Faculty of Bioscience Engineering, Ghent University, BE-9000 Gent, Belgium ... The Mediterranean flour moth, Ephestia Kuehniella Zeller (Lepidoptera: ...
Comm. Appl. Biol. Sci, Ghent University, 70/4, 2005
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COMPARATIVE TOXICITY OF THREE ECDYSONE AGONIST INSECTICIDES AGAINST THE MEDITERRANEAN FLOUR MOTH M. HAMI, F. TAIBI, G. SMAGGHE* & N. SOLTANI-MAZOUNI Laboratoire de Biologie Animale Appliquée, Faculté des Sciences, Université d’Annaba 23000-Annaba, Algérie * Laboratory of Agrozoology, Department of Crop Protection Faculty of Bioscience Engineering, Ghent University, BE-9000 Gent, Belgium
ABSTRACT The Mediterranean flour moth, Ephestia Kuehniella Zeller (Lepidoptera: Pyralidae), is an important pest in stored products worldwide, and is one of the major pests in flour mills in Algeria. Because environmental consideration, alternative approaches to neurotoxic insecticides, as well as safe, effective, and sound integrated pest management strategies are developed pest control agents such as the insect growth regulator (IGRs). Among these IGRs, the bisacylhydrazine derivatives are nonsteroidal ecdysteroid agonists that mimic the action of moulting hormones and induce a precocious and incomplete moult in several insect orders. In topical bioassays using the pupae of E. kuehniella, three ecdysteroid agonists: RH-5849, the first bisaclhydrazine ecdysone agonist and two analogs, RH-5992 (tebufenozide) and RH-0345 (halofenozide), were evaluated on the reproduction under laboratory conditions. In a first series of experiments, the efficacy of these compounds was tested. These compounds exhibited insecticidal activity and the duration of pupal development was reduced with a doseresponse relationship. Among the three tested compounds, tebufenozide (LD50 = 0.005 µg) appeared the most potent ecdysteroid agonist against E. kuehniella (RH-5849: LD50 = 0.05 µg and RH-0345: LD50 = 5.10 µg). In a second series of experiments, the effects of the ecdysone agonists (LD50) were investigated on the reproduction. Data showed that the three compounds affected growth of ovaries as evidenced by morphometric measurements of the ovaries from newly emerged adult females. In addition, the thickness of the chorion from basal oocytes was reduced only by RH-5992 and RH0345. However, electron microscopic observations revealed that the three compounds had no significant effect on the fine structure of chorion. Finally, measurements of ovarian ecdysteroids' production by an enzyme immunoassay showed an increase in the hormonal amounts recorded in treated series compared to control series.
INTRODUCTION In insects, ecdysteroids regulate many developmental and physiological processes (Gäde et al., 1997) and are considered as potential specific target sites for pest control (Dinan, 1989). In the past decade, RH-5849 has been discovered as the first nonsteroidal ecdystroid agonist leading to a new class of insect growth regulators (IGRs), namely benzoylhydrazines, that induce a precocious and incomplete molt in several insect orders. RH-5992 has great potential for use as chemical protectant in the stored product industry, especially against Lepidoptera. However, further research is necessary to determine the best method for assessing the dose-response of the different development insect pest stages. These compounds mimic the biological activity of the natural insect molting hormone 20-hydroxyecdysone (20E) by binding to the ecdysteroid receptor complex in a competitive manner with ecdysteroids (Wing, 1988). More recently, RH-0345 (halofenozide), a new
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nonsteroidal ecdysteroid agonist, has been considered more coleopteran selective than other members of this class of insecticides (Dhadialla et al., 1998). In order to extend these findings, the present study was designed to compare the insecticidal activity of three ecdysteroid agonists (RH-5849, RH5992, RH-0345) applied topically on newly ecdysed pupae of E. kuehniella. In a second series of experiments, we tested the effect of these compounds on reproductive events and the thickness of the chorion from basal oocytes. Moreover, the ovarian ecdysteroid production was measured by enzyme immunoassay. MATERIALS AND METHODS Experimental animals. Last instar larvae of E. kuehniella were collected from stock colony and reared on wheat flour at 27°C and 80% relative humidity. Pupae were staged according to their age in days from pupal ecdysis. Insecticides and treatments. RH-5849, RH-5992 and RH-0345 of technical grade were kindly provided by Dr. G.R. Carlson (Rohm & Haas, Spring House. PA). All other chemicals were of analytical grade. The compounds were dissolved in acetone (2 µl/insect) and topically administered at various doses: (0.03, 0.06, 0.12, 0.25 µg/insect for RH-5849; 0.007, 0.01, 0.03, 0.06 µg/insect for RH-5992; 2, 4, 6, 8 µg/insect for RH-0345). Toxicity bioassay. These compounds were tested at different doses on newly ecdysed pupae. The toxicity bioassay was carried out with three replications each containing 10 pupae. The rate of growth and development was examined and mortality was scored daily until adult emergence. The mortality percentage obtained was corrected (Abbott, 1925) and toxicity data were studied by probit analysis (Finney, 1971). LD50 values (50% doses of exuviations inhibition), 95% confidence limits and slope of the concentrationmortality lines were calculated (Swaroop et al. 1966). Ovary measurements. The compounds were tested topically (in 2 µl) on newly emerged females at the following doses corresponding to LD50: 0.05 µg for RH-5849, 0.005 µg for RH-5992, and 5.10 µg for RH-0345. The fresh weight of each paired ovaries, the number of oocytes and the size (length, width) and the volume of basal oocytes were determined, respectively. Thickness of chorion. Basal oocytes were collected and fixed according to Friend and Faquhar (1967), dehydrated and embedded in an Epon-araldite mixture (Andersen & Ellis, 1965). Thin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963) and then examined in Hitachi H330 electron microscope. The thickness of the chorion was measured from microphotographs made to the same scale. Ecdysteroid quantification. Ovaries were collected and extracted with methanol by sonication. After centrifugation (5000xg, 10 min), the supernatants were taken and evaporated. The extracts were suitably resuspended in phosphate buffer (0.1 M, pH 7.4) and each individual sample was analyzed by an enzyme immunoassay (EIA) using a conjugate of 20E coupled to
Comm. Appl. Biol. Sci, Ghent University, 70/4, 2005
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peroxidase as enzymatic tracer, tetramethyl benzidine as a color reagent and a rabbit B polyclonal antibody. Statistics. Data from insecticidal tests were subjected to analysis of variance (ANOVA) after angular transformation of observed mortality percentages. The age and the number tested per series are given with the results. The comparison of mean values (±SD) between control and tested series was done by Student’s t-test at 5% level. RESULTS Insecticidal activity. Three non-steroidal agonists were tested in vivo by topical applications. The compounds induced an inhibition of adult exuviation with a dose-response relationship. The inhibition doses, the fudicial limits and the slope were presented in Table 1. RH-5849 (LD50 = 0.05 µg/insect; 95% fiducial limits=0.03-0.07 µg/insect; slope = 3.80), RH-5992 (LD50 = 0.005 µg/insect; 95% fiducial limits = 0.002-0.006 µg/insect; slope = 5.85), RH-0345 (LD50 = 5 µg/ insect; 95% fiducial limits 3.75-695 µg/insect; slope = 3.54). Among the three compounds tested, RH-5992 appeared the most potent ecdysteroid agonist against E. kuehniella as evidenced by the LD50. Table 1. Comparative toxicity of three ecdysteroid agonists (RH- 5992, RH-5849 and RH-0345) applied topically to newly emerged pupae of E. kuehniella. Compounds RH-5992 RH-5849 RH-0345
Regression curve Y= 1.17 X + 4.28 Y= 1.97 X + 1.82 Y= 1.88 X + 3.73
Slope 5.85 3.80 3.45
LD50 (µg/insect) 0.005 0.05 5.10
95% fiducial limits 0.002 – 0.006 003 – 0.07 3.75 – 6.95
Effect on morphometric measurements of ovaries. The effect of LD50 of three compounds RH-5992 (0.005 µg/insect), RH-5849 (0.05 µg/insect) and RH-0345 (5.10 µg/insect) has been examined on some biometrical parameters of E. kuehniella ovaries sampled from newly emerged adult females. Data showed that the three treated compounds reduced the weight of ovaries, the number of oocytes per paired ovaries and the size of basal oocytes as compared to control females. Moreover, a significant reduction (p
