陳敏 張靜 陳禮浪 葉火春 閆超 馮崗

摘? 要：室內(nèi)測定了黃蟬花素對斜紋夜蛾幼蟲的抑制生長發(fā)育活性。結(jié)果表明，黃蟬花素對斜紋夜蛾幼蟲的抑制生長發(fā)育活性與其處理濃度具有一定的相關(guān)性。與對照相比，處理組食物消耗量減少，幼蟲生長發(fā)育被抑制，發(fā)育歷期延長。處理組幼蟲在蛹期不能正?；级纬苫斡迹鸹蟮某上x表現(xiàn)為形態(tài)畸形。在預(yù)蛹期和蛹期由于不能正常蛻皮導(dǎo)致死亡率較高。研究顯示黃蟬花素作為一類新型的昆蟲生長發(fā)育控制劑或害蟲田間種群管理的先導(dǎo)化合物值得進(jìn)一步研究。

關(guān)鍵詞：黃蟬花素;軟枝黃蟬;幼蟲生長發(fā)育;斜紋夜蛾;殺蟲活性

中圖分類號：S482.4????? 文獻(xiàn)標(biāo)識碼：A

Spodoptera litura （Fabricius） （Lepidoptera： Noc?t?ui?dae） is an important polyphagous insect pest infesti?ng cotton， vegetable， oilseed and fiber crops[1]. In rec?e?nt years frequent outbreaks have been more common in subtropical and tropical agriculture in Asia， and ha?ve the potentiality to be a serious pest of forage crops[2-3]. The effective method used for controlling S. litura was primarily dependent upon repeated applica?tions of synthetic insecticides[4]. However， the resista?nce of S. litura to conventional pesticides is getting more and more serious[2， 5]. In addition， the improper use of several pesticides has caused serious eco-envir?onme?ntal and human defects due to resid?ues[6]. Therefore， in the current scenario， there is an urgent need for developing safer， more environment-friendly and more efficient pe?sticides， which represent ideal alternatives substit?uteto conventional pesticides in integrated pest management.

In the screening assay for finding natural insecticides from plants， we have tested the insecti?cidal activities of 250 different species of tropical plants collected from Hainan， China. Some tropical pl?a?nts showed strong and promising insecticidal activities and one of them was Allamanda catha?rti?ca Linn.， which belongs to Apocynaceae family， Allam?a?nda genera. The plant grows in tropical areas and is used as decoction in various areas[7-8]. In recent years， some important pharmacologic activit?ies， including antihypertensive activity[9]， antifer?tili?ty activity[10]， antinematodal activity[11] were report?ed from A. cathartica. A number of iridoid lactones have been separated from this plant[12-14]. In rural areas of Wanning City （Hainan， China）， branches and leaves of A. cathartica were usually used to control maggots. In our previous work， we found that the extracts of A. cathartica possessed potent insecticidal activities against many pests. The ethanol extracts of the aerial part of A. cathartica exhibited antifeedant and stomach toxic effects to the fifth instar larvae of Brontispa longissima[15] and a prominent toxicity against the larvae and the adult of Aleurodicus disperses Russell[16]. Recently， the insecticidal activity of iridoid lactones from A. cathartica was assessed， and allamdin was found to exhibit strong insecticidal activity against Pieris rapae[17] and S. litura. In this paper， we investigated the insecticidal properties of allamdin against S. litura in vivo and conjectured its mode of action.

1? Materials and Methods

1.1? Materials

1.1.1? Chemicals

Allamdin was afforded in our previous work[17] and its chemical structure （Fig. 1） was determined by direct comparison of an authentic sample and spectroscopic data reported previously.

1.1.2? Insects

Spodoptera litura （Fabricius） eggs were collected from Ricinus communis （Castor） that were grown in the pesticide-free fields in Danzhou， Hainan， China.

Fig. 1? Structure of allamdin

The eggs were hatched at 25±1?℃ and 70%–80% re?l?a?tive humidity （RH） under a 12/12 h light/dark cycle in the laboratory. Freshly hatched larvae were fed with artificial diet. After 6 days， the third-instar of larvae were placed individually in Petri dishes （6 cm diameter） to supply artificial diet. Artificial diet was prepared by the method described by Zhu et al[18].

1.2? Methods

To examine the insecticidal properties of allamdin， an artificial diet feeding assay was used. Diet containing allamdin was prepared at the final concentrations of 10， 25， 50， 125 and 250 mg/kg [19]. Three instar larvae of S. litura （weights ranged from 5 to 10 mg per larva） were chosen and placed individually on portions of the diet in Petri dishes as described above. Larvae fed with artificial diet without allamdin were used as the control. Exper?iments were repeated three times with 30 larvae per treatment. The weight of each larva was measured until pre-pupation or dead. Mortality was calculated during larval and pupal development. The emergence of the adult insects was measured. Ten days later， the amount of food consumed by each larva was determined and the efficiency of food conversion （ECI） was calculated[20] by the index：

ECI = A ×100/B

where A is the weight increase of the insects during the testing， B is the feeding amount.

1.3? Statistical analysis

Analysis of variance was performed by using the PROC GLM procedure （SAS Institute， Cary， NC， USA）. If P>F less than 0.01， means were separated with the least significant different （LSD） test at the P=0.05 level.

2? Results

Allamdin was tested in an artificial diet method against S. litura. After 12 days， the average weight of the control larvae was 762?mg （Fig. 2）. In comparison， the average weight of the treated larvae was 320?mg and 416 mg， occupied 42.11% and 54.16% of that of the control larvae， with allamdin concentration at 250?mg/kg and 125?mg/kg， respe?c?ti?vely. Most treated larvae continuously had kept low weight for more than 18 days.

Fig. 2? Growth curve of S. litura larvae on artificial diet containing different concentrations of allamdin

As can be seen from Fig. 3， a dosage dependent manner in the food intake was found. Larvae consu?me?d less when they were exposed to the food treated with allamdin. The percentage of food inge?sted by S. litura larvae was strongly depended on the concentration of allamdin. At 250?mg/kg of alla?mdin， the amount of diet consumed was 0.31 g and the percentage of food ingested was only 22.83%， respectively， which was significantly （P<0.01） lower than that of the control larvae.

**： Significantly different from control at P<0.01， the same below.

Fig. 3? Amount of diet consumed corresponded to the weight gain of larvae at different allamdin concentration

Analogously， the pupae weight decreased with increasing allamdin concentration （Fig. 4）. At 125?mg/kg allamdin， the pupae weighed only 65.96% compared to that of the control. The treated larvae were not able to reach the pupal stage at a higher concentration of 250 mg/kg.

Fig.4? Weight of S. litura pupae after feeding on diet at different concentrations of allamdin

When the 6-day-old-larva was supplied with diets containing different concentration of allamdin， the larval period increased steadily corresponding to the allamdin concentration in the diet. The shortest larval period （13.520.68） d was observed without allamdin treatment while the longest larval period （21.142.05）?d was observed at 250?mg/kg of allam?din （Fig. 5）. The pupal period was ranged from 16 days for the control to 22 days at 125?mg/kg allam?din. At 250?mg/kg allamdin， no larvae were devel?op?ed to pupation.

Fig. 5? Length of larval and pupal period of S. litura

Fabricius after feeding on diet at different

concentrations of allamdin

Larval mortality increased with higher allamdin concentration （Fig. 6）. All larvae feeding on diet containing 250?mg/kg of allamdin died befo?re pupated. At 125?mg/kg of allamdin， the mortality rate of larvae was 82.5%. In addition， considerable mor?tality occurred in the pupal stage at lower con?centrations of allamdin. Whats more， the surviving adults were also affected after allamdin treatment.

Fig. 6? Toxic effects at different concentrations of

allamdin incorporated into a diet on S. litura

In the larval stage， we observed that the insects treated exhibited exuviating disturbances and/or malformations. Compared to the control （Fig. 7a）， some insects died slowly with slim and wrinkled bodies after consuming the treated diets （Fig. 7b）. Moreover， the molting process of the survived larvae was prevented or was not carried out to com?p?letion. In the pupal stage， compared to the control， pupal weight reduced obviously after allamdin treatment （Fig. 7c）. In addition， some insects were not able to remove the trunk exuviae and molted to malformed pupae （Fig. 7d）， which only lived for a few days and died quickly. After treatment of allamdin， several adults were not able to remove their pupal skin and form pupae-adult intermediates （Fig. 7e）. Malformed moths after emergence were observed to have abnormal wings （Fig. 7f）.

a： Normal larvae; b： Larvae showing moulting disorders; c： Normal pupae; d： Malformed pupae; e： Pupae-adult intermediates; f： Moths with abnormal wings.

.Fig. 7? Selected examples of S litura affected after uptake of diet containing allamdin

3? Discussion

Allamdin showed chronic and potent insecti?ci?d?al effects against S. litura in a time-depe?ndent manner to restrain S. litura population growth， whi?ch was different from the conventional neurotoxic insecticides， such as organophosphates， carbamates and pyrethroids. The development of larvae was retarded， the weight of pupae was reduced and the morphology of adults was also affected. Our present experiments revealed that larvae consumed less when they were exposed to diet with allamdin， but the efficiency of conversion of ingested food （ECI）， which measures the overall ability of the insect to convert ingested food into body matter， was not significantly affected. It was clear that allamdin had no effect on the absorption of food and consequ?en?tly on its conversion into larval tissue. The similar results would be obtained if the agent acted simply as a feeding deterrent. Therefore， the antifeedant effects of allamdin against the third-instar larvae of S. litura Fabricius were examined at the exposure time of 24 h and 48 h by the method of leaf dipping. We found that the allamdin caused a very small effect as antifeedant at the highest concentration.

Previously， some potential insecticidal compo?unds were found to have strong growth inhi?b?ition on the insect larvae， such as （E）-5-（2-bromo?vinyl）-2'-deoxyuridine （BVDU）[21]， ribavirin[22]， and aglaroxin A[23]. Breuer et al.[21] recently reported that the antiherpetic compound （E）-5-（2-bro?mov?inyl）-2' -deoxyuridine （BVDU） had remarkable insecticidal effects on Spodoptera frugiperda， which probably acted as growth inhibitors. More experiments in insect cell cultures revealed that the effects were due to the cytostatic action of BVDU in the S-phase. In this paper， we found that allamdin was able to inhibit the development of S. litura larvae， which was similar to the results published in Breuers study. Therefore， we conjectured that the mode of action of allamdin may be connected with the cytostatic action， although the exact mode of action was unknown.

In conclusion， the present work exhibited that allamdin isolated from A. Cathartica had strong growth inhibition against the larvae of S. litura. Although the toxic effects on mammals had not been done， this compound had great potential to act as lead chemicals for modification and derivation， and could be used as potential sources for novel insecticides development in integrated pest management. Therefore， the mode of insecticidal action and the molecular mechanisms of allamdin against S. litura need further study to explain.

References

Shankarganesh K， Walia S， Dhingra S， et al. Effect of dihydrodillapiole on pyrethroid resistance associated esterase inhibition in an Indian population of Spodoptera litura （Fabricius） [J]. Pesticide Biochemistry and Physiology， 2012， 102（1）： 86-90.

Su J， Lai T， Li J. Susceptibility of field populations of Spodoptera litura （Fabricius） （Lepidoptera： Noctuidae） in China to chlorantraniliprole and the activities of detoxification enzymes [J]. Crop Protection， 2012， 42： 217-222.

Nathan S， Kalaivani K. Efficacy of nucleopolyhedrovirus and azadirachtin on Spodoptera litura Fabricius （Lepidoptera： Noctuidae） [J]. Biological Control， 2005， 34（1）： 93-98.

Ahmad M， Sayyed A， Saleem M， et al. Evidence for field evolved resistance to newer insecticides in Spodoptera litura （Lepidoptera： Noctuidae） from Pakistan [J]. Crop Protection， 2008， 27（10）： 1367-1372.

Shad S， Sayyed A， Saleem M. Cross-resistance， mode of inheritance and stability of resistance to emamectin in Spodo?pt?e?ra litura （Lepidoptera： Noctuidae） [J]. Pest Management Science， 2010， 66（8）： 839-846.

Meng X， Hu J， Xu X， et al. Toxic effect of Destruxin A on abnormal wing disc-like （SLAWD） in Spodoptera litura Fabric?ius （Lepidoptera： Noctuidae） [J]. PLoS One， 2013， 8（2）： e57213.

Kosei Y， Tohru M， Irmanida B. Isolation， identification and tyrosinase inhibitory activities of the extractives from Allama?nda cathartica[J]. Natural Resources， 2011， 2（3）： 167-172.

Akah P， Offiah V. Gastrointestinal effects of?Allamanda cathartica leaf extracts[J]. International Journal of Pharmac?o?g?n?osy， 1992， 30（3）： 213-217.

Balunas M， Kinghorn A. Drug discovery from medicinal plants[J]. Life Sciences， 2005， 78（5）： 431-441.

Singh A， Singh S. Reversible antifertility effect of aqueous leaf extract of Allamanda cathartica L. in male laboratory mice[J]. Contraception， 2008， 40（6）： 337-345.

Alen Y， Nakajima S， Nitoda T. Antinematodal activity of some tropical rainforest plants against the pinewood nematode， Bursaphelenchus xylophilus[J]. Zeitschrift für Naturforschung C， 2000， 55（3-4）： 295-299.

Kupchan S， Dessertine A， Blaylock B， et al. Isolation and structural elucidation of Allamandin， an antileukemic iridoid lactone from Allamanda cathartica[J]. The Journal of Organic Chemistry， 1974， 39（17）： 2477-2482.

Tiwari T， Pandey B， Dubey N， et al. Plumieride from?Allamanda cathartica?as an antidermatophytic agent[J]. Phytotherapy Research， 2002， 16（4）： 393-394.

Kosei Y， Tohru M， Irmanida B. Isolation， identification and tyrosinase inhibitory activities of the extractives from Allamanda cathartica[J]. Natural Resources， 2011， 2（3）： 167-172.

Zhang J， Feng G. Insecticidal activity of extract from Allamanda Cathartica Linn against Brontispa longissima[J]. Chinese Journal of Tropical Crops， 2010， 31（7）： 1152-1156.

Feng G， Yan C， Zhang J. Insecticidal activity of extract from Allamanda cathartica Linn against Aleurodicus disperses Russell[J]. Chinese Journal of Tropical Agriculture， 2013， 33（7）： 54-57.

Feng G， Ye H C， Yuan E L， et al. Isolation and identification of insecticidal composition of Allamanda Cathartica Linn. Chinese Journal of Tropical Agriculture， 2013， 33（12）： 61-65.

Zhu L M， Ni Y P， Cao X Y. A method for artificially rearing to the cotton leafworm Prodenia litura Fabricius. Entomological Knowledge， 2001， 38（3）： 227-228.

Fraenkel G. Inhibition effects of sugars on the growth of the mealworm Tenebrio molitor L[J]. Journal of Cellular and Comparative Physiology， 1955， 45（3）： 399-408.

Waldbauer G. The composition and utilization of food by insects[J]. Advances in Insect Physiology， 1968， 5（C）： 229- 288.

Breuer M， Loof A， Balzarini J， et al. Insecticidal activity of the pyrimidine nucleoside analogue （E）-5-（2-bromovinyl）-2'- deoxyuridine （BVDU）[J]. Pest Management Science， 2005， 61（8）： 737-741.

Liu YQ， Zhang J， Feng G， et al. Ribavirin， a nucleoside with potential insecticidal activity[J]. Pest Management Science， 2012， 68（10）： 1400-1404.

Opender K， Gurmeet S， Rajwinder S， et al. Bioefficacy and mode-of-action of aglaroxin A from Aglaia elaeagnoidea （syn. A. roxburghiana） against Helicoverpa armigera and Spodoptera litura[J]. Entomologia Experimentalis et Applicata， 2005， 114（3）： 197-204.