Abstract: The effects of Anacyclus cyrtolepidioïdes flowerheads (Compositae family) on Tribolium confusum du Val (Coleoptera: Tenebrionidae) adults and larvae were determined. Insecticidal activity of the flowers essential oil, four crude extracts as well as twenty one fractions deriving from solid-liquid chromatographic separation was assessed using direct contact application method. The ethylacetate crude extract and eight fractions (A3, A4 P8, P10, F2, F3, F5 and F7) showed a significant inhibitory effect of the test material on Tribolium confusum du Val growth. One hundred percent mortality of the adults was achieved twelve days after treatment using fractions A4, P8 and F7, respectively. This preliminary study suggested that A. cyrtolepidioïdes may be considered as a potential source of insecticidal compounds.
INTRODUCTION
To circumvent the problems of human and animal health, pollution and disturbance of ecological balances generated by the massive use of synthesis pesticides, some alternative methods of pest management are developed. Natural pesticides have the advantage of being less phytotoxic and more degradable in the environment (Cooping and Menn, 2000). Thus, the development of new insecticides from plant extracts offers a very promising ground and especially going in the wake of the new concepts of sustainable development and the protection of natural resources. Over 2000 species of plants are known to posses some insecticidal activity (Klocke, 1989). They are still used to kill or repel insects. Traditionally, some plants belonging to the Compositae family are used as insect controllers or insecticides. For example Flaveria bidentis L. is used as external insecticide and Schkurhia pinnata (Lam) twigs are put below beds or among clothes for repelling insects particularly fleas. Essential oils and their constituents have also been shown to be a potent source of botanical pesticides (Singh and Upadhyay, 1993). Oil extracted from various parts of Tagetes minuta L. (Compositae) are used in the tropics as a dressing for livestock to control blowfly. The terthienyl (2,2`:5`,2`=-terthiohene) present in the oil has been identified as an active phototoxic compound against mosquitoes. Its high level of activity makes possible its commercialisation as a mosquito larvicide (Klocke, 1989). In Tunisia, research aiming the discovery and the development of new agent for pest control based on natural products has been undertaken (Barbouche et al., 2001; Ben Jannet et al., 2000, 2001, 2002; Hammami et al., 2006; Saïdana et al., 2005, 2007; Hoaous et al., 2006).
Continuing our contribution to the biological and chemical study of Tunisian plants (Oueslati et al., 2006; Bergaoui et al., 2007; Hammami et al., 2007; Tekaya-Karoui et al., 2007; Ben Jannet and Mighri, 2007; Chaieb et al., 2007). We describe here on, larval growth inhibition, toxicity and antifeedent effects of Anacyclus cyrtolepidioïdes flower heads essential oil, petroleum ether, chloroformic and ethyl acetate extracts against the major pest of stored products Tribolium confusum du Val (Rees, 1995; Jerraya, 2003).
MATERIALS AND METHODS
Plant Materials
Anacyclus cyrtolepidioïdes was collected in March 2005
at full flowering stage around Gabes city, located in the Southeast of
Tunisia. Samples were identified and voucher specimens were deposited
in the herbarium of Institut Supérieur Agronomique de Chott-Mariem,
Université de Sousse, Sousse, Tunisia. Flowerheads were manually
separated from the stems and leaves.
Preparation of Crude Extracts and Fractions
A. cyrtolepidioïdes fresh flowers (2000 g) were extracted
with a steam distiller apparatus during 4 h. Organic solution was dried
over anhydrous sodium sulphate then the solvent was removed by evaporation
under reduced pressure to give an extract weighing 300 mg. Water residue
was extracted three times with ethyl acetate and butanol for 96 h. The
crude extracts were obtained after solvents evaporation and were indexed
as follows: E1 for ethyl acetate extract, E2 for
the butanolic extract. The ethyl acetate extract was dissolved in water
then extracted successively with petroleum ether (E3) and chloroform
(E4) The essential oil, petroleum ether (E3) and
chloroformic (E4) extracts were simplified using silica gel
column chromatography (sds, 70-200 μm/2100027) and regrouped in four
fractions (from A1 to A4) using as eluent CH2Cl2;
CH2Cl2/AcOEt, AcOEt gradients for the essential
oil and in ten fractions (from P1 to P10) for E3
and eight fractions (from F1 to F7) for E4
using as eluent EP; EP/AcOEt, AcOEt gradients and stored in sealed glass
vials in a refrigerator at 4-5°C prior to analysis.
Insects Cultures
Larvae (3 mm of length) and young adults (10-15 days old) of the pest
T. confusum were obtained from same-age cultures. Insect was fed
with white wheat flour and beer yeast (95:5) and incubated at a constant
temperature of 30°C and 70% r.h., in darkness. Parent adults were
provided by the laboratory of Entomology, High School of Horticulture
and Animal production, Chott-Meriem, Sousse University, Tunisia.
Bioassay
Antifeedent, toxicity and insect growth inhibition effects of A.
cyrtolepidioïdes flowers essential oil, of its crude extracts
and some fractions of the chloroformic extract were evaluated. All bioessays
were carried out using the method described by Bloszyk et al. (1995).
In fact, 5 μL from each sample of the extracts, the essential oil or the twenty one mentioned fractions were deposited separately on diet disks (1 cm diameter) weighing about 30 mg. The disks are then dried and conserved at 30°C during 24 h and weighed before being offered to larvae and young adults. Three replications of ten insects of each stage kept in 4 cm diameter glass Petri dishes were performed. A control was prepared in the same way. Control and treated disks were placed in separate Petri dishes under no choice tests. Seven days after treatment, the diet disks were reweighed.
Nutritional Indices
The feeding deterrent action was calculated as Feeding-Deterrent Index
(Isman et al., 1990).
(FDI%) = [(C-T/C)]x100 |
where, C is the relative consumption rate of control disks and T is the relative consumption rate of treated disks.
The Relative Growth Rate (RGR) was calculated (Farrar et al., 1989) with some modification:
RGR = (A-B)/Bxday |
| Where: |
| A | = | Length of alive larvae (mm) on the 12th day/number of alive larvae |
| B | = | Original length of larvae (3 mm)/original number of larvae |
Mortality was determined in Petri-dishes, every four days during the essay (20 days),
Percentage of larval mortality = (n/N)x100 |
| Where: |
| n | = | No. of died larvae |
| N | = | Original No. of larvae (Tapondjou et al., 2005) |
Statistical Analysis
Statistical comparison was performed with SPSS version 11.0. Analysis
of variance (one way ANOVA), was followed by means comparison (at p>0.05)
and Duncan test
RESULTS
Antifeedant Activity
We tested the bioinsecticidal activity of the four extracts, essential
oil and 21 fractions constituents against T. confusum adults
and larvae (7 days old), results of the bioassay are showed in Table
1 and 2. The ethyl acetate and butanolic extracts,
the crude oil and the two fractions A2 and A3 appeared
to be attractive to T. confusum by presenting a negative
Feeding-Deterrent Index against adults at the 7th day old.
The extracts, the essential oil and most of the fractions showed a mild phagostimulant activity against larvae especially the fractions F2 (-283%), F4 (-287%) and F7 (-486%) presented a potent phagostimulants activity. Only fractions P8 and P10 seemed to be antifeedant (95 and 77%) against larvae of T. confusum. Those data showed how chromatographic fractionation of the crude extract can modify the specificity of the activity and allow the localisation of the antifeedant and phagostimulant activities in some fraction.
| Table 1: | The Feeding-Deterrent Index (%FDI) (larvae and adults) and the Relative Growth Rate (RGR) for larvae of Tribolium confusum caused by essential oil of A. cyrtolepidioïdes |
| *Each value represents mean±SD from the three different glass Petri dishes, Means followed by the same letter(s) within a column are not significantly different in Duncan`s Multiple Range Test at p>0.05% | |
| Table 2: | The Feeding-Deterrent Index (% FDI) (larvae and adults) and the Relative Growth Rate (RGR) for larvae of Tribolium confusum caused by plant extracts and fractions of petroleum and chloroformic extracts of A. cyrtolepidioïdes |
| Means followed by the same letter(s) within a column are not significantly different in Duncan`s Multiple Range Test at p>0.05% | |
The Inhibitory Activity of the Larvae Growth
The relative growth index of crude extracts, essential oil and fractions,
calculated after 12 days of experience were shown in Table
1 and 2. Larvae kept on Anacyclus diet disks
added with butanolic extract gave no significant differences in growth
length between the control and the ethyl acetate extract caused a high
significant reduce of larval growth after 12 days. The difference in RGR
calculated over a period of 12 days between larvae supplied with Anacyclus
cyrtolepidioïdes flowerheads petroleum ether and chloroformic
extracts and those kept on control (0.068) was highly significant (p>0.05),
whereas larvae fed on disks treated with fractions A4, P8
and F7 never showed any increase in body growth (RGR = 0) and
caused larval mortality. Those data revealed that those fractions have
substantial toxic effects as was shown by the relatively low growth rates
of larvae fed on treated diets.
Toxicity Test
Percentages of T. confusum mortality were calculated
every four days during a twenty days essay. Figure 1
shows the results of the toxicity test. In the preliminary studies carried
out to evaluate hand ling of A. cyrtolepidioïdes specie, it
was found that all the crude extracts prepared from the indicated plant
showed significant toxicity since the percentage of larvae mortality varied
from 54% till 88% 20 days after starting the test. A. cyrtolepidioïdes
ethyl acetate crude extract appeared as the most active one inducing a
percentage of 88% larval mortality.
In the other hand, we noticed that percentages of larval mortality caused by petroleum ether and chloroformic extract derived fractions (P1-P10; F1-F7) were less than 35% during the 4th day and started to increase from the 8th day, to achieve 100% within 12 days after treatment especially for larvae fed on diet disks impregnated with P8 and F7, while only 25% of larvae fed on control disks died 12 days after starting the essay. This shows that P8 and F7 can be considered of significant activities against T. confusum.
| Fig. 1: | Percentage mortality of T. confusum larvae exposed to different plant extracts, fractions of essential oil, petroleum ether and chloroformic extracts of A. cyrtolepidioïdes at concentration of 1% |
Other significant toxicity effects were observed by A. cyrtolepidioïdes essential oil as well as the fractions deriving from its chromatographic separation (A1-A4) within 20 days, the essential oil caused the dead of 80% of Tribolium larvae. Fraction A4 can be considered as the most active one inducing a percentage of 100% of larval mortality 12 days after treatment.
DISCUSSION
In this study, 4 natural extracts, essential oil and 21 fractions were tested for their bio-insecticidal activities against T. confusum larvae and adults. Antifeedant effect, toxicity and insect growth inhibition were followed up. Responses varied with plant material, extract type, fractions, insect stage and time exposition.
Previous studies searching for Natural products which can be useful as biologically active insecticides mentioned that the most promising botanical insect control agents are present in plants belonging to Compositae family (Jacobson, 1989). Anacycline is a natural insecticide isolated from Anacyclus clavatus was reported to have an inhibitory effect on Tribolium castaneum growth (Secoy and Smith, 1983; Pascual-Villalobos and Robledo, 1998).
Within the objective of searching Natural insecticide effective for the protection of stored food from insect infestation, we have been interested to the study of A. cyrtolepidioïdes insecticidal effects. The essential oil (A), four crude extracts (E1-E4) as well as 21 fractions derived from chromatographic separation of Anacyclus extracts (A1-A4; F1-F7 and P1-P10) were evaluated for their bio-insecticidal activities against T. confusum larvae and adults.
We noticed that A. cyrtolepidioïdes ethyl acetate crude extract was attractive against adults; it presents a high toxicity effect and shows a significant T. confusum larval growth inhibition when applied at 1%.
Fractions P8 and P10 deriving from the chromatographic separation of the petroleum ether crude extract presented the potent antifeedent activity (95 and 77%, respectively) and produced a significantly shorter larvae growth in comparison with the control. This suggests that this plant may contain nauseous constituents which can be responsible for antifeedent activity. On the other hand, fractions F2, F4 and F7 deriving from A. cyrtolepidioïdes chloroformic extract showed a highly phagostimulant constituents reducing the growth and inducing mortality of T. confusum larvae. This toxic effect may be due to the reversible competitive inhibition of acetylcholinesterase by occupation of hydrophobic site of enzyme`s active centre (Ryan and Byrne, 1988). Fraction A4 deriving from A. cyrtolepidioïdes essential oil exhibited the greatest toxic effect against larvae (100% of mortality).
This research work permitted us to conclude that Anacyclus cyrtolepidioïdes specie can be considered as an interesting plant for investigation in pest control.
ACKNOWLEDGMENTS
We are grateful to Dr. F. Harzallah-Skhiri for plant identification. Laboratoire de Botanique, Institut Supérieur de Biotechnologie de Monastir-UR: Agrobiodiversity, Institut Supérieur Agronomique de Chott-Meriem, Tunisie.