Sains Malaysiana 34(1): 7-16 (2005)

Teknik Pengesanan Spora Nosema bombycis dan Kesan

Jangkitan pada Larva Plutella xylostella

(Detection Techniques for Spores of Nosema bombycis and Effects

of Infection to the Larva of Plutella xylostella)

Zainal-Abidin B.A.H., Rosnizar M. Jarnil, Noran Majid, Idris A.B.

Pusat Pengajian Biosains & Bioteknologi

Pusat Pengajian Sains Sekitaran & Sumber Alam

Fakulti Sains & Teknologi

Universiti Kebangsaan Malaysia

43600 UKM Bangi, Selangor D.E. Malaysia

Sajap A.S.

Fakulti Perhutanan Universiti Putra Malaysia

43400 UPM Serdang, Selangor D.E.

Hussan A.K.

Malaysian Agricultural Research and Development Institute (MARDI)

P.O. BOX 12301, G.P.O

50774 Kuala Lumpur Malaysia

ABSTRAK

Beberapa teknik pewarnaan biasa telah digunakan untuk mengesan peringkat hidup Nosema bombycis (khususnya spora) yang terdapat pada larva Plutella xylostella (rama-rama belakang intan, DBM) yang terjangkit begitujuga kesan jangkitan spora ini ke atas larva telah dikaji. Larva instar 1,2,3 dan 4 DBM yang bebas daripadajangkitan spora telah dijangkiti dengan empat kepekatan spora (407150, 41420, 4260 dan 420 spora/μl) secara berasingan dengan membiarkan larva ini memakan makanan buatan yang telah dicemari dengan kepekatan spora tersebut. Pemprosesan tisu dengan teknik pewarnaan, penentuan kepekatan spora daripada larva dan cerapan langsung telah dilakukan dalam tempoh 24, 48 dan 72 j selepas jangkitan. Didapati teknik pewarnaan Gram, Giemsa, hematoksilin dan trikrom lebih sesuai untuk mengesan spora, sporon dan meron daripada tisu larva yang terjangkit berbanding teknik 'good Pasteur'. Kesan utama jangkitan spora ialah kematian larva yang bergantung kepada kepekatan spora yang digunakan dan didapati ins tar yang lebih muda adalah lebih rentan terhadap jangkitan ini berbanding instar yang lebih matang. Kepekatan spora 407150, 41420 dan 4260 spora) μl boleh menyebabkan kematian manakala kepekatan spora terendah (420 spora/)1.l) tidak menyebabkan kematian. Kadar kematian instar yang lebih muda lebih tinggi (p<0.001 ) berbanding instar yang lebih matang. Nilai LC₅₀ dan LC₉₅ bagi instar 1 dan 2 lebih rendah berbanding instar 3 dan 4, selepas 48 dan 72 j jangkitan. lni menunjukkan bahawa kepekatan spora yang lebih tinggi diperlukan untuk membunuh instar yang bersaiz lebih besar dan lebih matang. Bilangan spora terendah diperolehi pada tempohjangkitan 24 j manakala bilangan spora tertinggi diperolehi pada tempoh jangkitan 72 j. Kajian histologi ke atas tisu larva terjangkit mendapati kerosakan terjadi pada tisu lemak dan usus yang menyebabkan kematian. Hasil kajian ini menunjukkan bahawa peringkat hidup N bombycis khususnya spora dapat dikesan dengan baik menerusi teknik pewarnaan yang biasa dan kebolehjangkitan parasit ini dan kesannya kepada larva DBM telah diketahui.

Kata kunci: Nosema bombycis, Plutella xylostella

ABSTRACT

Several conventional staining techniques were employed to detect the life stages (in particular the spores) of Nosema bombycis from infected larvae of Plutella xylostella (diamondback moth, DBM) and the effects of the infection of N. bombycis on the larvae were studied. Larval instars 1, 2, 3 and 4 of the DBM were infected with four different spore concentrations (407150, 41420, 4260 and 420 spores/μl) accordingly by allowing them to feed on artificial diet previously inoculated with the respective spore concentrations. Larval tissues were processed for staining and the number of spores were counted and direct observations on the larvae were carried out at 24, 48 and 72 h post infection. The Gram s, Giemsa's, haematoxylin and trichrome staining techniques were more superior in detecting the spores, sporonts and meronts than the good Pasteurs. The main effect of the infection was mortality which was dose-dependent and that the younger ins tars were more susceptible to infection than the older ones. Spore concentrations of 407150, 41420 and 4260 spores/μl) caused death to the instars whereas the dose of 420 spores)μl was unable to kill the larval instars. The mortality rate of the younger instars was significantly higher (p<0.001) than the older ones. The LC₅₀ and LC₉₅ values of instar 1 and 2 were lower than those of the instar 3 and 4 after 48 and 72 h post infection. This showed that high spore concentrations were needed to kill the bigger size and matured instars. Histological studies on the infected larvae indicated that the infection caused severe cellular damages in fat tissues and the intestine leading to death. Results of this studies showed that life stages of N. bombycis in particular the spores were detected effectively using conventional staining techniques and the infectivity and the effects of the infection on the larval tissues of DBM were also established.

Keywords: Nosema bombycis, Plutella xylostella

RUJUKAN/REFERENCES

Balavenkatasubbaiah, M., Datta, R.K., Baig, M., Nataraju, B. & Iyengar, M.N.S. 1994. Efficacy of bleaching powder as a disinfectant against the pathogens of silkworm, Bombyx mori. Indian Journal of Sericulture 33: 23-26

Butt, B.A. 1990. Review of diamondback moth, Plutellaxylostella and plans for F-I sterility research. Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. pp. 1-59.

Canning, E.U. & Lorn, J. 1986. The microsporidia of vertebrates. London:Academic. Press Inc.

Canning, E.U., Curry, A., Cheney, S.A., Lafranchi- Trestem, .N.J., Kawakami, Y.,Hatakeyama, Y., Iwano, H. & Ishihara, R. 1999. Nosema tyriae n. sp. and Nosema sp. Microsporidian parasites of Cinnabar Moth Tyriajacobaeae. J: Inverb. Pathot. 74: 29-38.

Cantwell, G.E. 1970. Standard methods for counting Nosema spores. American Bee Journal 10:222-223.

Chapman, R.E & Joem, A. 1990. Biology of Grass hoppers, London: John Wiley & Sons Inc. London: Academic Press. Cheng, T.C. 1973. General Parasitology.

Croft, B.A. 1990. Arthropod biological control agents and pesticides. London: John Wiley &. Sons Inc.

Finney, D.J. 1971. Probit Analysis. (Third Edition). London. Cambridge University Press.

Garcia, L.S. & Bruckner, D.A. 1997. Diagnostic medical parasitology. USA: John Wiley & Sons Inc.

Ho, T .H. 1965. The life history and control of the diamondback moth in Malaysia. Bulletin Div. Agric. Min. Agric. and Cooperatives Malaysia 118: 1-25.

Husan, A.K. 1992. Potential of several baculoyiruses for the control of diamonback moth and Crocidolomia binotalis on cabbages.ln N.S. Talekar ed., Diamondback moth and other crucifer pests, pp.185-192. Proceedings of the Second International Workshop, Asian Vegetables and Development Centre (AVRDC), A VRDC Publications No. 92-368.

Ibrahim, Y.B. & Low, W 1993. Potential of mass production and field efficacy of isolates of entomopathogenic fungi Beauveria bassiana and Paecilomyces fumosoroseus against Plutella  xylostella. 1nternational Journal of Pest Management 39: 288-­292.

Idris, A.B. & Grafius E.J. 1999. Possible sources of inoculum for horizontal transmission of Nosema bombycis in diamondback moth Plutella xylostella (L.). Sains Malaysiana 28:41-49.

Idris, A.B. & Sajap, A.S. 1999. Bancian awal terhadap kejadian jangkitan Nosema bombycis pada rama-rama belakang intan, Plutella xylostella (L.) di lapangan dan kultur makmal. Prosiding Seminar IRPA-UKM, hlm. 629-634.

Idris, A.B. & Sajap, A.S. 2003. Prevalence of Nosema bombycis in Malaysian field populations of the diamondback moth Plutella xylostella. International Journal of Pest Management 49(1): 71-73.

Idris, A.B., Zainal-Abidin, B.A.H. & Norhayati, A.M. 1997. Detection of Nosema bombycis (Naegeli) in diamondback moth using Giemsa stain. Malaysian Applied Biology 26(1): 105-107.

Idris, A.B., Zainal-Abidin, B.A.H., Sajap, A.S. & Hussan, A.K. 2002. Can Nosema bombycis be a potential biopesticide of diamondback moth, Plutella xylostella L.? Abs. 3rd International Conference on Biopesticides Kuala Lumpur pp. 41.

Iwano, H. & Ishihara. 1991. Dimorphism of spores orNosema spp. in cultured cells. Journal Invertebrate Pathology 57:211-219.

Iwano, H. & Kurtti, T.J. 1995. Identification and isolation of dimorphic spies from Nosemafurnacalis (Microspora: Nosematidae). 1: Journal Invertebrate Pathology 65: 230-­236.

Jurand, A., Simoes, L.C.G., & Pavan, C. 1967. Changes in ultrastructure of salivary gland J cytoplasm in Sciara ocellaris (Chomstock, 1882) due to microsporidian infection. Journal Insect Physiology 13: 795-803.

Kudo, R.R. 1971 Protozoology (Fifth Edition). USA. Charles C. Thomas.

Lian, L. Y. 1991. Silkworm diseases. Food and-griculture Organization of the United Nations: Roma.

Lim, G.S. 1986. Biological control of diamondback moth. Diamondback Moth Management. In Telekar N.S. & T.D. Griggs eds., Proceedings of the First International Workshop A VRD Cpp. 154-171.

Loke, WH., Syed, A.R., Sivapragasam, A., Fauziah, I., Md Jusoh, M. & Hussan, A.K. 1997. Qynamism in diamondback moth IPM development: The Malaysian experience. In A. Sivaprasagam, Loke, W.H., Hussan, A.K. & Lim, G.S. eds. The Management of diamondback moth and other crucifers pests, pp. 249-252. Proceedings of the the 3^rd International Workshop Lumpur.

Marquardt, WC. 7 Demaree, R.S. 1985. Parasitology. USA. Macmillan Publishing Company USA.

Me Laughlin. 1971. Use of protozoan for microbial control of insects. 151-172. In H.D. Burges & N.W. Hussey eds. Microbial Control of Insects and Mites. London: Academic Press.

Nataraju, B., Datta, R.K., Sivaprasad, V., Baig, M., Gupta, S.K. & Sharnin, M. 1994. Protein A linked latex antisera (pallas) test for the detection of nuclear polyhedrosis in silkworm, Bombyx mori. Indian Journal of Sericulture 33: 27-30.

Ooi, PAC. 1986. Diamondback moth in Malaysia pp.25-34. In N.S. Talekar & T.G. Briggs eds., Diamondback Moth Management. First Proceedings of International Workshop, AVRDC Taiwan 11-15 March 1985.

Sajap, A.S. & Lewis L.c. 1992. Chronology of infection of European com borer (Lepidoptera: Pyralidae) with the micro sporidium Nosema pyrausta: effect on development and vertical transmission. I: Environ. Entomol. 21: 178-182.

Siegel, J.P., Maddox, v. & Ruesink, W.O. 1986. Impact of Nosema pyrausta on a braconid Macroscentrus grandii in Central 1l1inois. 1: Invertebr. Pathol. 47: 271- 276.

Syed, A.R., Sivapragasam, A., Loke, W.H. & Fauziah. I. 1997.Classical biological control of diamondback moth: the Malaysian experience. In Sivapragasam, A., Loke, W.H., Hussan, A.K. & Lim, O.S., The management of Diamondback Moth and Other Crucifers Pests. Proceedings of the 3^rd  International Workshop Kuala Lumpur 1996, Mardi Malaysia.

Tanada, Y. & Kaya, H.K.1993. Insect Pathology. USA: Academic Press Inc.

Talekar, N.S. & Shelton, A.M. 1993. Biology, ecology and management of Diamondback Moth. Annu. Rev. Entomol. 38: 275-301..

Undeen, A.H. 1997. Micosporidia (protozoa): A Handbook of Biology and Research.

Weiser, J. 1969. An Atlas of Insesct Diseases Czechoslovakia. Irish University Press.

Wigglesworth, W.B. 1984. Insect Physiology (Eighth Edition). New York: Chapman & Hall.

Yang, L.C. & Chow, Y.S. 1978. Spermatophore formation and morphology of the reproductive system of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) .Bull. Inst. Zool. Academia Sinica 17: 109-115.

Yasunaga, C, Funakoshi, M., Kawarabata, T., Aratake, Y Iwano, H. 1992. Isolation and characterization of Nosema bombycis (Microsporidia: Nosematidae) from larvae of beet armyworm Spodoptera exigua. Japanese Journal of Applied Entomology and Zoology 36(2): 127-134.

Zainal-Abidin, B.A.H., Rosnizar, Sharul Shahari, Norairin Ibrahim & Idris, A.B. 2002. Detection, infection and distribution of Nosema bombycis on DBM, parasitoid and related insects. Abs., 3rd International Conference on Bio­pesticides Kuala Lumpur pp.74.