Variasi Tipe Alginat Sargassum siliquosum: Kajian Aktivitas Immunomodulator dan Ekspresi Gen Immun pada Udang Vaname (Litopenaeus vannamei, Boone 1931) untuk Penanggulangan White Spot Syndrome Virus

ERVIA YUDIATI, Dr. Ir. Alim Isnansetyo, M.Sc.;Dr. Ir. Triyanto, M.Si.;Dr. Ir. Murwantoko, M.Si.

2016 | Disertasi | S3 Ilmu Pertanian

Penanggulangan penyakit White Spot Syndrome Virus pada budidaya Litopenaeus vannamei dapat diatasi dengan pemberian immunostimulan untuk meningkatkan kekebalan non spesifik udang. Metode enzimatis digunakan untuk mengukur aktivitas Phenoloxidase (PO), Superoxide Dismutase (SOD) dan Total Protein Plasma (TPP) dengan alat spektrofotometer. Metode mikroskopis dilakukan untuk mengukur Total Haemocyte Count (THC) dan Aktivitas/Indeks Fagositosis (AF/IF). Ekspresi gen-immun dan jumlah copi WSSV dihitung dengan qRT-PCR. Percobaan telah dilakukan pada udang dengan suplementasi dengan asam, sodium dan kalsium alginat secara oral pada dosis 0, 1,0 dan 2,0 g kg-1 dan sodium alginat pada dosis 0 (kontrol), 2,0; 4,0 dan 6,0 g kg-1 selama 14 hari. Udang yang diberi pakan dengan sodium alginat pada dosis berbeda telah diinjeksi dengan WSSV pada dosis 1 x 105 copi udang-1. Hasil menunjukkan bahwa paramater ketahanan seluler yaitu THC, AF/IF dan humoral (PO dan SOD) meningkat pada semua perlakuan apabila dibandingkan dengan kontrol. Tingkat ekspresi gen LGBT, TLR, Lektin dan proPO pada semua perlakuan udang yang diberi pakan dengan suplementasi sodium dan asam alginat 2 g kg-1 meningkat apabila dibandingkan dengan kontrol. Tingkat ekspresi gen Toll, Lektin pada udang yang diberi pakan dengan suplementasi sodium alginat juga mengalami up regulated. Setelah ditantang dengan WSSV, gen Lektin mengalami up regulasi pasca infeksi dan gen TLR mengalami down regulated, kecuali pada udang yang diberi pakan dengan suplementasi alginat pada dosis 6,0 g kg-1 yang mengalami up regulated (48 hpi). Pada 48 hpi, udang yg diberi pakan suplementasi alginat pada dosis 6,0 g kg-1 adalah paling resisten dengan jumlah copi WSSV terendah yaitu 3,3 x 103 mikrogram-1 total DNA. Pada set percobaan lainnya, udang putih L. vannamaei yang diberi pakan suplementasi sodium alginat selama 28 hari, diinjeksi dengan WSSV pada dosis 1 x 105 copi udang-1 . Laju sintasan udang yang diberi penambahan pakan meningkat sebanyak 560% apabila dibandingkan dengan kontrol. Pemberian pakan secara oral dengan dosis 2,0 g kg-1 pada asam dan sodium alginat memberikan pertumbuhan yang lebih baik. Sedangkan pemberian pakan secara oral dari sodium alginat tidak berpengaruh terhadap pertumbuhan. Nilai TPP dari kedua penelitian tersebut tidak berbeda nyata. Setelah pemeliharaan 28 hari dan diinjeksi dengan WSSV, udang yang diberi pakan dengan sodium alginat pada dosis 6,0 g kg-1 memberikan laju sintasan terbaik. Dapat disimpulkan bahwa suplementasi alginat Sargassum siliquosum pada pakan L. vannamei mampu meningkatkan sistem immun bawaan dan tingkat ekspresi gen immmun. Suplementasi sodium alginat juga mampu meningkatkan laju sintasan dan juga resistensi terhadap infeksi WSSV.

The White Spot Syndrome Disease in Litopenaeus Vannamei culture can be countered by enhancing the non-specific defence system with immunostimulant. Enzymatic methods was administered to determine the Phenoloxidase (PO), Superoxide Dismutase (SOD) dan Total Protein Plasma (TPP) activities, spectrophotometrically. Microscopic methods was applied to count the Total Haemocyte Count (THC) and Phagocytic Activity/Index (AP/IP). The related-immune genes and WSSV copy number was determined by qRT-PCR. The rearing of Litopenaeus vannamei with supplemented acid, sodium and calcium alginate at 0 (control), 1.0, 2.0 g kg-1 and sodium alginate at 0 (control), 2.0, 4.0, and 6.0 g kg-1 for 14 days were done. Results indicated that the immune parameters and LGBP, Toll, Lectin and proPO expression of all shrimps were increased. The WSSV at 1 x 105 copies shrimp-1 was injected to the shrimp at 14 day and the copy number of virus were examined quantitatively (qRT-PCR). Immune�¢ï¿½ï¿½related genes expression was evaluated by quantitative Real Time PCR (qRT-PCR). During rearing of the shrimps fed with supplemented sodium alginate at 2.0, 4.0, and 6.0 g kg-1 and before WSSV infection, the two immune-related genes expression i.e. TLR, Lectin were up regulated. The Lectin gene were up-regulated post infection, and the TLR gene in all treatments were down regulated, except the shrimp fed with sodium alginate at 6.0 g kg-1 were up-regulated at 48 hour post infection (hpi). At the same hpi, the shrimp fed the 6.0 g kg-1 sodium alginate was the most resistence and gave the least WSSV copy number (3.3 x 103 microgram-1 of total DNA). In a separate experiment, white shrimp L. vannamei, which had been fed diets supplementing the extract for 28 days, were injected with white spot syndrome virus (WSSV) at 1 x 105 copies shrimp-1 and then placed in seawater. The survival rate of shrimp fed the sodium alginate supplemented diets was significantly increased by 560% comparing to the control treatment. Oral administration of acid and sodium alginate at 2.0 g kg-1 enhanced the growth but were not affect the Total Protein Plasma. While oral administration of sodium alginate was not affect the growth and TPP. At 120 h postchallenge, the shrimp with diets supplemented sodium alginate at 6.0 g kg-1 treatment exhibit the best results on survival rate. It is therefore concluded that the supplementation of alginate of S. siliquosum on the diet of L. vannamei enhanced the innate immunity and the expression of immune-related genes. The supplementation of sodium alginate were also able to improve the survival as well as the resistance against WSSV infection. It is the first report on the simultaneous evaluation on the capability of sodium alginate to enhance innate immune parameters, immune-related genes expression as well as the resistance against WSSV infection in L. vannamei.

Kata Kunci : alginate, immune response, genes expression, Litopenaeus vannamei, Sargassum siliquosum, White Spot Syndrome Virus