dari serangga akan
sering mengacu pada A. mellifera.
Alignment dari sekuen asam amino ekson ketiga pada gen PBP dari
berbagai spesies menunjukkan adanya enam sistein konservatif, namun, sekuen
asam amino Since_PBPexpected hanya memiliki satu sistein konservatif.
Berdasarkan analisis filogenetik, Since_PBPexpected dapat diklasifikasikan ke
dalam kelompok PBP dan memiliki kekerabatan paling dekat dengan gen PBP
pada Ostrinia furnacalis dan Ostrinia nubilalis. Since_PBPexpected memiliki
jarak genetik terdekat dengan gen PBP dari O. furnacalis (distance = 3,366) dan
O. nubilalis (distance : 3,366). Tiga ikatan disulfide dan kantong hidrofobik pada
gen PBP dibentuk oleh adanya enam asam amino sistein konservatif. Dari keenam
asam amino sistein konservatif tersebut, tiga diantaranya berada pada ekson
ketiga. Hal ini menyebabkan bagian ekson tiga lebih konsrvatif dibandingkan
dengan ekson lainnya pada gen PBP. Oleh karena itu, meskipun pada
Since_PBPexpected hanya ditemukan satu sitein konservatif, namun tetap
memberikan sinyal filogentik yang mengelompok bersama gen PBP lain pada
anggota satu famili Crambidae.

 
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© Copyright of IPB, year 2010

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IDENTIFICATION OF PHEROMONE BINDING PROTEIN
GENE OF YELLOW RICE STEM BORER
Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae)

JAZIROTUL FITRIYATI

A Thesis
As Partial fulfillment of the Requirement to obtain
Master of Science Degree in Animal Biosciences

GRADUATE SCHOOL
BOGOR AGRICULTURAL UNIVERSITY
BOGOR
2011

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External Member Supervisor Committee:
Dr. Ir. Purnama Hidayat, M.Sc

 xix
 

Title

: Identification of Pheromone Binding Protein Gene of
Yellow Rice Stem Borer Scirpophaga incertulas
(Walker) (Lepidoptera:Crambidae)

Name

: Jazirotul Fitriyati

Registration Number : G352090151
Major

: Animal Biosciences

Approved:
Advisory Committee

Dr. Ir. I Made Samudra, M.Sc
(Member)

Dr. Ir. Rika Raffiudin, M.Si
(Chair)

Agreed:

Coordinator of Animal Biosciences Major

Dr. Bambang Suryobroto

Examination Date: June 22nd, 2011

Dean of Graduate School

Dr. Ir. Dahrul Syah, M.Sc. Agr

Date of completing studies:

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ACKNOWLEDGEMENT

This research was financially supported by Kerja Sama Kemitraan
Penelitian Pertanian Dengan Perguruan Tinggi (KKP3T) from Ministry of
Agriculture Indonesia Republic and Bakrie Center Foundation (BCF) for Masters
Scholarship funding.
I would like to express my gratitude for Dr. Ir. Rika Raffiudin, M.Si and Dr.
Ir. I Made Samudra, M.Sc as supervisory committee, for all of guidance and
encouragement as well as invaluable academic advices for the whole period of my
study and research at IPB.
I am very grateful to all the invaluable lecturing staff (Especially for Dr.
Achmad Farajallah) and technical staff of Animal Biosciences Study Program
who have imparted knowledge and help and also for the full support given to
enable the successful completion of this research
Moreover, I would like to express my appreciation to all fellow students of
Animal Bioscience especially for class of 2009. Of these fellows, I would like to
express my deepest thanks to close friends Ruth Martha Winnie, Ibu Taruni Sri
Prawasti,

Sanou

Faye,

Kaleem

Saleem,

Petlane

Molafe,

and

Princy

Razafimandimby who always providing a helping hand and good advices. I
wholeheartedly thank to research team of Dr. Rika Raffiudin and Dr. Achmad
Farajallah supervisees such as Cahyo, Made, Rindi, Raisa, Bisri, Dea, Rijal, and
Chyntia who made my master research a great experience of team working.
Further, I am highly indebted to my affectionate parents, young brothers
(Reza, Kiki, and Agil) and other family members who always inspire and
encourage me for higher education, and finally to Yonan Arman who contributes
immensely in providing supports and good constructed advice during busy time of
my study and research.

Bogor, August 2011
Jazirotul Fitriyati

xxii
 
 

xxiii
 
 

BIOGRAPHY
Jazirotul Fitriyati was born on January 5th, 1988 in Kendal, Central Java as
the first daughter of Mr Jazuri and Mrs Sri Nuriyati among other three children.
In 2005, Ms fitriyati finished Senior High School from State Senior High
School 6 Semarang, she continued to study for Bachelors Degree in Department
of Biology, Mathematic and Natural Sciences Faculty, Bogor Agricultural
University and graduated in June 2009. Ms Fitriyati was awarded by the Bakrie
Center Foundation to continue her study to Masters Degree in Bogor Agricultural
University, majoring in Animal Biosciences in 2009.
During the period of her study in Bogor Agricultural University, she has
become a research assistant for several lecture subjects, i.e. Basic of Biology,
Development of Animal, Invertebrate, Animal Structure, Vertebrate, Animal
Function and Behavior, Molecular genetics for undergraduate students and
Zoogeography for graduate students.

xxiv
 
 

xxv
 
 

TABLE OF CONTENTS

Page
LIST OF TABLES ............................................................................................ xxvii
LIST OF FIGURES ......................................................................................... xxviii
LIST OF APPENDICES ......................................................................................xxx
1. INTRODUCTION................................................................................................1
Background .........................................................................................................1
Research Objective ..............................................................................................2
Research Output ..................................................................................................2
Hypotheses ..........................................................................................................2
2. LITERATURE REVIEW ....................................................................................3
Classification and Distribution of Scirpophaga incertulas .................................3
Morphological Structure and Life Cycle of S. incertulas ...................................4
The Damages Are Caused by S. incertulas .........................................................5
Sex Pheromone and PBP Proteins Function........................................................6
in Moth Mating Behavior ....................................................................................6
The Structure of Pheromone Binding Protein .....................................................9
3. MATERIAL AND METHOD ...........................................................................11
Time and Place of Research ..............................................................................11
Material .............................................................................................................11
S. incertulas Imagoes Collection .......................................................................11
S. incertulas Imagoes Rearing ...........................................................................12
DNA Molecular Analysis ..................................................................................13
DNA Extraction..........................................................................................13
DNA Amplification ....................................................................................14
DNA Sequencing........................................................................................19
PCR Product Visualization.........................................................................19
Data Analysis .............................................................................................19
4. RESULT.............................................................................................................21
Imagoes Emergence and the Life Cycle Estimation of S. Incertulas ................21
Visualization of PBP Expected Amplicons of S. incertulas from Several PCR
Modifications.....................................................................................................22

xxvi
 

Standard PCR .................................................................................................... 22
Polyacrilamide gel cutting – RePCR method ............................................ 22
Nested PCR ................................................................................................ 23
Touchdown PCR ........................................................................................ 24
Touchdown-nested PCR ............................................................................ 24
Identification of the PBP Expected Sequences from S. incertulas ................... 25
Phylogenetic Analysis of Third Exon ............................................................... 27
PBP Sequence of S. incertulas.......................................................................... 27
5. DISCUSSION.................................................................................................... 31
Rain exposure as a key factor that influenced the emergence of S. incertulas
imago ................................................................................................................ 31
Optimalization amplicon of S. incertulas PBP gene by using degenerate
primers and touchdown-nested PCR method.................................................... 32
Clustering Since_PBPexpected to the PBP of Crambidae based on
Phylogenetic Analysis....................................................................................... 34
Future Research ................................................................................................ 35
6. CONCLUSION AND SUGGESTION.............................................................. 37
Conclusion ........................................................................................................ 37
Suggestion ......................................................................................................... 37
REFERENCES ...................................................................................................... 38
APPENDICES ....................................................................................................... 43

 

 
xxvii
 

LIST OF TABLES

Page
1 Sex pheromone compounds ratio from several moths with different functional
group ................................................................................................................... 7
2 Several pheromone binding proteins gene data of Lepidoptera member in
Genbank............................................................................................................ 10
3 PCR and sequencing degenerate primers for PBP Gene of Scirpophaga
incertulas .......................................................................................................... 16
4 Primer combinations list that were used to amplify PBP gene of Scirpophaga
incertulas .......................................................................................................... 17
5 List of PBP, OBP, GOBP, and OR sequences used for phylogenetic analysis. 20
6 Life cycle of reared Scirpophaga incertulas ..................................................... 21
7 Top one BLASTN result of two fragments sequenced from Scirpophaga
incertulas polyacrilamide gel cutting rePCR amplicon ................................... 26
8 Top one BLASTN result of two fragments sequenced from Scirpophaga
incertulas nested PCR amplicon ...................................................................... 26
9 BLASTP result from third exon putative amino acid sequencess of Scirpophaga
incertulas touchdown-nested PCR amplicon ................................................... 27
10 Genetic distance of Scirpophaga incertulas PBP putative amino acid
sequences with published GOBP, OBP, and OR amino acid sequence from
various species in Lepidopteraa ........................................................................ 30

 
xxviii
 

LIST OF FIGURES

Page
1 Praecinctorium: absent in Pyralidae (A & B) and present in Crambidae ............ 3
2 Life cycle of the yellow rice stem borer (YRSB) Scirpophaga incertulas ......... 4
3 Diagrammatic section through a sensillum of Lepidoptera ................................. 8
4 Diagrammatic of the binding protein capturing pheromone molecules in lymph
sensilla .............................................................................................................. 8
5 Binding of two different pheromones to the Antheraea polyphemus PBP
binding site ....................................................................................................... 9
7 Equipments to capture imago of Scirpophaga incertulas in the field ............... 12
8 Geenhouse condition was used for Scirpophaga incertulas rearing process .... 12
9 Rearing method of rice stem borer .................................................................... 13
10 Position of eight degenerate primers in alignment of five PBP sequences ..... 15
11 Primer position used to amplify Scirpophaga incertulas based on genomic
PBP gene of Ostrinia furnacalis .................................................................... 16
12 Expected PBP amplicons of Scirpophaga incertulas using twelve primer
combinations .................................................................................................. 22
13 Expected PBP amplicons of Scirpophaga incertulas using polyacrilamide gel
cutting – RePCR modification ....................................................................... 23
14 Expected PBP amplicons of Scirpophaga incertulas using nested PCR
modification.................................................................................................... 23
15 Expected PBP amplicon of Scirpophaga incertulas using touchdown PCR
modification.................................................................................................... 24
16 Expected PBP amplicons of Scirpophaga incertulas using touchdown- nested
PCR modification ........................................................................................... 25
17 DNA sequence and predicted amino acid sequence of Scirpophaga incertulas
PBP expected from Touchdown-nested amplicon ......................................... 26
18 BLASTP alignment result of Since_PBPexpected amino acids sequence with
GOBP of Apis mellifera from Genbank database........................................... 27

xxix
 

19 Alignment of the amino acid sequences from translated third exon of
Scirpophaga incertulas PBP expected. .......................................................... 28
20 Conserved cysteine amino acid position in exons of three moths PBP gene and
the schematic of intron and exon size from three moths PBP gene ............... 28
21 Phylogenetic analysis of Since_PBPexpected PBP with other OBP, GOBP,
PBP, and OR genes of Crambidae moths based on amino acid sequences
using Maximum likelihood method implemented WAG model, with 1000
Bootstrap 1000 replication. ............................................................................ 29
22 Schematic diagram of the three disulfide linkages in PBP of Bombyx mori .. 35

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
xxx
 

LIST OF APPENDICES

Page
1 Chromatogram of Scirpophaga incertulas polyacrilamide gel cutting – RePCR
amplicon using pair of sequencing primer F2.1 and R3.1 ................................ 44
2 Chromatogram of Scirpophaga incertulas polyacrilamide gel cutting – RePCR
amplicon using a pair of sequencing primer F2.2 and R3.1 ............................. 45
3 Chromatogram of Scirpophaga incertulas nested PCR amplicon from 700 bp
fragment using reverse primer R3.1 ................................................................. 46
4 Chromatogram of Scirpophaga incertulas nested PCR amplicon from 500 bp
fragment using reverse primer R3.1 ................................................................. 47
5 Chromatogram of Scirpophaga incertulas nested PCR amplicon from 300 bp
fragment using forward primer F2.1 ................................................................. 48
6 Chromatogram of Scirpophaga incertulas touchdown-nested PCR amplicon
from 600 bp fragment using reverse primer R3.1 ............................................. 49

 1
 
 

1. INTRODUCTION

 

Background
The yellow rice stem borers (YRSB) moth, Scirpophaga incertulas
(Walker) (Lepidoptera: Crambidae), is known as the most major rice stem borer
pest in tropical Asia (Kumar et al. 2001). In China, India, and Southeast Asia,
annual losses to yellow rice stem borers average 5 to 10%, but losses in individual
fields may reach 50-60% (Taylor 1988). In Karawang, Indonesia, S. incertulas is
dominant pest among other rice stem borers, and it caused 20,5% losses (Suharto
and Usyati 2005).
S. incertulas is monophagous in rice (Pathak and Khan 1994), its larva
bore into the rice stem and complete the larva and pupa stages within the rice
stem. During development, larva feed voraciously causing tremendous yield
losses (Kumar et al. 2001). Larva of S. incertulas are difficult to be controlled
with insecticides because after hatching, the larva are exposed only for a few
hours before they enter the rice stem. In addition, the use of insecticides can
contaminate rice field and diminish the other non-target insect such as pollinator
and predator (Pathak and Khan 1994, Cork and Hall 1998).
Currently, Sustainable biocontrol such as mating disruption has
implemented to control the population of S. Incertulas moth. Artificial sex
pheromone trap as an example of mating disruption that using the analogue of sex
pheromone which is produce by female moth to attract the male come to the trap.
This method can lower the copulation behavior hence the pest population will
decrease (Cork and Hall 1998). The idea of artificial sex pheromone trap is
influenced by natural mating behaviour of moth that using sex pheromone as
communication signal. Female moth produce an airbone signal consisting of a
single chemical compounds or a particular blend of compounds called sex
pheromone (Reelofs et al. 1995). Moreover, male moth has pheromone binding
protein (PBP) that selectively bind the sex pheromone release from the female
moth for a signal to find the female position to mate (Willett and Harrison 1999).
The active compound of S.incertulas sex pheromone is already known that consist

2
 

of cis-9-heksadecenyl aldehyde, cis-11-heksadecenyl aldehyde, and cis-9octadecynyl aldehyde (Tatsuki et al. 1985).
PBP gene consists of two introns and three coding regions (exons) (de
Santis et al. 2006; Xiu and Dong 2007; Xiu et al. 2008), as shown in PBP gene of
Crambidae Family i.e Chilo supressalis (Striped rice stem borer) (Genbank
EU825762), Ostrinia furnacalis (Asian corn stem borer) (Genbank AF133629)
and Ostrinia nubilalis (European corn borer) (Genbank AF133643). PBP plays a
role in the recognition of sex pheromone in male moth antennae. By binding
selectively to different components of pheromone blends, therefore, PBP can be a
selective filter to bind the sex pheromone (Willett and Harrison 1999). Out of all
the PBP gene found in Lepidoptera, none was reported from S. incertulas.
Identification of S. incertulas PBP gene can be used as an initial step to determine
protein structure and to developing new insect control using the PBP inhibitor to
decrease capability by the insect to detect new incoming pheromone; hence,
prevent them to mate (Riba et al. 2001).

Research Objective
These researches were aimed to identify and characterize the genome of
PBP gene in S. incertulas.

Research Output
Pheromone Binding Protein genomic data of S. incertulas will be used as
basic information for studying PBP expression in female or male S. incertulas;
moreover, for initial step to developing new insect biocontrol.
 

Hypotheses
This current study proposed several hypotheses are:
1.

Pheromone binding protein gene of S. incertulas might have high similarity
with Crambidae’s PBP gene due to their close relationship.

2.

Intron and exon position on O. furnacalis PBP gene is suppose to be similar
with that of S. incertulas PBP gene.

 

3 
 

2. LITERATURE REVIEW
 

Classification and Distribution of Scirpophaga incertulas
Scirpophaga incertulas is classified in Order of Lepidoptera, Family  of
Crambidae, and Subfamily of Schoenobiinae (Kristensen 2007). Crambidae has
17 subfamilies and more than 11.600 species known today (Solis 2007). Many
Crambidae moth involved as pest and usually called the grass moth family. Chilo
and Ostrinia are the best known genus for biology and genetics data in
Crambidae. Previously, Crambidae have been treated as a subfamily of the
Pyralidae or snout-moths, but currently, Crambidae is separated from Pyralidae.
The principal difference between Crambidae and Pyralidae are the structure in the
tympanic at the front leg called the praecinctorium, which joins two tympanic
membranes in the Crambidae; on the contrary, it is absent in the Pyralidae
(Figure 1).(Solis 2007).
 
Front leg

praecinctorium

Figure 1 Praecinctorium: absent in Pyralidae (A & B) and present in Crambidae
(C & D) (Solis 2007)
Distribution of S. incertulas is throughout of Asia such as Afghanistan,
Pakistan, India, Nepal, Burma, China, Thailand, Vietnam, Cambodia, Malaysia,
Indonesia, Sabah, Philippines, Hongkong, Taiwan, Japan, and Okinawa (Grist and

4
 

Lever 1969, Pathak and Khan 1994). Chilo distribution was recorded as a pest in
Portugal, Spain, Iraq, Pakistan, India, Sri Langka, Vietnam, Cambodia, Laos,
Malaysia, Indonesia, Thailand, Sabah, Philippines, New Guinea, Taiwan, Korea,
China, and Japan (Grist and Lever 1969).
Morphological Structure and Life Cycle of S. incertulas
Scirpophaga incertulas undergoes metamorphosis from egg, larva, pupae,
up to adult. The eggs hatch in 5 to 9 days, and the optimum egg hatching
temperature is 24-29 °C. After hatching, the first instar larva feed on leaf tissue
before bore the rice stem. Larva of S. incertulas usually undergoes four to seven
larval instars stages to become full grown larva. The larval development take 20
to 30 days, followed by a pupal period of 9-12 days. The average imago life is
only 5 to 7 days for mating including oviposit their eggs. The entire life cycle of
S. incertulas range from 39 to 58 days (Figure 2). (Pathak and Khan 1994).

 

YRSB

YRSB
Adult
Adult/Imago

Egg mass
Pupa

5-9 days
5‐9 days 

9-12
days
9‐12 days 

4-7 Larval stages
20-30 days

20‐30 days

Larva

Figure
2 Life
cycle
of the
yellow
ricerice
stemstem
borer
(YRSB)
Figure
2. Life
cycle
of the
yellow
borer
(YSB)Scirpophaga
Scirpophagaincertulas
incertulas(Pathak
(Pathakand
andKhan
Khan1994)

1994)

5
 

The eggs of S. incertulas are laid in a flat compact mass covered with
brown scales measuring 0.14 x 0.11 mm length; color translucent at first,
darkening during development. An egg mass contain 50 to as many as 150, with
an average total of 600 eggs per female. The larva of S. incertulas is ivory-colored
to greenish and rather worm-like in appearance, posterior tapering from the first
abdominal segment. There are four instars; length 18 to 22 mm. The pupa of S.
incertulas have a straw colored within a white silken cocoon measuring 18 to 20
mm, the pupa itself being 13 to 15 mm long. The adult male has a forewing
brownish ochre with a rather indistinct black spot at the lower angle of the cell, an
opaque brown band and marginal row of black dots, hindwings white, and span 18
to 23 mm. The adult female has an orange-yellow forewing with a prominent
black spot, hindwing light fuscous, and span 24 to 36 mm in female (Grist and
Lever 1969, Pathak and Khan 1994).
The Damages Are Caused by S. incertulas
Being the most important insect pest in the world, S. incertulas is
responsible for destroying an average of about 0.8 million tons of rice a year,
while crop losses in Taiwan vary from 20 to 40 percent (Grist and Lever 1969). In
Indonesia, the infestation intensity of rice borer and the damage area in 1988 were
20.5% and 151.577 ha, respectively (Suharto and Usyati 2005). Crop losses which
are caused by yellow rice stem borer occurred in China, India, and Southeast Asia,
annual losses to rice borer average 5 to 10%. Recovery or prevention of 5% of the
losses to stem borers could feed 140 million people for 1 year (Taylor 1988).
S. incertulas is a stem borer which monophagous on rice (Stevenson et al.
2005). This stem borer feed on the crop during the vegetative and reproductive
stages of the rice plant. Excessive boring through the sheath can destroy the crop.
The first larvae of stem borer bore at the base of the plants during the vegetative
stage. This early infestation causes deadheart, which is showed by the central leaf
whorl does not unfold, but turns brownish and dries off. If the larva bore during
reproductive stage of rice plants, the larvae will bore through the upper nodes and
feed toward the base. This late infestation causes whiteheads where the emerging

6
 

panicles are whitish and unfilled or empty (Pathak and Khan, 1994; Cork and Hall
1998).
Yellow rice stem borer are difficult to control with insecticides because
after hatching, the first instar larva are exposed only for a few hours before they
enter a tiller or enter other part of the rice stem. In addition, the use of insecticides
has side effect such as water contamination and indiscriminate killing (Pathak and
Khan 1994; Cork and Hall 1998). Currently, Sustainable biocontrol such as
mating disruption has implemented to control the population of S. Incertulas
moth. Artificial sex pheromone trap as an example of mating disruption that using
the analogue of sex pheromone which is produce by female moth to attract the
male come to the trap. This method can lower the copulation behavior hence the
pest population will decrease (Cork and Hall 1998).
The idea of artificial sex pheromone trap is influenced by natural mating
behaviour of moth that using sex pheromone as communication signal. Female
moth is release sex pheromone into the air to attract male moth (Reelofs 1995).
Moreover, male moth has pheromone binding protein (PBP) that selectively bind
the sex pheromone release from the female moth for a signal to find the female
position to mate (Willett and Harrison 1999). In the same case, male moth will be
attracted to artificial pheromone as a presence of female insect and they will be
able to respond by flying to the trap (Willett and Harrison 1999; Tatsuki et al.
1985).

Sex Pheromone and PBP Proteins Function in Moth Mating Behavior
Female moth produce an airbone signal consisting of a single chemical
compounds or a particular blend of compounds called sex pheromone (Reelofs
1995). A typical Lepidoptera or moth’s sex pheromone consists of two or three
structurally related compounds in a specific ratio. The compounds often contain
an oxygenated functional group that function as terminal groups such as acetates,
alcohols or aldehydes (Cork et al. 2007) (Table 1).
The mechanism by which male moths specifically recognize the sex
pheromone produced by conspecific females is just beginning to be understood.
The first step in the recognition process involves PBP, a small, abundant protein

7
 

in the male moth’s antennae (Willet and Harrison 1999). Pheromone binding
protein was first identified by its ability to bind the sex pheromone of the silk
moth Antheraea polyphemus (Vogt and Riddiford 1981).

Table 1 Sex pheromone compounds ratio from several moths with different functional
group

No Species

1

S. incertulas

2

O. furnacalis

3

O. nubilalis

4

Grapholita molesta

Family

Sex
pheromone
compounds

E9-16: Ald
E11-16: Ald
E9-18: Ald
E12-14:OAc
Crambidae
Z12-14:OAc
E11-14:OAc
Crambidae
Z11-14:OAc
Z8-12:OAc
Tortricidae E8-12:OAc
Z8-12:OH
Crambidae

Ratio
(%)

Functional
Reference
group

17
66
17
53
47
99
1
95
4
1

Aldehyde
Aldehyde
Aldehyde
Acetate
Acetate
Acetate
Acetate
Acetate
Ac