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Penelitian: Imunohistokimia Jaringan Larva Spodoptera Litura

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Penelitian: Imunohistokimia Jaringan Larva Spodoptera Litura LAPORAN AKHIR PENELITIAN DISERTASI DOKTOR IMUNOHISTOKIMIA JARINGAN LARVA SPODOPTERA LITURA YANG TERINFEKSI SPODOPTERA LITURA MULTIPLE NUCLEAR POLYHEDROSIS VIRUS (SPlTMNPV) IN VITRO DALAM FORMULA FOTO- PROTEKTAN Tahun ke 1 dari rencana 1 tahun Dra. Mahanani Tri Asri, M.Si NIDN: 0024076703 UNIVERSITAS NEGERI SURABAYA Desember, 2013 iii iv DAFTAR ISI Halaman HALAMAN JUDUL i HALAMAN PENGESAHAN............................................................................... ii DAFTAR ISI....................................................................................................... iii DAFTAR GAMBAR............................................................................................ v DAFRTAR TABEL ............................................................................................ ix DAFTAR LAMPIRAN.......................................................................................... x RINGKASAN....................................................................................................... xi BAB I. PENDAHULUAN............................................................................. 1 1.1. Latar Belakang ................................................................... 1 1.2. Rumusan Masalah ............................................................. 8 BAB II. KAJIAN PUSTAKA 10 2.1. Spodoptera litura................................................................. 10 2.2. Anatomi dan Histologi Larva Spodoptera litura ................... 15 2.3. Kultur Sel Insekta ................................................................ 18 2.4. SpltMNPV (Spodoptera litura Nuclear Polyhedrosis Virus) Sebagai Bioinsektisida pada larva S. litura ......................... 20 2.5. Potensi Agen Hayati SpltMNPV Sebagai Bioinsektisida ..... 28 2.6. Formulasi Bioinsektisida SpltMNPV..................................... 30 2.7. Bahan Pelindung SpltMNPV: Tabir Surya (Foto-Protektan) 32 2.8. Radiasi Sinar Matahari ........................................................ 35 BAB III. TUJUAN DAN MANFAAT PENELITIAN........................................ 41 3.1. Tujuan penelitian ................................................................. 41 3.2 Manfaat Penelitian............................................................... 42 BAB IV. METODE PENELITIAN .................................................................. 43 4.1. Tempat dan Waktu Penelitian ............................................. 43 4.2. Definisi Operasional........................................................... 43 4.3. Kerangka Operasional penelitian ........................................ 44 4.4. Bahan dan Alat Penelitian ................................................... 45 4.5. Prosedur Penelitian.............................................................. 46 a. Pemeliharaan dan Perbanyakan Larva S. litura........... 46 b. Perbanyakan SpltMNPV Secara in Vivo pada larva Spodoptera litura.......................................................... 47 c. Pemurnian SpltMNPV dari larva S. litura...................... 48 d. Perhitungan konsentrasi SpltMNPV ............................ 48 v e. Pembuatan Kultur Sel Epithel Midgut Larva S. 50 litura.......... f. Perbanyakan Virus Secara In Vitro ............................. 50 g. Ekstraksi EPMS dari Rimpang Kencur (Kaemferia galanga)....................................................................... 52 h. Tahap Formulasi .......................................................... 52 i. Pembuatan Preparat Histologis dari SpltMNPV yang Menginfeksi Larva S. litura (Metode parafin dan imunohistokimia)........................................................... 53 4.6 Analisi Data.......................................................................... 55 BAB V. HASIL DAN PEMBAHASAN........................................................... 56 5.1. Histopatologi Larva S.litura yang terinfeksi SpltMNPV Dilihat Dengan Menggunakan Metode Parafin.................... 56 5.2. Jaringan dan Organ Larva S. litura yang terinfeksi SpltMNPV Dalam Formula Foto-Protektan pada Tubuh 60 Larva S. litura....... 5.3. Gambar Histopatologis dari Berbagai Organ dan Jaringan yang Terinfeksi SpltMNPV In Vitro....................................... 64 5.4. Imunohistokimia Larva S. litura yang Terinfeksi SpltMNPV in Vitro Dalam Formula Foto-Protektan 76 5.5. Jaringan dan Organ larva S. litura yang terinfeksi 85 SpltMNPV dalam Formula Foto-Protektan pada tubuh larva S. litura dilihat dengan Imunohistokimia .......................................................................................... BAB VI. KESIMPULAN DAN SARAN ......................................................... 91 7.1. Kesimpulan ......................................................................... 91 7.2. Saran ................................................................................. 92 DAFTAR PUSTAKA ......................................................................................... 93 LAMPIRAN......................................................................................................... 99 iv DAFTAR GAMBAR No Judul Halaman 2.1. a Kumpulan telur Spodoptera litura Fabr. b).Neonate 14 (larva) kecil)…………..…………. ………………………...................... 2.2. b Larva Spodoptera litura instar 2, b) larva Spodoptera litura instar 3, c). Larva Spodoptera litura instar 4……………. 14 2.3. Pupa Spodoptera litura Fabr. …………………………………… 14 2.4. a) Imago S. litura jantan, b) Imago S. litura betina ……………… 15 2.5. Anatomi eksternal dari larva Lepidoptera (Spodoptera litura) … 15 2.6 Anatomi internal dari larva Lepidoptera (Spodoptera litura) …… 16 2.7 Potongan melintang dari midgut larva Lepidoptera (Spodoptera 17 litura) ……………………………………………..……………........... 2.8 (A) Ekstrak Lemak tubuh (FBX) menyebabkan proliferasi sel di 18 epithel midgut, terbentuknya lapisan multiseluler (panah) dan terjadi hiperplasia sel induk (B) sel epithel midgut tanpa perlakuan (C). Pemotongan integumen thoraks secara melintang pada larva yang diberi perlakuan FBX menunjukkan adanya kutikula ganda dengan sintesis dari kutikula baru (Cu2) oleh sel-sel epidermis (Epid) di bawah ruang ecdysial (ES) dan kutikula lama (Cu1), FBX merangsang induksi molting / metamorphosis, (D) fase intermolt terlihat hanya ada satu kutikula (Cu1).................................................................................. 2.9 Rumus molekul dari Ethyl P-metoksinamat (C12H13O3)……..… 34 2.10 Klasifikasi cahaya matahari …………………………………… 36 4.1 Kerangka Operasional Penelitian Efektivitas SpltMNPV in vitro 44 dalam formula Foto-protektan terhadap mortalitas larva Spodoptera litura ....................................................................... 4.2. Kotak besar haemocytometer.................................................... 49 v No Judul Halaman 5.1 Gambar histopatologi ulat sehat dan ulat terinfeksi virus pada 57 berbagai lama waktu infeksi SpltMNPV..................................... 5.2. Organ yang potensial terinfeksi oleh SpltMNPV pada penampang 59 melintang larva S. litura........................................ 5.3. Tubuh larva S.litura Kontrol (tanpa SpltMNPV) dipotong 64 melintang (40 x) : a. Lumen midgut, b. epithel midgut............. 5.4. Trakhea (perbesaran 1000 x): (a) Saluran pernafasan ulat dengan bentukan seperti sisir yang teratur. (b) Lapisan sel epithel yang menyelimuti trakhea 65 (c) Sel trakhea mulai berlobang ( skala mikrometer 1 = 1 µm )........................................... 5.5. Trakhea yang terinfeksi SpltMNPV (perbesaran 1000 kali) 65 (a) PIB SpltMNPV ditengah trakhea yang sel epithelnya telah lepas ( skala 1 mikrometer = 1 µm) ...................................... 5.6. A dan B. Pembuluh darah/haemolimfa terinfekasi SpltMNPV (perbesaran 1000x). Anak panah menunjukkan Polyhedra 66 SpltMNPV pada pembuluh darah ( skala mikrometer 1 = 1 µm )....................................................................................... 5.7. Midgut larva S. litura. a. Lumen (a), membran peritrofik (b) dan epithel midgut (c) yang potensial terserang SpltMNPV (pada lumen terlihat banyak Polyhedra SpltMNPV) perbesaran 400 x b. Polyhedra SpltMNPV terlihat di dalam sel epithel midgut larva S. litura (panah hitam). Perbesaran 1000x ( skala mikrometer 1 = 1 µm berukuran selitar 1- 3 µm) . c. Membran peritrofik terbuka dan PIB SpltMNPV masuk ke membran basal (panah hitam) d. Lapisan sel epithel yang tidak terinfeksi SpltMNPV (a= sel 68 silindris/kolumner, b= sel goblet).......................................... 5.8. a. Lemak tubuh di lumen midgut yang terinfeksi SpltMNPV 69 perbesaran 400x PIB SpltMNPV di membran basal perbesaran 1000x b. PIB SpltMNPV di lumen perbesaran 1000x ( skala mikrometer 1 untuk perbesaran 1000 x = 1 µm ).......... 5.9. a.Tubulus Malpighi dengan lapisan sel yang tebal, perbesaran 71 1000x b. Tubulus malpighi berisi PIB SpltMNPV (tanda panah hitam) Perbesaran 1000x (perbesaran 1000x, skala mikrometer 1 = 1 µm ) .......................................................... vi No Judul Halaman 5.10. A. lemak tubuh yang normal (perbesaran 400x, skala mikrometer 71 1=2.5 µm ) B= Lemak tubuh terinfeksi SpltMNPV (perbesaran 400x)........................................................................................... 5.11. A dan B Lapisan kutikula perbesaran 1000x, Epi = epikutikula, 73 Bm = membran basal, Ep= Epidermis, a =pidermis yang rusak, b= Kutikula yang lobang dan perlihat ada polyhedra SpltMNPV ( skala mikrometer 1 = 1 µm )................................................... 5.12. Sel syaraf pada larva S.litura (panah hitam) Perbesaran 1000x ...
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