Insecticidal activity of alkaloids extract of Pergularia tomentosa (Asclepiadaceae) against fifth instar larvae of Locusta migratoria cinerascens (Fabricius 1781) (Orthoptera: Acrididae)

To assess bioinsecticidal effects of Pergularia tomentosa on Locusta migratoria cinerascens, crude alkaloids were extracted from aerial part of the plant. Newly emerged fifth instar larvae were treated by forced ingestion. Results showed that the test compound exhibited a considerable larvicidal effect with a dose-dependent mortality. On the other hand, it also caused antifeeding effect, weight loss of larvae with a reduction in protein and carbohydrate contents. These results indicated that P. tomentosa may be a promising naturally occurring agent for locust larval control. KeywordsPergularia tomentosa; Locusta migratoria cinerascens; alkaloids; toxicity; development; energy reserves 1. INTODUCTION The migratory locust Locusta migratoria in its various gregarious forms is the most important crop pests in Africa and Asia. Like the desert locust: Schistocerca gregaria, L. migratoria is characterized by the pronounced ability to exhibit a continuum of forms between the extreme solitarious and gregarious phases [1]. In the gregarious phase, the single swarm can comprise many millions of locusts, and be more than 100 km long and several km wide [2]. In this phase, the migratory locust cause substantial damage to crops and grazing. The control of grasshoppers and locusts has been based exclusively on chemical insecticides, however chemical control of pest insects has three major disadvantages: pollution of the environment by insecticide residues, development of insect resistance and potential toxicity to non target organisms. Plants synthesize a wide array of compounds that are generally thought to be involved in plant-insect interactions [3, 4]. Such compounds of secondary metabolism as alkaloids, terpenoids, phenols, flavonoids, steroids, etc. are important for mediating interactions between plants and their biotic environment and do not have apparent function in physiological or biochemical processes [3, 5, 6, 7, 8]. Most of these compounds have potent effects on insect pests, low mammalian toxicity, lack of neurotoxic activity, low persistence in the environment, and high biodegradability [9, 10]. Thus, organic molecules of botanical origin may offer a safe source of compounds for pest management, being environmentally friendly, and an excellent alternative to persistent synthetic insecticides [5, 11, 12]. Alkaloids are cyclic organic compounds containing nitrogen in a negative oxidation state; they are widely distributed in plant kingdom [13] and constitute an interesting family synthesized and accumulated by several plants [14]. Alkaloids have been reported as the most important group of natural substances [14], playing an important role in the ecology of organisms which synthesize them. For instance, it has been suggested that they constitute part of the plant defenses against phytophagous animals [15] together with terpenoids, phenols, flavonoids, steroids, etc. [16, 14, 17, 18]. Those compounds are insecticidal at low concentrations, their mode of action varies, but many affect acetylcholine receptors in the nervous system (e.g. nicotine) or membrane sodium channels of nerves (e.g. veratrin) [19]. In an effort to identify novel pest-control products that are both environmentally acceptable and effective for the management of the migratory locust, L. migratoria, we assessed the effects of a crude extract of alkaloids from the aerial part of the plant Pergularia tomentosa (Asclepiadaceae) on growth and development of the fifth instar larvae of the migratory locust. This plant is common International Journal of Science and Advanced Technology (ISSN 2221-8386) Volume 3 No 6 June 2013 http://www.ijsat.com 9 in the arides regions of Algeria, where it escapes feeding damage by L. migratoria (unpublished observations). II. MATERIALS AND METHODS A. Insect rearing Mass rearing of L. migratoria cinerascens was carried out in the laboratory according to the method of Pener et al. (1989) [20], using adults collected in the field in the Adrar region, Algeria. Larvae and adults were held in 45 x 50 x 50 cm cages at 30–32 °C, 50–70% RH, and under a 12 h:12 h, L:D photoperiod. Locusts were fed an Avena sterilis-based diet, complemented with wheat germ. B. Plant and extraction of crude alkaloids extract The Asclepediaceae P. tomentosa was collected in the biotope of the migratory locust, in the Adrar region (South of Algeria). The crude alkaloids extract of the aboveground portion of the plant was prepared from leaves and stems dried in the shade and ground into a fine powder. The extraction was carried out by macerating the powder in aqueous acid solution for 2 h, followed by degreasing with n-hexane and extraction with chloroform until Mayer’s test was negative. The extracts were dried over anhydrous sodium sulphate and the solvent evaporated to afford a crude extract of alkaloids. The dried extract was kept at 4 °C and resuspended in ethanol just before being used for treating insects. C. Insecticidal activity against fifth instar larvae The bioassay for insecticidal activity against fifth instar larvae of L. migratoria was carried out using five concentrations (60, 120, 180 and 240 μg of the crude extract/larvae). Test compounds were dissolved in 50 μl of ethanol. For treatment, newly emerged larvae fifth instar (0–12 h post emergence) were treated by forced feeding of an ethanol solution using a micropipette, without anesthesia. Controls were given absolute ethanol only. Ten larvae were used for each concentration and five replicates were done for treated and control larvae. D. Post embryonic development and feeding assay The effects of alkaloids extract of P. tomentosa on the development and the food consumption were investigated by exposing the newly emerged larvae (0-1 day old) fifth instar to sublethal dose of alkaloids 60 μg/larvae. To induce a higher feeding rate, the larvae were starved for 6 h prior to use. The larvae (n = 50) were treated by forced ingestion. Observations including weight, food intake, duration of 5th instar larvae and adult emergence, were recorded until adult emergence. E. Sample collection and metabolites quantification For biochemical analyses, hemolymph samples (10 μl/larva) were obtained from five larvae in each of the control and treated groups at 2, 6, 10, 14 and 18 days posttreatment. Methanol (300 μl) was added to hemolymph collected from metathoracic legs using a graduated capillary pipette. Before analysis, haemolymph samples were centrifuged at 5000g for 10 min and the supernatant was recovered. Soluble protein and carbohydrates contents were quantified spectrophotometrically. The amount of proteins was measured according to the method of Bradford [21] using bovine serum albumin as a standard. Carbohydrates content was quantified by the method of Duchateau and Florkin using anthrone reagent [22]. F. Statistical analysis Results are expressed as means ± standard deviation (SD). To identify significant effects of the treatments on the variables measured, data were submitted to a monofactorial ANOVA using XLSTAT 7.5.2. For comparison of treatment groups, we used Tukey’s test with a significance threshold of 0.05. III. RESULTS A. Larvicidal activity The alkaloids extract of P. tomentosa exhibited a potent larvicidal effect against the fifth instar larvae of L. migratoria. The mortality began one day after treatment for the doses of 120, 180 and 240 μg. In larvae treated at 60 μg, the mortality appeared after two days. The alkaloids extract showed the highest mortality percentage with 96% at 180 μg after 10 days and above 240 μg it showed a mortality of 100 % (Fig. 1). The result indicated that the larvicidal efficiency of the extract was concentration dependent. Mortality of control larvae did not exceed 2% during the observation period. Alkaloids toxicity was demonstrated by morphological deformities and exuviations difficulties causing larval mortality at the end of stage and increased percentage of abnormal imagos emerging. These difficulties were due to the persistence of the larval cuticle and the impossibility to reject the old integuments. The statistical analysis showed that the treatment at both doses had a highly significant effect on larval mortality (P < 0.05). Figure 1. Effect of alkaloids extract of P. tomentosa on mortality (%; mean ± SD, n = 50) of fifth instar larvae of L. migratoria. 0 20 40 60 80 100 1 3 5 7 9 11 13 C u m u la ti v e m o rt a li ty ( % ) Days after treatment Dose 1: 60 μg/L5 Dose 2: 120 μg/L5 Dose 3: 180 μg/L5 Dose 4: 240 μg/L5 International Journal of Science and Advanced Technology (ISSN 2221-8386) Volume 3 No 6 June 2013 http://www.ijsat.com 10 B. Effect on development -Effect on weight The results of weight change in treated and control larvae are shown in Figure 2. A progressive increase in control larvae weight was observed during the experiment. After 10 days, control larvae gained 110.41 % in weight compared to their initial weight. In contrast, a slight increase of weight of treated insects was recorded. Thus, 18 days after treatment, the weight gained was 0.28 g. The ANOVA analysis showed that the treatment had a significant effect on weight (P < 0.05). Figure 2. Effect of alkaloids extract of P. tomentosa on weight (g; mean ± SD, n = 50) of fifth instar larvae of L. migratoria. Each point represents the mean ± standard error of five replicates. Different letters denote significant differences (Tukey’s test, p < 0.05). Effect on duration of 5th instar larvae Treatment of 5th instar larvae with alkaloids of P. tomentosa resulted in significant increase of the larval duration, from 9 ± 0.96 to 18 ± 0.21 days (Fig. 3). Adult emergence was also affected in treated larvae. Alkaloids caused a significant inhibition of adult emergence. Figure 3. Effect of alkaloids extract of P. tomentosa on larval duration (days; mean ± SD, n = 50) of fifth instar larvae of L. migratoria. Different letters denote significant differences (Tukey’s test, p < 0.05). Effect on food intake Alkaloids extracted from P. tomentosa exhibited a significant decline in food intake on fifth instar treated larvae compared to control (Fig. 4). Food consumption in the treated groups remained at a significantly lower level throughout the experimental period, only 0.45 ± 0.05 g were consumed from treated larvae at day 6 compared to 1.31 ± 0.09 g eaten from control larvae. Figure 4. Effect of alkaloids extract of P. tomentosa on food intake (g; mean ± SD, n = 50) in L. migratoria fifth instar larvae. Different letters denote significant differences (Tukey’s test, p < 0.05). C. Effect on biochemical parameters Biochemical results showed clearly that alkaloids of P. tomentosa induced a decrease in protein and carbohydrates contents compared with control larvae (Figs. 5 & 6). A rapid rise in protein level was seen in control larvae between the 2nd and 6th days, when it increased from 84.99 ± 9.7 to 158.33 ± 12.8 μg/μl. On day 10, the protein contents decrease due to the use of the hemolymph metabolites in the moult processes. This rate of proteins was severely reduced in treated larvae. It was between 45.3 ± 1.5 and 53.9 ± 0.8 μg/μl, respectively, for the 2nd and 18th days. Carbohydrates content was severely reduced in treated larvae. In control larvae, this rate was increased from 38.08 ± 11.32 to 78.73 ± 3.6 μg/μl between the 2nd and 10th days. a a a