BIOCHEMICAL STUDIES OF THE MIXTURES OF SOME INSECTICIDES WITH SURFACTANTS ON THE 4TH INSTAR LARVAE OF COTTON LEAFWORM, SPODOPTERA LITTORALIS BOISD.

BIOCHEMICAL STUDIES OF THE MIXTURES OF SOME INSECTICIDES WITH SURFACTANTS ON THE 4^TH INSTAR LARVAE OF COTTON LEAFWORM, SPODOPTERA LITTORALIS BOISD.

Hala  M. Mead^*; B. A. Soliman^**; A. A. El-Sheakh^*;   A. H. Abo-Ghalla^** and  W. M. H. Desuky^*

*Plant Protection Research Institute, Agricultural Research Center, Giza, Egypt.

**Faculty of Science, Suez Canal University, Egypt.

ABSTRACT

The effect of spinosad and triflumuron either alone or as mixtures with two surfactants, Triton X-100 and Tween-20 on some biochemical response of 4^th instar larvae of S. littoralis was studied after 24, 48 and 72 hours of treatment. All the tested treatments significantly decreased the effect of total soluble protein more than control at different time intervals especially after 24 hr. Also, the mixtures reduced effect compared to spinosad and triflumuron alone. The mixture of spinosad at the level of (LC₄₀) with Triton X-100 gave the highest reduction in effect. As for transaminase enzymes, all treatments caused decrease in the activates of glutamic oxaloacetic transaminace (GOT) and glutamic pyruvic transaminase (GPT) through the tested time intervals as compared to control. Moreover, the mixtures recorded a great reduction in the activities than the selected agents alone. The mixtures of spinosad (LC₄₀) and triflumuron (LC₄₀) were the most effective.

Key words: Spodoptera littoralis, spinosad, triflumuron surfactants, biochemical, total soluble protein, transaminase enzymes, GOT, GPT, Triton X-100, Tween-20.

INTRODUCTION

The cotton leafworm, Spodoptera littoralis is the most injurious and wide spread pest of different crops in Egypt. Spinosad is the first product in the new naturalyte class of insect control,  that gave a promise effect against S. littoralis. Similarly, insect growth regulators proved to be diversely affect against S. littoralis.

            Protein is among the most important compounds of insect that bind with foreign compounds. The decrease of the total protein in treated larvae may reflect the decrease in the activity of various enzymes (El-Kordy et al., 1995). The building block for protein synthesis comes from the amino acids pool maintained by transaminase enzymes (Meister, 1957).

GOT and GPT in insects are the most active transaminase enzymes (Kilby and Neville, 1957 and Carbetree and Newsholme, 1970). The amino transaminase especially GPT is one of the components of oxidative metabolism of protein (Bursell, 1963).

MATERIALS AND METHODS

1. Tested compounds

1.1. Insecticides:

A. Trade name: Tracer.

Common name: Spinosad 24% Suspension Concentrate (SC). Spinosad is comprised primarily of two macro cyclic lactones, Spinosyn A and D, secondary metabolites produced by the actinomycete, Saccharopolyspra spinosa under natural fermentation condition.

Rate: 50 cm³ / feddan.

Action: Naturalyte insecticide.

      Basic Product: DowElanco.

B. Trade name: Alsystin (48% SC).

Common name: Triflumuron.

Chemical name: 2-chloro–N-[[[4-(trifluoromethoxy) phenyl] amino] carbonyl] benzamide.

Rate: 100 cm³ / feddan.

Action: insect growth regulators (benzophenyl urea).

Basic product: Bayer.

1.2. Surfactants:

A. Triton  X-100.

      Octyle phenoxy poly ethoxy ethanol.

B. Tween-20.

     Poly oxi ethylene (20) sorbitan monolaurate.

2. Rearing technique of S. littoralis :

Egg masses were reared on leaves of castor bean oil, Ricinus communis according to El-Defrawi et al., (1964) under laboratory conditions 27±1 °C and 70±5% R.H.

3. Biochemical assays:

The preparation of samples involved the use of the 4^th instar larvae after 24, 48 and 72 hr. of treatment with two different concentrations of triflumuron and spinosad at the levels of (LC₃₀ and LC₄₀) alone and as mixtures with surfactants. Each of them was used at 2.11 ppm. when tested with triflumuron and at 0.397 ppm.  with spinosad.

The healthy 4^th instar larvae of S. littoralis were picked up from each concentration and control applied and placed in clean jars, and then starved for 4 hours The starved larvae were homogenized in distilled water (10 larvae/10 ml) using a teflon homogenizer surrounded with jacket of crushed ice for 3 minutes. The homogenate was centrifuged at 3500 r.p.m. for 10 minutes at 5 °C to remove the haemocytes. The samples were divided into small portions and kept in a deep freezer at (-20 °C) until required. The supernatant was immediately assayed to determine total soluble protein; the activity of transaminase enzymes, (GOT) and (GPT).

Colourimetric determination of total soluble protein in total homogenate of larvae was carried out as described by (Gornall et al., 1949) and The activities of GOT and GPT enzymes according to the method of Reitman and Frankle (1957).

4. Statistical analysis:

Data were analyzed using commercial statistical software (SPSS for Windows, release 6.1; SPSS Inc., Chicago, TL). One way analysis of variance (ANOVA) was used to test for significant differences between mean values. Multiple compares of means using Tukey’s honestly significant difference was applied as a posterior test to locate the source of each significant difference (P<0.05).     

RESULTS AND DISCUSSION

1. Total soluble protein:

The changes in the level of total soluble protein in the supernatant of the homogenated larvae were generally decreased as affected by all tested treatments at different time intervals as compared to control. In addition, the mixtures of both spinosad and triflumuron with Triton X-100 and Tween-20 caused decrease in total soluble protein than the selected agents alone     (Table 1). The mixture of spinosad at concentration LC₄₀ with Triton X-100 gave the highest reduction that recorded  -56.79 %, -53.61% and -46.23 %, respectively after 24, 48 and 72 hr. post application. While spinosad alone at LC30and LC₄₀ were recorded (-33.33 %, -23.71 % and -19.81 %) and          (-40.74 %, -34.02 % and -27.36 %) at 24, 48 and 72 hr., respectively. In all treatments, the higher reduction in total soluble protein recorded after 24 hr. then decreased.

The same trend was obtained in the case of triflumuron and its mixtures, except mixture of triflumuron (LC₃₀) with Triton X-100 that gave the higher reduction after 48 hr (-34.02 %). Triflumuron (LC₄₀) with Tween-20

         Table 1: Changes in the effect of total soluble protein (gm./100ml.) in S. littoralis larvae treated with spinosad, triflumuron and their mixtures at different time intervals.

*Data expressed as (Mean ± SE).

 Effect (%) = Treated – Control / Control  × 100 = increase or decrease in the effect relative to control.

caused the greatest decrease (-51.85 %, -50.52 % and -43.40 %) at 24, 48 and  72 hr,  respectively.  Triflumuron at LC₃₀ and LC₄₀ were recorded (-23.46%, -20.62% and –17.92%) and (-39.66%, -25.77% and –23.58%) at different time intervals, respectively.

All the tested treatments caused significant reduction in the total soluble protein than control and triflumuron (LC₃₀) after 24 hours, P = 0.000. While, after 48 and 72 hr, another significant decrease was obtained as affected by all the treatment when compared to control, triflumuron (LC₃₀) and spinosad (LC₃₀)_, P = 0.005 and 0.001, respectively.

These results are supported by the works of (El-Kordy et al., 1995; Sokar, 1995 and Mohamady, 2000) when observed reduction in the level of total soluble protein at all interval times in S. littoralis larvae treated with different IGRs. Also, Abd El-Aziz (2000) and Tawfik et al. (2002) found that pronounced decrease in the total soluble protein with all concentrations used of Dipel-2x. However, Khedr (2002) noticed an increase in the total soluble protein after two days of treating 2^nd and 4^th instar larvae with IGRs, except Cascade followed by marked decrease after five days of treatment.

2. Transaminase enzymes (GOT and GPT):

            Data represented in Tables (2 & 3), show the changes in glutamic oxaloacetic transaminase (GOT (and glutamic pyruvic transaminase (GPT) activities of the supernatant cotton leafworm larvae as a result of treating with spinosad and triflumuron at two different concentrations (LC₃₀ and LC₄₀) and its mixtures with Triton X-100 and Tween-20. Data also indicate that the selected agents alone or as mixtures reduced GOT and GPT activities at the different time intervals comparing to the control. Moreover, the mixtures caused great reduction in the activities than the selected agents alone.

Spinosad and its mixtures revealed that the highest reduction in GOT activity recorded at 72 hr. post application except spinosad at LC₃₀ when mixed with Tween-20 that caused a great reduction in the activity after 24 hr. (-29.17%). The highest reductions were recorded (-37.50%, -37.45% and -37.50%) for the mixture of spinosad (LC₄₀) with Triton X-100 after 24, 48 and 72 hr. post treatment, respectively (Table  2).

            The same trend was obtained in the case of triflumuron and its mixtures with the exception of triflumuron at LC₃₀ when mixed with Triton X-100 that recorded -32.14% after 48 hr. The highest decrease were recorded -33.33%, -35.71% and – 37.50% for triflumuron (LC₄₀) when mixed with Tween-20 at different time intervals, respectively.

The mixtures of spinosad (LC₄₀) and triflumuron (LC₄₀) with the surfactants significantly decreased the activity of GOT comparing with the remaining treatments after 24 hr, P = 0005. Moreover, all the treatments caused significant decrease in GOT more than control after 72 hr, P = 0.0003  (Table  2).

With regard to GPT activity, the higher decrease in the activity were recorded -31.58%, -33.33% and -30.00% for spinosad (LC₄₀) with Triton X-100 after 24, 48 and 72hr., respectively followed by spinosad (LC₄₀) with Tween-20 that recorded –28.07%, -30.16% and -25.715% after different time, respectively. While, spinosad (LC₃₀)  and  spinosad  (LC₄₀)  were  manifested

Table 2: Changes in the activity of transaminase enzyme (GOT, ųg pyruvate-L.) in S. littoralis larvae treated with spinosad, triflumuron and their mixtures at different time intervals.

*Data expressed as (Mean ± S. E.).

 Activity (%) = Treated – Control / Control  × 100 = increase or decrease in the activity relative to control.

(-10.53%, -17.46 % and -14.29 %) and (-12.28 %, -20.63 % and -20.00 %) after 24, 48 and 72 hr., respectively (Table 3).

Triflumuron at two different concentrations LC₃₀ and LC₄₀ caused decrease in GPT activities than control, that recorded (-10.53%, -15.87% and    -12.86%) and (-11.07%, -19.05% and -20.00%) after 24, 48 and 72 hr, respectively. When Tween-20 mixed with triflumuron (LC₄₀) great  reduction

Table 3: Changes in the activity of transaminase enzyme (GPT, ųg pyruvate-L.) in S. littoralis larvae treated with spinosad, triflumuron and their mixtures at different time intervals.

*Data expressed as (Mean ± S. E.).

 Activity (%) = Treated – Control / Control  × 100 = increase or decrease in the activity relative to control.

in the activity comparing to other treatments were obtained and recorded        -29.82 %, -26.98 % and -28.57 % at different time intervals, respectively.

Multiple comparison of means showed significant decrease in GPT activity when mixtures of spinosad (LC₄₀) with Triton X -100 were applied after 24 hr, P = 0.000, and mixtures of spinosad with both Triton X-100 and Tween-20 were used after 48 hr. of treatment, P = 0.001. Furthermore, all the mixtures as spinosad (LC₄₀) and triflumuron (LC₄₀) caused significant reduction in the activity more than the other treatments after 72 hr, P = 0.004 (Table  3).

The decrease in the activities of GOT and GPT may be explained by (Gilbert, 1967 and Abdel Hafez et al., 1988) who found the changes in GOT and GPT activities were in harmony with the changes in protein and amino acids. Many searches studied the effect of different insect growth regulators and biological insecticides on the transaminase enzymes of S. littoralis larvae. The results showed irregular effects, where it fluctuated between increases and decreases through the tested interval times (Salem et al., 1995, Mohamady, 2000 and Tawfik et al., 2002). In addition, Zidan et al., (1996) found that Dipel-2x caused great inhibition in GPT within 24 hr. While Khedr (2002) showed increase in the activities of GOT and GPT after two and five days of treatment S. littoralis larvae with different IGRs. This contradiction may be due to differences in treatments, concentrations and times used by those authors.

In conclusion:

            It could be concluded that spinosad and triflumuron either alone or as mixtures with two surfactants caused highly decreased in total soluble protein and transaminase enzymes in cotton leaf worm larvae.

REFERENCES

Abdel-Hafez, M. M.; Shaaban, M. N.; El-Malla, M. A.; Farag, M. and Abdel-Kawy, A. M. (1988). Effect of insect growth regulators on the activity of transaminase enzymes with reference to protein and amino acids in the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.). Minia Journal of Agriculture Research & Development, 10 (3): 1375-1372.

Bursell, E. (1963). Aspects of the metabolism of amino acids in the tsetse fly Glossina sp. (Diptera). Journal of Insect Physiol., 9: 439-452.

Carbetree, B. and Newssholme, E. A. (1970). The activity of protein dehydrogenase and aspartate oxoglutarate amino transferase in some insect flight muscles. Biochem. Journal, 117: 1019-1021.

El-Defrawi, M. E., Toppozada, A.; Mansour, N. and Zeid, M. (1964). Toxicological studies on the Egyptian cotton leafworm, Prodenia litura F. susceptibility of different larval instars of Prodenia to insecticides. Journal of  Econ.  Entomol., 57: 591-593.

El-Kordy, M. W.; Gadallah, A. I.; Abbas, M. G. and Mostafa, S. A. (1995). Effect of pyriproxyfen, flufenoxuron and teflubenzuron on some biochemical aspects of Spodoptera littoralis. Al-Azhar Journal of  Agriculture Research, 21 (6): 223-238.

Gilbert, L. I. (1967). Biochemical correlations in insect metamorphosis. Comp. Biochem., 28l: 199-252.

Gornall, A. G.; Bardawil, C. D. and David, M. M. (1949). Determination of serum protein by means of bruit reduction. Journal of  Biochemistry, 177: 751-766.

Khedr, M. M. (2002). Effect of some plant extracts and insect growth regulators applied to control cotton leafworm on honeybees, Apis mellifera, L. M. Sc. Thesis, Faculty of Agriculture, Zagazig University, Egypt, 204 pp.

Kilby, B. A. and Neville, E. (1957). Amino acid metabolism in insect tissues. Journal of   Exp. Biol., 3: 270-289.

Meister, A. (1957). Biochemistry of the amino acids. Academic Press, New York, 175-196 pp.

Mohamady, A. H. (2000). Biochemical  and toxicological studies on the effect of some insecticides on the cotton leafworm, Spodoptera littoralis (Boisd.). M. Sc. Thesis, Faculty of Agriculture, Zagazig University, Egypt, 278 pp.

Reitman, S. and Frankle, F. (1957). Colourmetric method for aspartate and alanine transaminases. Amer. Journal of  Clic. Pathol., 28-56.

Salem, I. E., El-Sheakh, A. A.; Khidr, A. A., Desuky, W. M. and Raslan, S. A. (1995) Biochemical effect of some IGRs on phosphatases and transaminases of the cotton leafworm. S. littoralis (Boisd.) (Lepidoptera). Zagazig Journal  of  Agric. Res., 22 (3): 895-899.

Sokar, L. A. (1995). Possible alternatives to classical insecticides in management program of Spodoptera littoralis(Boisd.). Ph. D. Thesis, Faculty of  Agric. Moshtohor, Zagazig University, Egypt, 209 p.

Tawfik, S. M.; Farghali, A. A.; Sokar, A. and Abd-El-Wahab, I. S. (2002). Biochemical studies of Dipel-2X and abamectin on the 4^th instar larvae of cotton leafworm, Spodoptera littoralis (Boisd). Egyptian  Journal of Applied  Science, 17 (3): 371-386.

Zidan, Z. H.; Moawad, G. M.; Gadallah, A. I. and Sweeki, F. E. (1996). Biochemical aspects of the cotton leafworm larvae Spodoptera littoralis(Boisd.). as affected by some soft nontoxic insecticides. Proc. 6^th Conf. of Agricultural Development Research, 17-19 Dec. Cairo, Annual of  Agric. Sci., Cairo, 233-244 pp.

دراسات بیوکیمیائیة لمخالیط بعض المبیدات مع المنشطات

على یرقات العمر الرابعلدودة ورق القطن

هالة محمد میعاد¹- بلال احمد سلیمان² - على عبد العزیز الشیخ¹-  أحمد حسن ابو غالیة²-وحید محمود حسین دسوقى¹  

1. معهد بحوث وقایة النباتات - مرکز البحوث الزراعیة – الدقى- جیزة - مصر.

2. کلیة العلوم -  جامعة قناة السویس -  مصر.

تمت دراسة تأثیر مرکبى سبینوزاد وترایفلومورون بصورة منفردة او کمخالیط مع اثنین من المنشطات وهما ترایتون اکس – 100 و توین – 20 على بعض الاستجابات الکیمیائیة الحیویة للعمر الیرقى الرابع لدودة ورق القطن وذلک على فترات زمنیة مختلفة (بعد 24 ، 48 ، 72 ساعة من المعاملة). بینت النتائج حدوث نقص معنوى فى البروتین الکلى الذائب فى کل الفترات المختبرة نتیجة للمعاملات المختلفة خاصة بعد 24 ساعة من المعاملة مقارنة بالتجربة الضابطة، کما سببت المخالیط ایضا خفضا فى الکفاءة وذلک مقارنة باستخدام سبینوزاد وترایفلومورون بصورة منفردة. وقد اعطى مخلوط سبینوزاد عند الترکیز المسبب لإماتة 40٪ من الیرقات مع ترایتون اکس – 100 اکبر انخفاض فى البروتین الکلى الذائب. اما بالنسبة للإنزیمات الناقلة للأمین فلقد سببت کل المعاملات المستخدمة نقصا فى نشاط هذه الإنزیمات خلال الفترات الزمنیة المختبرة مقارنة بالتجربة الضابطة. بالإضافة الى هذا أحدثت المخالیط نقصا کبیرا فى کفاءة الإنزیمات عنه فى حالة سبینوزاد وترایفلومورون بصورة منفردة. و کانت مخالیط سبینوزاد وترایفلومورون عند الترکیز المسبب لإماتة40٪ من الأفراد هى الأکثر نشاطا.