Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is an important polyphagous insect pest infesting cotton, vegetable, oilseed and fiber crops[1]. In recent years frequent outbreaks have been more common in subtropical and tropical agriculture in Asia, and have 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 applications of synthetic insecticides[4]. However, the resistance 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-environmental and human defects due to residues[6]. Therefore, in the current scenario, there is an urgent need for developing safer, more environment-friendly and more efficient pesticides, which represent ideal alternatives substituteto conventional pesticides in integrated pest management.
In the screening assay for finding natural insecticides from plants, we have tested the insecticidal activities of 250 different species of tropical plants collected from Hainan, China. Some tropical plants showed strong and promising insecticidal activities and one of them was Allamanda cathartica Linn., which belongs to Apocynaceae family, Allamanda genera. The plant grows in tropical areas and is used as decoction in various areas[7,8]. In recent years, some important pharmacologic activities, including antihypertensive activity[9], antifertility activity[10], antinematodal activity[11] were reported from A. cathartica. A number of iridoid lactones have been separated from this plant[12,13,14]. 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
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.The eggs were hatched at 25±1 ℃ and 70%-80% relative 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. Experiments 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, respectively. Most treated larvae continuously had kept low weight for more than 18 days.
As can be seen from Fig. 3, a dosage dependent manner in the food intake was found. Larvae consumed less when they were exposed to the food treated with allamdin. The percentage of food ingested by S. litura larvae was strongly depended on the concentration of allamdin. At 250 mg/kg of allamdin, 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.
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.
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 (13.52±0.68) d was observed without allamdin treatment while the longest larval period (21.14±2.05) d was observed at 250 mg/kg of allamdin (Fig. 5). The pupal period was ranged from 16 days for the control to 22 days at 125 mg/kg allamdin. At 250 mg/kg allamdin, no larvae were developed to pupation.
Larval mortality increased with higher allamdin concentration (Fig. 6). All larvae feeding on diet containing 250 mg/kg of allamdin died before pupated. At 125 mg/kg of allamdin, the mortality rate of larvae was 82.5%. In addition, considerable mortality occurred in the pupal stage at lower concentrations of allamdin. What’s more, the surviving adults were also affected after allamdin treatment.
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 completion. 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).
3 Discussion
Allamdin showed chronic and potent insecticidal effects against S. litura in a time-dependent manner to restrain S. litura population growth, which 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 consequently 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 compounds were found to have strong growth inhibition on the insect larvae, such as (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU)[21], ribavirin[22], and aglaroxin A[23]. Breuer et al.[21] recently reported that the antiherpetic compound (E)-5-(2-bromovinyl)-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 Breuer’s 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.