TOXICITY OF STREPTOMYCES LAVENDULAE (STREPTOMYCETACEAE) CULTURE FILTRATE TO SPODOPTERA LITTORALIS (BOISD.) LARVAE (LEPIDOPTERA: NOCTUIDAE)

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1 Egypt. J. Exp. Biol. (Zool.), 3: (2007) The Egyptian Society of Experimental Biology 23 RESEARCH ARTICLE Hanem H. Sakr TOXICITY OF STREPTOMYCES LAVENDULAE (STREPTOMYCETACEAE) CULTURE FILTRATE TO SPODOPTERA LITTORALIS (BOISD.) LARVAE (LEPIDOPTERA: NOCTUIDAE) ABSTRACT: The effect of Streptomyces lavendulae (Streptomycetaceae) culture filtrate (containing 1x104 CFU/ml) on Spondoptera littoralis larvae was studied in laboratory experiments. The thinfilm technique was employed. Third instar larvae were exposed to different concentrations (226 CFU/cm 2, 113 CFU/cm 2, 56.5CFU/cm 2 and 28.25CFU/cm 2 ) for 24h. The LC50 was found to be CFU/cm 2 (95 F. L. = CFU/cm CFU/cm 2 ; Slope = ± 0.089), obtained 7days post-treatment. The effects of S. lavendulae filtrate on survival, growth, pupation and emergence rates were investigated. Symptoms of exposure included: delay of pupation, formation of intermediate forms (larval-pupal and pupal-adult intermediates) and deformation of wings and mouth parts of the emerged adults. At a concentration of 226 CFU/ cm 2, the cumulative larval mortality was 82.8, while the percentage of adult moth emergence was reduced by Deleterious histological effects were observed in the mid-gut of the treated larvae five days post-treatment. Some epithelial cells showed histolysis and cytoplasmic vacuolation. The muscle fibers in the treated individuals were separated from each other. In the meantime, S. lavendulae culture filtrate caused a noticeable decrease in the proteins content especially in the cytoplasm of the larval mid-gut cells. KEY WORDS: Toxicity, Streptomyces lavendulae, Histopathology, Protein, Spodoptera littoralis, Lepidoptera and Noctuidae. CORRESPONDANCE: Hanem Hamed Sakr h_sakr2000@yahoo.com INTRODUCTION: In the search for new bioactive natural products as a safer source of insecticides, increasing attention is being given to the microorganisms such as bacteria and fungi (Hiort et al., 2004; Quesada-Morga et al., 2006). These can be cultured under fermentation conditions and the resulting insecticidal compounds can be purified, formulated and used effectively against major arthropod pests (Quesada-Morga et al., 2006). Actinomycetes are a large heterogenous group of bacteria tend to form branching filaments, usually Gram-positive, and grow on ordinary laboratory media. The main ecological role of this group is the decomposition of organic matter in and on soil, and production of the most antibiotics. These have found practical application in human and veterinary medicine, agriculture and industry (Madigan et al., 1997). The actinomycete, Streptomyces spp. provide an abundant source of bioactive secondary metabolites possessing biological activity against insects with low toxicity level to the non-target organisms (Koga et al., 1987; Thompson et al., 1997) and behave as nematocide (Ishikawa et al., 2003), antibiotic (Wang and Vining, 2003) or antitumor (Arai et al., 1985; Johnson et al., 1997). The cotton leaf worm, Spondoptera littoralis is a notorious pest of field crops in Egypt and other near east countries. The polyphagous larval stage of this insect feed on cotton, many ornamentals and vegetable crops, causing severe loss of crop productions (Hosny et al., 1986; Martinez and Van Emeden, 2001). Although chemical insecticides have been the primary means of controlling the cotton leaf worm for many years, increasing concerns about human safety, effects on the non target organisms, reduction efficacy due to insect resistance have created a dire need for alternative control strategies. Consequently, the purpose of the present study is to evaluate the bioactivity of the S. lavendulae crude filtrate against S. littoralis larvae. MATERIALS AND METHODS: Insect Tested: S. littoralis larvae were reared in the laboratory at 28 C ± 2 C and 65 ± 5 R. H.

2 198 Egypt. J. Exp. Biol. (Zool.), 3: (2007) The larvae were exclusively reared on castor bean leaves, Ricinus commtmis until they were used in the experiments. Only 3rd instar larvae were used in the present study. The Control Agent: The S. lavendulae culture filtrate containing 1x 10 4 CFU/ml (where, CFU = colony forming unit) was kindly provided by Dr. S. M. El-Sabagh, assistant Prof. of Microbiology, Botany Dept., Fac. Sci., Menuofia Univ., Egypt. Bioassay: Third instar larvae were used in this experiment. The Bioassay was performed by applying the tested filtrate as a thin-film on a glass Petri dish (177 cm 2 ). Different amounts of the stock filtrate (containing 1x 104 CFU/ml) were spread onto glass Petri dishes to obtain different concentrations as follows: 226 CFU/cm 2, 113CFU/cm 2, 56.5 CFU/cm 2 and 28.25CFU/cm 2. Control dishes were treated with water. The Petri dishes were left to dry at room temperature. Then 20 healthy 3rd instar larvae were introduced into the middle of each Petri dish. After six hours of treatment, a piece of castor bean leaves was supplied to these larvae. Each treatment was replicated three times. After 24 h post-treatment, these larvae were transferred to clean glass jars, provided with castor bean leaves and covered with muslin tied round the rim by rubber bands (to prevent the insect from escaping) and held in the laboratory. The larvae were allowed to feed on untreated castor bean leaves until pupation. The larvae were examined daily and the dead ones were recorded. The pupae were counted and placed in separate jars provided with saw dust, until adult emergence. The following parameters were measured: percentage larval mortality, percentage pupation, pupa-adult intermediates were scored as failing to successful emergence and percentage of adult emergence. Data Analysis: Mortality data were subjected to Finney's method (Finney, 1971) to estimate the LC50 and F. L. values. Larval mortality was corrected using Abbott's formula (Abbott, 1925). The reduction in the adult emergence was calculated according to khazanie (khazanie, 1979). Histopathological and Total Protein Determination: The effect of S. lavendulae culture filtrate on the histology of the mid-gut of the 3 rd larval instar of S. littoralis was determined using different concentrations (226 CFU/cm 2, 113 CFU/cm 2, 56.5 CFU/cm 2 and CFU/cm 2 ). The mid-guts of the experimented larvae were removed 1, 2, 3, and 5 days post-treatment. At each interval, parts of the larval mid-gut were fixed overnight in either Bouin's solution or 10 neutral formalin. After fixation, the specimens were dehydrated in graded series of ethanol, cleared in xylene and embedded in parablast. Transverse serial sections, 5µm in thickness were prepared and stained using hematoxyline and eosin. The mercury bromophenol blue stain technique (Bonhag, 1955) was used for protein demonstration. The Stained sections were mounted on glass slides in DPX-mounting medium under cover slips. Untreated larvae were included as controls. The photographs were made using Leitz photomicroscope. RESULTS: When S. lavendulae culture filtrate was used as a thin-film, it was generally toxic to the 3rd instar larvae of S. littoralis (Table 1) even at the lowest concentration. The LC 50 value was CFU/cm 2 (95 F. L. = CFU/cm CFU/cm 2 ); Slope = ± for the tested species 7days posttreatment. The filtrate induced mortality within 24 h post-treatment and mortality became apparent 5 days post-treatment (Fig. 1) in a dose dependent manner. At a concentration of 226 CFU/cm 2, 82.8 of larvae failed to pupate and died causing reduction in the percentage of pupation and adult emergence (Table 1). Table 1. Effect of S. lavendulae culture filtrate on larval mortality, pupation and adult emergence of the cotton leafworm S. littoralis treated as 3rd larval instar for 24h * *Thin-film method. 20 larvae were used / concentration/ **Conc. CFU/cm 2 corrected larval mortality Corrected larval mortality replicate. Three replicates were used. **CFU = colony forming unite. *** Relative to the number of larvae used. Reduction = (C T / C) 100 (Khazanie, 1979), Where C = number of emerged adults in control and T = number of emerged adults after treatment. The survivors eventually succumb or metamorphose into abnormal pupae and adults as compared to the controls (Figs 2, 3, and 4). Morphological changes appeared as: adults with fang-mask mouthparts, adults with unplasticized wing lobes seen as crippled wings, pupa-adult intermediates, pupae with deformed head region, larval-pupal intermediates, and larvae which had difficulty in shedding their cuticle and died during ecdysis pupation Dead pupae 1day 3day 5day 7day 9 day 11day 18day Time post-treatment (days) Total *** Adult emergence Malform ed Reduc control CFU/cm2 113 CFU/cm2 56.5CFU/cm2 Fig. 1. Susceptibility of the 3 rd instar larvae of S. littoralis to the culture filtrate of S. lavendulae.

3 Sakr HH, Toxicity of Streptomyces lavendulae (Streptomycetaceae) Culture 199 Figs 2-4. Morphological changes induced by S. lavendulae culture filtrate. 2a-d: larval stage of S. littoralis. a: healthy 6 th instar larva of S. littoralis. b-d: abnormal individuals resulted from treated larvae. b: permanent larva (over aged larvae). 2c-d: intermediate forms with larval-pupal characters. Fig. 3a-d: pupal stage of S. littoralis. a: healthy pupa of S. littoralis. b-c: deformed pupae resulted from the treated larvae. b: deformed pupae with soft body. c: pupae with abnormal head and thorax. Fig. 4a-c: adult stage of S. littoralis. a: healthy moth. b-c: deformed moth resulted from larvae treated with S. lavendulae. b: pupal-adult intermediate. c: deformed moth with crippled wings and a fang-mask mouth parts. Histopathology: The mid-gut of the control larvae is composed of a simple columnar epithelium including goblet cells and regenerative cells. It rests on a basement membrane and coated from the outside with longitudinal and circular muscle fibers. Some epithelial cells show vesicular secretions at their terminal ends. The food mass is separated from the brush border of the epithelial cells by the peritrophic membrane (Fig. 5). Fig. 5. Transverse section of the mid-gut of a control larva of S. littoralis stained with H/E. When the 3 rd larval instar of S. littoralis was exposed to thin film of 226 CFU/cm 2 of S. lavendulae culture filtrate, great variations were noted in the columnar cells. The majority of variations are shown in (Fig. 6a-d).

4 200 Egypt. J. Exp. Biol. (Zool.), 3: (2007) Fig. 7. Transverse section of the mid-gut of control larva and stained with mercury bromophenol blue, notice all cell structures exhibit positive stain reaction (bluish colouration). Fig. 6a-d. Transverse section of the mid-gut of 3 rd instar larva treated with S. lavendulae culture filtrate at a concentration of 226 CFU/cm 2 and stained with H/E; showing; a: histolysis of epithelial cells (arrow) and cytoplasmic vacuolation. b: empty epithelial vesicle. c: some cells with pyknotic nuclei (arrow). d: muscle fibers separated from each other leaving a degenerated area inbetween. mo = the microorganism S. lavendulae; vs = vesicle; cm = circular muscles; de = degenerated area; L = lumen; lm = longitudinal muscle; p = peritrophic membrane. The first noticeable histopathological signs appeared at 48h post-treatment, and became more obvious 5 days post-treatment. Few epithelial cells showed apparent histolysis and cytoplasmic vacuolation (Fig. 6a). The apical brush border of the epithelial cells is destroyed. Some of the epithelial cells lost their ability for secretion as indicated by the empty apical vesicles (Fig. 6b). Other cells exhibited ill-defined cell boundaries and lost their nuclei or appeared pyknotic (Fig. 6c). The muscle fibers of treated individuals were separated from each other leaving a degenerated area in-between. Moreover, this layer showed an obvious separation from the epithelial cells (Fig. 6d). In the epithelial cells of the mid-gut of the control larvae, the protein contents appeared as intensely bluish colouration by the mercury bromophenol blue method. All structures of the cell exhibited positive stainability with varying degrees, reaching its maximum in the nucleus (Fig. 7). Treated 3 rd instar larvae exhibited a marked decrease in the proteins of the mid-gut cells (especially in the cytoplasm) in a dose-dependant manner. At a concentration of 226 CFU/cm 2, there was an obvious decrease in the total mid-gut cell proteins at 5 days post-treatment (Fig. 8). Fig. 8. Transverse section of the mid-gut of 3 rd instar larva exposed to a thin film of culture filtrate of S. lavendulae at a concentration of 226 CFU / cm 2 and stained with mercury bromophenol blue, showing: an obvious decrease in the total protein content in the epithelial cells especially in the cytoplasm. cm = circular muscles; de = degenerated area; E = epithelial layer; L = lumen; lm = longitudinal muscle; p = peritrophic membrane. DISCUSSION: In the present study, larval growth was inhibited in response to S. lavendulae culture filtrate treatment. Mortality of the 3 rd instar larvae of S. littoralis exposed to a thin film of S. lavendulae culture filtrate was dose dependent. Mortality was caused mainly by moult disruption and morphogenetic defects. It is not clearly determined if the mortality was caused by direct contact with the secondary compound(s) released by the tested microorganism or by residues present on the surface of the larvae while walking on the treated Petri dish. However, inhibition of larval growth in the bioassay can arise through the chemical compounds in the culture filtrate released from S. lavendulae and their spores. The same finding was reported by Thompson et al. (1997). They found that, spinosyns A and D which were derived from Saccharopolyspora spinosa had strong insecticidal activity. When the 6 th instar S. littoralis larvae were exposed to sawdust treated with the entomopathogen, Serratia marcescens (Eubacteriales: Enterobacteria), this pathogen decreased adult emergence and increased the percentage of malformations (El- Sheikh, 2006). The strong insecticidal activity of S. marcescens was due to a metalloproteaselike insecticide (MPLI) released from this pathogen (Matsumoto et al., 2003). The present

5 Sakr HH, Toxicity of Streptomyces lavendulae (Streptomycetaceae) Culture 201 results also coordinate those of Abdel-Aal (2006), who reported that, B. thuringensis kurstaki administered orally to the 4th larval instar of S. littoralis decreased the percentages of pupation and adult emergence compared to the control. The mid-gut epithelial cells were affected by the used filtrate and showed signs of pyknosis manifested as presence of condensed chromatin, with some vacuolation. This process is generally considered as an important mechanism by cell against pathogenic and toxic agents (Dougherty et al., 2006). Similar histopathlogical findings have been reported by many authors on the toxicity action of other pathogenic toxins, indicating that this action in most cases was due to paralysis in the mid-gut of larvae. For instance, when Manduca sexta larvae were injected with exotoxin protein extracted from Photorhabdus luminescens, the mid-gut epithelium was disintegrated leaving space between the basal lamina and the disintegrated epithelium, which has pyknotic nuclei (Daborn, et al., 2002). Feeding S. littoralis larvae on artificial media mixed with soluble protein extracted from Metarhizum anisopliae, caused deterioration and destruction of the mid-gut epithelium (Quesada-Morga et al., 2006). Comparable histological changes have been reported in the mid-gut epithelium of S. littoralis larvae fed on a botanical extract of Artemisia monosperma (Sakr and Abo-El- Mahasen, 2006). In the 3 rd instar larvae of S. littoralis exposed to 5-aminopyrazolopyrazolone the mid-gut epithelium showed clear signs of pyknosis (Sakr, 2007). The obvious reduction of the protein content in the mid-gut epithelium of the treated larvae could be due to disturbance in protein metabolism that affected by the histological alterations. The present results find confirmation by Abdel-Aal (2006) and Abuo-El- REFERENCES: Abbott WS A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18: Abdel-Aal AE Effect of chlorfluzuron, nuclear polyhydrosis virus (SLNPV) and Bacillus thuringienesis on some biological and enzymes activity of cotton leaf worm, Spodoptera littoralis (Boisd.) Bull. Entmol. Soc. Egypt, Econ., 32: Abuo-El-Mahasen MM Histochemical effects of abamectin and Bacillus thuringiensis on the mid-gut and the fat bodies of the cotton leafworm, Spodoptera littoralis (Lepidoptera: Noctuidae) larvae. Sci. J. Fac. Sci. Menufia Univ., 21: Arai T, Yazawa K, Takahashi K, Maeda A, Mikami Y Directed biosynthesis of new saframycin derivatives with resting cells of Streptomyces lavendulae. Antimicrob. 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6 202 (Lepidoptera: Noctuidae). Bull. Entmol. Soc. Egypt, Econ. 32: Finney DJ Probit Analysis 3 rd ed. Cambridge University Press, New York. Hiort J, Maksimenka K, Reichert M, Perovic- Ottstadt S, Lin WH, Wray V, Steube K, Schaumann K, Weber H, Proksch P, Ebel R, Muller WEG, Beringmann G New natural products from the sponge-derived fungus Aspergillus niger. J. Nat. Prod., 67: Hosny MM, Topper CP, Moawad GG, El-Saadany GB Economic damage thresholds of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) on cotton in Egypt. Crop Prot., 5: Ishikawa M, Takahashi S, Takatsu T, Moriguchi T, Horiuchi N, Wanibuchi K , a novel nematocide from Streptomyces lavendulae SANK J. Antibiot., 56(3): Johnson DA, August PR, Shackleton C, Liu H, Sherman DH Microbial resistance to mitomycins involves a redox relay mechanism. J. Am. Chem. Soc., 199: Khazanie R Elementary Statistics. Good Year Publishing Co., California, USA. 488PP. Koga D, Isogai A, Sakuda S, Matsumoto S, Suzuki A, Kimura S, Ide A Specific inhibition of Bombyx mori chitinase by allosamidin. Agric. Biol. Chem., 51: Madigan TM, John MM, Jac P Brock Biology of Microorganisms (3 rd Ed.). Chapter 16: Prentice Hall Incorporation. Martinez SS, Van Emden HF Growth disruption, abnormalities and mortality of Egypt. J. Exp. Biol. (Zool.), 3: (2007) Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) caused by Azadirachtin. Neotrop. Entomol., 30(1): Matsumoto H, Tanaka K, Noguchi H, Hayakawa Y Cause of mortality in insects under severe stress. Eur. J. Biochem., 270: Quesada-Morga E, Carrasco-Diaz A, Santiago- Alvarez C Insecticidal and antifeedant activities of protein secreted by entomopathogenic fungi against Spodoptera littoralis (Lep., Noctuidae). J. Appl. Entomol., 130(8): Sakr HH, Abo-El-Mahasen MM Biological and biochemical studies on Spondoptera littoralis (boisduval) (lepidoprera: Noctuidae), treated with Artemisia monosperma (Compositae) extracts. Proc. 4 th Int. Con. Biol. Sci. (Zool.): Sakr HH. 2007: Insecticidal activity manifested by two new synthetic pyrazolone compounds on Spondoptera littoralis (Boisd) (Lepidoptera: Noctuidae). Eypt. J. Exp. Biol. (Zool.), 3: Thompson GD, Michel KH, Yao RC, Mynderse JS, Mosburg CT, Worden TV, Chio EH, Sparks TC, Hutchins SH The discovery of Saccharopolyspora spinosa and a new class of insect control products. Down to Earth, 52(1): 1-5. Wang L, Vining LC I- Control of growth, secondary metabolism and sporulation in Streptomyces venezulae ISP5230 by jadw1, a member of the afsa family of γ-butyrolactone refulatory genes. Microbiology, 149: (Streptomycetaceae) التا ثيرات السامة للمستخلص الخام لبكتيريا Streptomyces lavendulae على دودة ورق القطن الكبرى Spodoptera littoralis (حرشفية الا جنحة: نوآتويدى) هانم حامد صقر قسم علم الحيوان- آلية العلوم- جامعة النوفية السامة التا ثيرات تقييم البحث هذا تناول لترآيزات مختلفة من المستخلص الخام لبكتيريا Streptomyces lavendulae (Streptomycetaceae) على العمراليرقى الثالث لدودة ورق القطن الكبرى تحت وذلك Spodoptera littoralis (Boisd) الظروف المعملية وباستخدام طريقة الملامسة وآانت ولقد وجد أن الجرعة 24 ساعة. فترة المعاملة لمدة سم 2 وذلك وحدة/ المميتة لنصف العدد تساوى ولقد أظهرت النتاي ج أن أيام من المعاملة. 7 بعد 2 /سم من هذا 226 وحدة معاملة العمر اليرقى بترآيز الا فراد لموت تراآمية نسبة أعطت المستخلص آما انخفضت نسبة خروج 8 82 المعاملة بمقدار أحدث المستخلص بنسبة الا فراد الكاملة المستخدم العديد من التغيرات النسيجية للمعى 2 /سم 226 وحدة الوسطى للا فراد المعاملة بترآيز حيث حدث تحلل المعاملة. أيام من خمسة بعد ل بعض الخلاي ا الطلاي ي ة م ع ظه ور فج وات سيتوبلازمية. آما حدث انفصال للا ليا ف العضلية لطبقة العضلات. وفى ذات الوقت أدت معاملة اليرقات بالمستخلص البكتيرى الى نقص ملحوظ فى المحتوى البروتينى لخلايا المعى الوسطى وخاصة فى السيتوبلازم آما ظهر من القطاعات النسيجية المصبوغة بطريقة. mercury bromophenol blue وتوضح هذه الدراسة ولا ول مرة إمكانية إستخدام المستخلص الخام لهذا النوع من البكتيريا آمبيد حشرى لمكافحة دودة ورق القطن الكبرى. المحكمون: أ.د. صلاح عثمان قسم علم الحيوان علوم الا سكندرية أ.د. أمال إبراهيم سيف قسم علم الحيوان علوم طنطا