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APCBEE Procedia 4 (2012) 105 – 109
ICAAA 2012: July 23-24, 2012, Singapore
Effect of Nosema on Mortality and Histopathology of Nosemainfected Plutella xylostella (Lepidoptera: Plutellidae) Nadia M Kermania, Zainal-Abidin B A Hb, Noor Farehan Ismaila and Idris A Ba∗ a
School of Environmental and Natural Resource Sciences, University Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia b Faculty of Medicine, University Technology MARA UITM, 40450 Shah Alam Selangor
Abstract Biological control, using pathogenic microsporidia, may have the potential to be an alternative to chemical control against the diamondback moth (DBM), Plutella xylostella (Lepidoptera: Plutellidae). The microsporidium, Nosema bombycis (NB), is one of many disease agents that can be used in the Integrated Pest Management (IPM) of DBM. However, its pathogenicity or effectiveness can be influenced by many factors, especially temperature. As such, this study was conducted to investigate the effect of temperature on the NB infection of DBM larvae. Infection was performed on second instar larvae at different dose (spore number/concentration) levels (0, 102,103 104,105), at temperatures of 15°, 20°, 25°, 30° and 35°C, and at 65% RH, 12:12 (L: D) and larval mortality were recorded. The spore concentration had negatively affected larval mortality at all temperatures, although this effect was more pronounced (92%) at 35°C than at 20° and 30°C ( 50%) and 25°C (26%). Histological observations showed that Nosema preferentially infected adipose tissue and the epithelial cells of the midgut resulting in marked vacuolization of the cytoplasm. These findings suggest that Nosema exert their negative effect by damaging the midgut epithelial cells. © byby Elsevier B.V.B.V. Selection and/orand/or peer review under responsibility of Asia-Pacifi c Chemical, © 2012 2012Published Published Elsevier Selection peer review under responsibility of Asia-Pacific Chemical,&Biological & Environmental Engineering Society Biological Environmental Engineering Society Open access under CC BY-NC-ND license. Keywords: Plutella xylostella; Histopathology; Nosema sp; Mortality; Temperature
1. Introduction The diamondback moth (DBM) Plutella xylostella L. (Lepidoptera: Plutellidae) causes considerable
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2212-6708 © 2012 Published by Elsevier B.V. Selection and/or peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society Open access under CC BY-NC-ND license. doi:10.1016/j.apcbee.2012.11.018
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economic losses in Brassicaceous crops worldwide and occasionally other crops. Control of this pest is usually achieved through the application of synthetic insecticides [8], but their high cost, environmental contamination, development of resistance to chemicals, and pest resurgence [8;7;11] have encouraged the search for alternatives more compatible with the environment. Microbial control is an environmentally sound and a valuable option to apply of chemicals for controlling this pest. Nosema bombycis Negali, is one of several important mortality factors of DBM in the field [3]. DBM mortality was higher in younger instars (1st and 2nd generations) than in the older instars. Even at lower concentrations, infection was also significantly higher for both larvae and pupae in highlands than in lowlands [3]. Since temperature is one of the most important ecological factors for the development of insect populations, its effect on the biology of Nosema should be studied. Therefore, the purpose of this research is to study the effects of Nosema spore concentration on the different stages of DBM, reared at different temperatures. Result of this study will tell us about the kind of climatic conditions within which this pathogen might have more impact in a pest population. The second objective to add information on the histopathology effects of larval caused by Nosema. 2. Material and methods 2.1. Diamondback moth The disease-free DBM larvae of the University Putra Malaysia (UPM) strain were obtained from a colony maintained at the Malaysian Agriculture Research and Development Institute (MARDI). The stock-culture of DBM used throughout this study, were reared on potted cabbage, Brassica oleracea var capitata in screen cages (38cm x 26cm x 26cm) and maintained at 25° + 3°C temperature, with a photoperiod of 12L: 12D, and 60 to 80% RH. A 10% honey solution was offered to the adults as food, which had been reared over several generations in a laboratory, prior to experiments. 2.2. Nosema sp Spore Production Nosema sp. spores were isolated from infected DBM collected from Cameron Highlands, Pahang. A purified spore suspension was prepared by crushing infected DBM in sterile distilled water, filtering the resultant suspension through nylon mesh cloth, centrifuged and re-suspended using sterile distilled water. The spores were used to infect young larvae to produce fresh spores for experiments. Fresh spores were used immediately or stored at 4°C for no more than 1 month before use. 2.3. Inoculation of Larvae with Nosema sp. For all experiments, 2nd instar larvae were fed leaf discs (of 2cm diameter) of rape, (Brassica juncea) plants treated by Nosema sp. spore suspensions at 102,103, 103, 104 or 105 concentrations or sterile water as a control. Spore suspensions were spread evenly on the surface of leaf discs using the bulb end of a Pasteur pipette. Larvae were selected randomly from the uninfected colonies and transferred into wells of 24-cell plastic culture plates; at one larva per well. These wells were put into environmental chambers which were maintained at 15°, 20°, 25°, 30° or 35°C ± 0.5°C. The relative humidity (70 ± 10%) and photoperiod (12:12 h, light: dark) were kept similar for all the experiments. After 24 h, larvae were transferred to untreated diet (disease-free leaf disease) and reared under standard growth chamber conditions. The data of their mortality were recorded. Percent mortality was subjected to ANOVA and means were separated using Tukey’s multiple range test, with a reference probability of P 0.05 used to detect significance.
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2.4. Histological preparations In general, larvae were infected as late 2nd instars and then dissected at 24 hr of post treatment, fixed in Bouin’s solution for 24 hr, dehydrated in an ethanol series, and embedded in Paraffin wax (58- 60°C). Sections were cut with a rotary microtome (5 m thickness) and were stained with Haematoxylin-Eosin [2]. 3. Results and Discussion 3.1. Larval mortality of P. xylostella inoculated with Nosema Result showed that all Nosema concentrations tested against 2nd instar larvae of DBM caused various levels of mortality at all temperatures (Fig.1). Leaf dip bioassays showed that mortality of 2nd instar DBM larvae was significantly (F = 93.82, df = 4, P < 0.01) increased with increasing spore concentrations (Fig 1) with the highest accumulated larval mortality (92%) was observed as 105 spores concentration were applied at 35°C, followed by the 20°C, and 30°C ( 50%), with the lowest mortality (26%) was observed at 25°C.
Fig. 1. Mean percent (accumulative) mortality (SE) of diamondback moth larvae fed rape (Brassicajuncea) leaves treated at various concentrations of Nosema spores; 15 (A), 20 (B), 25 (C), 30 (D) and 35°C (E). Vertical bars = S.E
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These findings are in accordance with earlier results, which indicate that DBM larval mortality was high even at lower concentrations of Nosema spores [3] and for the gypsy moth (Lymantria dispar L.), where mortality varied between 79 and 99%, independent of spore concentration [1]. Also previous reports of [9] and [6] on silkworm have shown high mortality of Nosema sp. infected larvae. In contrast, minimal mortality (3%) was observed in infected southwestern corn borer Diatraea grandiosella larvae with Nosema sp. (isolate 506) even at high doses [4].The larval mortality was also affected significantly by the time (days) after ingested the spores at 15° (F = 17.85, d.f. = 3 & 80, P < 0.05), 20° (F = 42.50, d.f. = 3 & 80, P < 0.05) , 25° (F= 25.09, d.f. = 3 & 80, P < 0.05), 30°(F= 37.83, d.f. = 3 & 80, P < 0.05) and 35°C (F = 73.66, d.f. = 3 & 80, P < 0.05 (Fig. 1). However, there was no significant interaction between concentration and time for all temperatures (P > 0.05). Increasing the mortality through the days could be a result to the damage of cell cause during spread or disease profirelation in the body of larvae. [5] reported that Nosema sp. spores were found in the midgut of Antheraea mylitta on the beginning of infection then spread to fat body and during later stages, infection was observed in the tracheal epithelium, Malphigian tubules and gonads. 3.2. The effect of Nosema on the histopathology of midgut of P. xylostella larvae Normally, the midgut of DBM larvae is a long straight tube, which consists of columnar and goblet cells linked by well developed border of microvilli. The epithelial cells rest on a basement membrane and muscle fibers (Fig 2.A). However, at 72h after the treatment, the epithelial cells exhibited swelling and some microvilli were disrupted due to swelling of the cells and lysis of cytoplasmic material (Fig 2.B). In addition, vacuoles increased and some columnar cells are dislodged and sloughed into the lumen of the midgut (Fig. 2 B, arrows). The work presented here clearly shows that Nosema caused cytopathological changes in midgut epithelial cells. Similar cytotoxic effects have also been observed in the midgut of P. xylostella after treatment with B. thuringiensis [10]. A
B
Fig. 2. Histopathological effects of Nosema in on P. xylostella midgut: general aspects of the midgut larvae (A) and histopathological effects of Nosema on it (B) after 72 h of treatment. In B arrows indicate lysis of columnar cells and disrupted columnar cells (stars) are sloughed into the lumen of midgut. V, vacuoles; Lu, lumen; Gc, goblet cell, Cc, columnar cell; Mv, microvilli; Bm, basement membrane. Magnification 40 x.
Acknowledgements This research was supported by UKM grant No. (02-01- 02-SF0601). The authors wish to thank MARDI for providing DBM egg masses and all lab assistants in Entomology, MPG2 and parasitology labs.
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