Biochemical and Histological Variations Induced by IGRs in Spodoptera littoralis (Boisd.)

Glob. J. Environ. Sci. Toxicol., Vol. 1(2): PP. 163 - 178 (2014)

Massive Publisher House M.P.H. Egypt www.mphegypt.com ISSN 2356-6264

Biochemical and Histological Variations Induced by IGRs in Spodoptera littoralis (Boisd.) Sabry, H. M.* and Khedr, M. A. Plant Protection Research Institute, ARC, Giza, Egypt. *Corresponding author: Email: [email protected]

ABSTRACT

The susceptibility of the laboratory 4th instar larvae of cotton leafworm,

Spodoptera littoralis (Boisd.) to the treatments with four Insect Growth Regulators (IGRs) belonging to two different groups, chitin synthesis inhibitors (flufenoxuron and teflubenzuron) and molting hormone agonist (methoxyfenozide and tebufenozide) was evaluated. Flufenoxuron exhibited high level of toxicity with low LC50 value (2.05 ppm) followed by methoxyfenozide (4.25 ppm), teflubenzuron (8.95 ppm) and finally tebufenozide (66.04 ppm).The deleterious histological observations in the cuticle and mid-gut of S. littoralis showed that this instar is very sensitive to the all tested IGRs. Destruction in cuticle layers, fissures in endocuticle and irregular distribution of hypodermal cells, disintegration and destruction of the gut epithelium and its separation from the basement membrane were detected with different degrees depending on the sort of IGR. Treatment with teflubenzuron caused decrease in chitinase and increase in protease, phenoloxidase and trehalase activities, while the activity of chitenase was significantly elevated using Tebufenozide. The treatment with flufenoxuron reflected highest significant reduction in the activity of trehalase enzyme. Key words: Spodoptera littoralis, IGRs, biochemical, histological, toxicological.

1) INTRODUCTION

Undoubtedly, among more than 1300 pest species recorded on cotton, the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) is considered as the most important cotton pest that is found almost everywhere cotton is grown in Egypt (Hosny and Isshak, 1967). The control strategy was based mainly on uses of synthetic insecticides but most are toxic to all animals including human beings, and persistent in environment, number of them have carcinogenic and mutagenic effects on human, domestic animals, birds and predators.

Insect growth regulators (IGRs) generally have a good margin of safety for most nontarget biota including invertebrates, fish, birds and predators (Duffie et al.,

Sabry, H. M. and Khedr, M. A., (2014)

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1998 and Badawy and El-Arnaouty, 1999). They are relatively safe for human beings and domestic animals, thus its offer a good alternative for a selective insect pest control that is in harmony with existing IPM programs.

Insect growth regulators can be divided according to their mode of action into two major groups; chitin synthesis inhibitors (i. e. of cuticle formation) and substances that interfere with the action of insect hormones (i. e. Juvenile Hormones (JHs) and ecdysteroids) (Tunaz and Uygun, 2004).

The insect cuticle serves as an interface between the living organism and its environment, forms the exoskeleton, supporting the linings of the gut, respiratory systems, reproductive ducts and some gland ducts. It consists primarily of protein and chitin fractions. Chitin is a very important constituent of the cell walls in the integuments of invertebrates (arthropods). Most chitin synthesis inhibitors (CSIs) are primarily used as larvicides. Treated larvae develop until molting, but fail to ecdyse due to inhibition of the synthesis of new cuticle, specifically, chitin biosynthesis (Tunaz and Uygun, 2004). Another group of IGRs is the molting hormone agonist (diacylhydrazines) that exert the same action as 20-hydroxy-ecdysone such as tebufenozide and methoxyfenozide. The bisacylhydrazine derivatives are nonsteroidal ecdysteroid agonist that mimic the action of molting hormones and induce a precocious and incomplete molt in several insect orders (Khebbeb et al., 2008).

Therefore, the major goal of the present work is to clarify the role played by the sublethal dose (LC50) of four IGRs belonging to CSIs and molting hormone agonist on the 4th instar larvae of S. littoralis in order to better understand the action of these compounds on lepidopterous larvae through studying biochemical and histological parameters. This is attained by estimating activity of some enzymes affecting in cuticle formation, as well as histological changes in treated larvae.

2) MATERIALS AND METHODS 2.1. Rearing technique of S. littoralis:

S. littoralis strain used in this study is a laboratory susceptible strain reared in the Plant Protection Research Institute at Zagazig, Sharquia Governorate according to El-Defrawi et al., 1964. The culture was maintained under optimum conditions (27 ºC ± 2 and 70±5 R.H) and reared on fresh castor bean leaves until the 4th larval instar which used in this study.

2.2. Tested IGRs: Chitin synthesis inhibitors (CSIs): 2.2.1 Teflubenzuron, Nomolt® 15% (SC), N-[[(3,5-dichloro-2,4-difluorophenyl) amino]carbonyl]-2,6-difluorobenzamide). a trademark of BASF Co. Rate: 50 cm3 / 100 L 2.2.2 Flufenoxuron, Cascade® 10% EC, [4-[2-chloro 4-(trifluoromethyl phenoxy)] 2-flurophenyl 3-(2,6-difluorophenzoyl) urea a trademark of BASF Co. Rate: 200 cm3./feddan.

Biochemical and Histological Variations Induced by IGRs in Spodoptera littoralis (Boisd.)

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2.3. Molting hormone agonist: 2.3.1 Methoxyfenozide, Runner® 24% SC, (3-methoxy-2-methylbenzoic acid 2-(3,5-dimethylbenzoyl)-2-(1,1-dimethylethyl) hydrazide) a trademark of Dow Agro Science Co. Rate: 150 cm3 / feddan. 2.3.2 Tebufenozide, Mimic® 24% EC, (3,5-dimethylbenzoic acid 1-(1,1-dimethylethyl)-2-(4-ethylbenzoyl) hydrazide) a trademark of Dow Agro Science Co. Rate: 350 cm3 /feddan. 2.4. Bioassay:

Leaf-dipping bioassay method was used to determine the median lethal concentration (LC50) value of the tested IGRs. Series concentrations (in water) of the tested formulated compounds were prepared. Castor bean leaves were dipped for ten seconds in each concentration then left to dry. The treated leaves were offered to newly molted 4th instar larvae for 48 hr. then replaced by untreated ones for 24 hr. Mortality percentages were recorded after 72 hr. and corrected according to Abbott's formula, 1925 to estimate the LC50 values, the corrected mortality percentages were subjected to probit analysis according to Finney, 1971. Toxicity index was estimated by Sun, 1950.

Accordingly to the mode of action of IGRs, the mortality of larvae began after three days of treatment, thus in this paper we try to study the histological and biochemical changes. So samples for analysis been taken 48 hr. post treatment with LC50 of tested IGRs and before the onset of the mortality and the appearance of symptoms of molting process failure. 2.5. Histopathological studies:

All tested IGRs were applied to the 4th instar larvae using the leaf dipping technique method at LC50 values. The larvae were taken after 48 hrs post treatment then transferred into alcoholic Bouin’s fixative followed by dehydration through a series of ethanol, cleared in xylene and imbedded in wax. Serial sections at five microns were made by microtome and mounted on clean slides using Mayer’s albumin. Sections were mounted on glass slides and stained with Haematoxylene and Eosin prepared for observation and photomicroscopy Humason and Freeman (1979). 2.6. Preparation of samples for biochemical studies:

The preparation of samples involved the use of the 4th instar larvae of S. littoralis after 48 hr of all treatments at LC50 level and control. The larvae placed in clean jars away of food and starved. The starved larvae were homogenized in distilled water using a Teflon homogenizer surrounded with jacket of crushed ice for three minutes. Homogenates were centrifuged at 3500 r.p.m for ten minutes at 5ºC to remove haemocyte and the supernatants were used directly for the biochemical analysis.


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2.7. Enzyme measurements: - Trehalase enzyme was determined using the method of Ishaaya and Swiriski

(1976) using trehalose as substrate. - Chitinase activity was determined using 3, 5- dinitrosalicylic acid reagent to

determine the free aldehydic groups of hexoamines liberated on chitin digestion according to the method described by Ishaaya and Casida (1974).

- Phenoloxidase was determined according to a modification of Ishaaya (1971). - Protease enzyme was measured according to the method described by Tachell et

al., (1972) with some modifications by measuring the increase in free amino acids split from substrate protein (albumin), during one four incubation at 30 ºC.

3) RESULTS AND DISCUSSION 3.1. Toxicological studies:

The toxicological studies of four insect growth regulators, the CSIs (flufenoxuron and teflubenzuron) and molting hormone agonist (methoxyfenozide and Tebufenozide) were represented in Table (1) which showed the levels of sensitivity of S. littoralis larvae to the tested IGRs three days post treatment. Treatment of S. littoralis larvae with each tested compound revealed that, flufenoxuron was more toxic to the tested cotton leafworm than the other IGRs; teflubenzuron, methoxyfenozide and tebufenozide, the LC50 values were 2.05, 8.95, 4.25 and 66.04 ppm, respectively. The same trend was obtained in LC90 values except that teflubenzuron and methoxyfenozide that change their places and recorded 50.14 and 149.38 ppm, respectively. Table (1): Toxicity data of different IGRs against 4th instar

larvae of laboratory S. littoralis strain.

Tested insect growth regulators (IGRs)

LC50 (ppm)

(Lower-Upper)

LC90 (ppm)

(Lower-Upper) Slope

*Toxicity index

Flufenoxuron (Cascade)

2.05 (0.84-5.42)

30.57 (20.41-60.76)

1.83 100 Chitin

synthesis inhibitors

(CSIs) Teflubenzuron (Nomolt)

8.95 (3.19-12.02)

50.14 (39.45-128.24)

1.76 22.91

Methoxyfenozide (Runner)

4.25 (1.81-6.84)

149.38 (85.84-421.39)

0.80 48.24 Molting hormone agonist

Tebufenozide (Mimic)

66.04 (50.08-85.29)

989.77 (672.31-1820.51

1.12 3.10


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*Toxicity index = x100

Growth and development of insects are under the control of hormones

including ecdysteroids and juvenile hormones (JH). Ecdysone is responsible for cellular programming and together with JH, initiating for the molting process. Any disturbance in the normal hormone balance may cause a crucial disorder in the growth and development of insects that leading to death. JHs control a number of processes such as molting, metamorphosis and reproduction. Dhadialla et al., 2005 found that tebufenozide has a similar hormonal activity with the natural hormones in the insects and particularly in Lepidoptera of which the principal effect is to cause incomplete and lethal molts.

Most CSIs are primarily used as larvicides. Treated larvae develop until molting, but fail to ecdyse due to inhibition of the synthesis of new cuticle, especially, chitin biosynthesis, Khedr (2002) manifested the symptoms of death due to the exposure of S. littoralis larvae to the tested IGRs (flufenoxuron, chlorfluazuron, benzoylphenyl urea, lufenuron and tebufenozide), larvae became inactive, alive without feeding, appeared to be wet, completely paralyzed, dark color and viscous excretion comes out the larval body, then it became unable to ecdyse.

Moreover, Zidan et al., (2013) found that lufenuron, chlorfluazuron and emamectin benzoate exhibited distinguished high preventive effect on reducing fecundity and ovicidal effectiveness of S. littoralis. 3.2. Histopathological studies: 3.2.1. Cuticle and muscles:

The normal integument basically composed of three layers, one cellular layer, the epidermis (hypodermis) and two non cellular layers; the outer cuticle above the epidermis and the inner basement membrane underneath the epidermis, Plate I (A). Histological features resulting from the treatment of the cotton leaf worm, S. littoralis larvae with the tested compounds were observed. Flufenxuron revealed a separation of the hypodermal cells from endocuticle, destruction of basement membrane and appearance of vacuoles between endocuticle and hypodermis Plate I (B). Teflubenzron showed severe damage on the integument, the epidermal cells were highly impaired being irregular and complete disintegration in the hypodermis were obvious Plate I (C). The two chitin synthesis inhibitors compounds have dystrophic changes on larval muscles where the myofibrils grouped into masses separated by vacuoles and fissure Plate I (B & C).

Methoxyfenozide and tebufenozide appear to cause abnormalities in the shape of epicuticle, distortion in the endocuticle this distortion revealed blockage of its formation the hypodermal cells separated from the endocuticle leading to abnormal endocuticular deposition and abortive molting and the thickness of the cuticle decreased Plate I (D & E, respectively). The effect of the molting hormone agonist on muscles ranged between slight degeneration as the occurrence of fissures to complete destruction of the whole tissue, this is attributed to the destruction of the sarcolemma Plate I (D & E).

LC50 of the most efficient compound LC50 of the other compound


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The result are in agreement with the findings of Hassan (2009) where the 6th larval instar of S. littoralis resulting from the 4th larval instar treated with LC50 of methoxyfenozide revealed a separation of the hypodermal cells from the endocuticle and the hypodermal cells showed some fissures. Distortion in the endocuticle was also quite visible. This distortion revealed blockage of its formation showed destruction in the cuticle layers, fissures in the endocuticle and irregular distribution of the hypodermal cells. Elbarky et al. (2008) showed that, the amount of total carbohydrates, total proteins, carbohydrate hydrolyzing enzymes were significantly decreased when S. littoralis treated with spinetoram. These components are necessary for building cuticle and lake of them may lead to abnormal endocuticular deposition and abortive molting. These results accordance with the demonstrated by El-Sheikh et al. (2005).


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cm: circular muscle dm:degenerated muscles en: endocuticle ep: epicuticle g: cytoplasmic granules hy: hypodermis m: muscles v: vacuoles

E


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b: basement membrane cb: ciliated border cm: circular muscles dec: destroyed epithelial cells ec: epithelial cells f: food materials l: lumen lm: longitudinal muscles p: papillary crypt pt: peritrophic membrane re: regenerative cells v: vacuoles


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3.2.2. Mid gut: The normal mid gut epithelium consists of tall columnar cells which contain

large nuclei in the middle of apical region and is interspersed apically with the goblet cells and basally with regenerative cells. The columnar cells possesses a fine brush border facing towards lumen. Large number of vesicles discharging into extra peritrophic region of the lumen. The peritrophic membrane is well evident in the lumen of the mid gut Plate II (A). The histological structure of the mid gut of treated larvae shows many changes, the mid gut epithelial cells in most areas appeared to be completely disorganized while cell boundaries disappeared and separated from the basement membrane.

The peritrophic membrane was not loosely lying to the epithelial cells and the space in between the epithelium and peritrophic membrane was filled with few cytoplasmic vesicles. Slight vacuolation was apparent in some cases. In addition, the surrounding muscles appeared to be detached from the epithelium in some areas with different degrees using flufenoxuron, teflubenzuron, methoxyfenozide and tebufenozide (Plate II, B-E, respectively).

These results are well agreed with the findings obtained by Federici (1993) who found that the ingestion of toxicant by the insects releases a toxic peptide which binds to sites on the microvillus membrane of the mid gut causing cytolysis. Hussein and Hamed (2004) reported that histopathological features resulting from the treatment of herbicides, plant growth regulators and their potential interactions with insect growth regulators against potato tuber moth Phthorimaea operculella the epithelium cells being destroyed and exfoliated from basement membrane, the apical zone of the epithelium cells appeared empty, the basement membrane and muscular layer were degenerated. Dahi et al., (2011) mentioned that the epithelial cells were completely ruptured after treatment with pyridal and separated from the basement membrane. Abdel-Aal et al., (2012) when added Flufenxuron (LC10) to SpliMNPV (LC50) showed histopathological effects to midgut e.g. loss of the compact appearance of the muscular layer, exfoliation and vacuolization of the midgut epithelium. The peritrophic membrane was completely disrupted. 3.3. Biochemical studies:

The changes in activities of different enzymes related to the cuticle synthesis of 4th instar larvae of S. littoralis (laboratory strain) as response of treatment with the LC50 of the tested compounds, data were expressed as percentages in the changes in the activities of enzymes relative to control. 3.3.1. Chitinase enzyme:

The response in larval chitinase activity to the treatment with the LC50 values of the tested compounds tabulated in Table (2). The results obtained showed remarkable significantly increase in the enzyme activities in the larvae treated with tebufenozide 30.83% compared to control. In reverse effect, teflubenzuron caused significant reduction in chitinase activity (-3.82%). 3.3.2. Protease enzyme:

Table (2) shows the changes in protease enzyme. In general, the treatment with tested IGRs caused different levels of significant decrease in protease activity, except teflubenzuron that gave the highest significant increase (8.29% relative to


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control). The highest decrease in the activity was recorded in larvae treated with methoxyfenozide (-15.68%) followed by tebufenozide (-8.78%) and finally flufenoxuron (-7.34%). 3.3.3. Phenoloxidase enzyme:

Treated 4th instar larvae of S. littoralis with IGRs recorded elevation in phenoloxidase activity in the case of CSIs that gave (49.69, 1.34% relative to control) for flufenoxuron and teflubenzuron, respectively. On the other hand, molting hormone agonist decreased phenoloxidase activity comparing to control as follows: methoxyfenozide (-20.91%) and tebufenozide (-3.84%), Table (2). 3.3.4. Trehalase enzyme:

Data in Table (2) revealed the changes in trehalase enzyme, only flufenoxuron gave significant reduction in trehalase activity (-18.28% relative to control). The rest IGRs can be ordered dissentingly as follows: 23.72; 14.19 and 5.75% for methoxyfenozide, teflubenzuron and lastly tebufenozide treated larvae.


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Table (2): Changes in tested enzyme activities of 4th instar S. littoralis larvae treated with LC50 of insect growth regulators.

Data expressed as Mean ± S. E. ***= p≤ 0.01 Mean under each variety having different letters in the same raw denote a significant different (p≤ 0.05).

% of Control = X100Test – Control

Control


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Generally, the entire used IGRs caused disturbance in the enzyme balances either decrease or increase comparing to control. The significant elevation in the activities of chitenase, protease, phenoloxidase and trehalase enzymes caused by tebufenozide, teflubenzuron, flufenoxuron and methoxyfenozide, respectively. On contrary, the significant reduction in chitinase and trehalase was observed by teflubenzuron and flufenoxuron. Methoxyfenozide gave significant reduction in both protease and phenoloxidase enzymes.

The reason behind deformations and mortality may be a result of changes in chitinase, protease, phenoloxidase and trehalase enzymes. Ecdysis is initiated by apolysis, the process that separates epidermal cells from the old cuticle by molting fluid secretion and ecdysal membrane formation. Molting fluid contains protease and chitenase enzymes that digest the main constituents of the old endocuticle (Reynolds and Samuels, 1996). Synthesis of chitin depends on the action of the cellular enzyme chitin synthesis attached to the plasma membrane. However, this enzyme is produced as a zymogen (inactive) in the endoplasmic reticulum of the epidermis and has to be activated by proteases of chitin synthesis (Hepburn, 1985). The enzyme phenoloxidase (PO) is one of the first immune molecules that was identified in invertebrates. Recently, the immune function of PO has been challenged. Bidla et al., (2009) tested how PO is activated following injury in two insects (the fruit fly, Drosophila mealnogaster and the wax moth, Galleria mellonella). Rapid PO activation in Drosophila was limited to discrete areas of the haemolymph clot that forms after injury. The elevations of chitinase and phenoloxidase were indicated by Verloop (1977) as secondary effect of acylureas (diflubenzuron), and in 1994, Lee et al., found that, the treatment of Hyphantria cunea with the LC50 of tebufenozide and other insect growth regulators caused increase in chitinase activity compared to untreated larvae. Amin and Mohamady (2009) found that, methoxyfenozide had no effect on chitin production in the newly ecdysed 4th instar larvae of S. littoralis but endocuticle chitin degradation during molting might be depressed as illustrated by the presence of higher amount of N-acetylglucosamine in control larvae than methoxyfenozide treated. It is suggested that methoxyfenozide might induce a precocious molt by initiating molting cycle, but its main toxic effect is due to the fact that its level remains high during molting and do not decrease for allowing other hormones necessary for successful molting to be released.

Trehalase enzyme has the important function for liberating glucose for energy and activated during molting to generate glucose for chitin build up (Ishaaya and Ascher, 1971 and Meisner et al., 1978), also it is an important physiological process for various organisms such as insect flight and the resumption of growth in resting cells. Khedr et al., (2005) noticed an activity increase of trehalase enzyme when 4th instar larvae of S. littoralis were treated with Consult, Atabron, Match, Mimic and Cascade.

Data obtained in the present work could help to suggest the lethal effect of IGRs may be occurred through the alternation of enzyme balances of chitinase, protease, phenoloxidase and trehalase depending on the kind of used IGRs (either chitin synthesis inhibitor or molting hormone agonist) since these enzymes played a known role in ecdysis and metamorphosis.


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4) CONCLUSION

In the light of the present findings, it could be concluded that the four tested IGRs belonging to two different groups; CSIs and MHAs exhibited pronounced histological and biochemical effects against 4th instar larvae of S. littoralis. Additionally, flufenoxuron (CSI) showed high level of toxicity comparing to other treatments, such knowledge may help in the control strategies of S. littoralis.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Hala Mead and Dr. Samah Nour, Plant Protection Research Institute, Sharquia Governorate for their valuable help and support in this work.

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التغیرات البیوكیمیائیة و التشریحیة الحادثة بمنظمات النم�و الح�شریة ف�ى دودة

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هند حممد صربى و حممد حممد امحد خضر

مصر– جیزة – الدقى –معھد بحوث وقایة النباتات

تم دراسة حساسیة العمر الیرقى الرابع لساللة ح�ساسة م�ن دودة ورق القط�ن معملی�ا وذل�ك

المعاملة بأربع منظمات نم�و ح�شریة تنتم�ى ال�ى مجم�وعتین مختلفت�ین ھم�ا مثبط�ات تخلی�ق عن طریقمیثوك��سى فینوزی��د و (و م��ضادات ھرم��ون اإلن��سالخ ) فلوفینوك��سیرون وتیفل��وبنزیرون(الكیت��ین

٢,٠٥(أظھر مركب فلوفینوكسیرون سمیة عالیة مع التركیز القاتل لنصف تعداد األف�راد ). تیبوفینوزید ٨,٩٥(ث�م تیفل�وبنزیرون ) ج�زء ف�ى الملی�ون٤,٢٥(یلی�ھ مرك�ب میثوك�سى فینوزی�د ) ى الملیونجزء ف

أدت المعامالت الى ظھ�ور ). جزء فى الملیون٦٦,٠٤(و أخیرا مركب تیبوفینوزید ) جزء فى الملیونتأثیرات ھستولوجیة واض�حة ف�ى ج�دار الج�سم خاص�ة منطق�ة الجلی�د و الع�ضالت حی�ث ت�شققت طبق�ة

و انفصلت ع�ن طبق�ة تح�ت الجلی�د باالض�افة ال�ى تحل�ل خالی�ا الغ�شاء القاع�دى و ظھ�ور بع�ض الجلیدالفجوات و كذلك تحلل األلیاف العضلیة كما أثرت المعامالت فى منطقة القناة الھضمیة خاص�ة منطق�ة المع�ى األوس�ط حی�ث اختف�ت بع�ض الخالی�ا وظھ�رت بع�ض الفج�وات ف�ى الخالی�ا الطالئی�ة العمودی�ة

ق�ات المعامل��ة موض��حة الت�أثیر الق��وى لمنظم��ات النم�و الح��شریة الم��ستخدمة ب�درجات مختلف��ة تبع��ا للیرأدت المعاملة بمركب تیفلوبنزیرون الى خفض فى نشاط انزیم كیتینی�ز و زی�ادة ف�ى . للمركب المستخدم

دة معنویة فى نشاط انزیمات بروتییز وفینول اكسیدیز و تریھالیز بینما أدى مركب تیبوفینوزید الى زیاكما أحدثت المعاملة بمرك�ب فلوفینوك�سیرون ال�ى خف�ض معن�وى كبی�ر ف�ى ن�شاط . نشاط انزیم كیتینیز

.انزیم تریھالیز

Biochemical and Histological Variations Induced by IGRs in Spodoptera littoralis (Boisd.)

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Transcript of Biochemical and Histological Variations Induced by IGRs in Spodoptera littoralis (Boisd.)