Microsoft Word - 2-Dr Vazirian zadeh .doc Iranian J Arthropod-Borne Dis, 2007, 1(2): 7-13 B Vazirianzadeh et al.: The Effects of Oral Application … 7 *Corresponding author: Dr B Vazirianzadeh, Tel: +98 611 3782956, Fax: +98 611 3332036, E-mail: babakvazir@yahoo.co.uk Original Article The Effects of Oral Application of Cyromazine and Triflumuron on House-Fly Larvae *B Vazirianzadeh1, MA Jervis2, NAC Kidd2 1Department of Medical Parasitology and Mycology, School of Medicine, Ahwaz Jundishapoor Medical Sciences University, Ahwaz, Iran 2Department of Ecology and Biodiversity, School of Biosciences, Cardiff University of Wales, UK (Received 26 Nov 2006; accepted 13 Oct 2007) Abstract Accumulations of large quantities of wastes (manure, used litter, dead birds) which are excellent medium for fly-larvae over poultry houses provide breeding places for different groups of fly pests, with house-flies being the dominant species. This project is a comparative lab study. In this research project the larvicidal effects of cyromazine and triflumuron were studied as two Insect Growth Regulators (IGRs) to reduce the fly population using oral application. Both IGRs had a signifi- cant effect on larval mortality compared with their controls among the concentrations (P< 0.01, Fisher’s LSD with Bonf- feroni correction) including a dose-dependent relationship. Comparisons among LC50 and LC90 values, using fiducial limits, showed that cyromazine was significantly more toxic to the larvae of the two strains than triflumuron. It is concluded that cyromazine should be used in a larvicidal programme to control house-fly rather than triflumuron. Keywords: Housefly, Cyromazine, Triflumuron, Oral application Introduction Commercial poultry houses are rapidly ex- panding worldwide to meet the needs of the increasing human population (Axtell 1999). In modern production systems, poultries are housed in high densities with, consequently, accumula- tions of large quantities of wastes (manure, used litter, dead birds) which are excellent medium for fly-larvae, consequently adult fly production (Axtell 1986, 1999). This phenomenon provides breeding places for different groups of fly pests, with house-flies being the dominant species. IGRs are a diverse group of insecticides, with a range of effects on insect specific phe- nomena, disrupting the growth and development of insects and other arthropods. They mainly affect the development of immature stages, and disrupt metamorphosis and reproduction (Graf 1993, Retnkaran et al. 1985) and are becoming more important in the management of insect pests (Grenier and Grenier 1993). IGRs include various chemical categories including: juvenile hormones, chitin synthesis inhibitors, and triazine derivatives (Retnakaran et al. 1985) with differ- ent modes of action. Cyromazine (CGA 72662, N-cyclopropyl-1, 3, 5-triazine-2, 4, 6-triamine) represents a new class of IGRs derived from aziodotriazine her- bicides (Shen and Plapp 1990). It was discov- ered by Ciba-Giegy Ltd. in the mid 1970s and originally developed under the trade name of ‘Vetrazine’, a blow-fly control agent. Cyromaz- ine is now also applied topically to control house- fly larvae in manure (‘Neporex’), as a feed– through in poultry (‘Larvadex’), as well as in crop protection (‘Trigard’) (Moreno-Mari et al. 1996, Graf 1993). Ultrastructural studies on larvae of L. cu- prina have suggested that the pesticide works at the cuticular level (Jimenez-Pyedro 1995). How- ever, Binnington (1985) and Friedel et al. (1988) Iranian J Arthropod-Borne Dis, 2007, 1(2): 7-13 B Vazirianzadeh et al.: The Effects of Oral Application … 8 suggested that cyromazine did not inhibit chitin formation but might be acting by disruption of the endocrine system. Triflumuron is the common name for the chemical 2-chloro-N-[[[4-(trifluoromethoxy) phenyl] amino] carbonyl] benzamide. It was first intro- duced by Bayer in 1979 at the International Plant Protection Congress in Washington (Senior 1998). Triflumuron has been shown to be effective against a variety of insects of medical and agri- cultural significance, under laboratory and field conditions, belonging to the orders of Diptera, Orthoptera, Siphonaptera, Coleoptera and Lepi- doptera. It has been used to control house-flies, mosquitoes, fleas and cockroaches in public and animal health (Retnakaran and Wright 1987, Weaver et al. 1984, Main and Mulla 1982, Miura and Takahashi, 1979). ‘Alystin’ and ‘Stary- cide’ are two of its trade names. Triflumuron is a benzoylphenyl urea that acts primarily as a stomach poison, but may also have a contact action, depending on the insect species, the developmental stage of the insects, the dose applied, and the method of application (Senior 1998). Its effects are typical of benzoyl phenyl ureas, it acts to inhibit the deposition of chitin in the cuticle of arthropods. It has both ovicidal (Smith and Grigarik 1989, Broadbent and Pree 1984) and larvicidal (Smith and Grigarik 1989, Hejazi and Granett 1986, Asher and Nemny 1984) properties, depending on the species studied. In addition, it induces sterility in adult female house-flies (Howard and Wall 1995, Weaver and Begley 1982). There have been several previous investiga- tions using cyromazine, of triazine derivatives, as a larvicide against house-fly larvae (Keiding et al. 1991, Keiding et al. 1990, Kelly et al. 1987, Iseki and Georghiou 1986) but no comparisons have ever been made with triflumuron. The benzoylphenyl ureas, to which triflumuron belongs, are mainly used as larvicides (Reynolds 1987), but very little work has been done on the lar- vicidal effects of triflumuron on house-fly larvae (Weaver and Begley 1982). In this study the toxicity effects of cyromaz- ine and triflumuron were evaluated and com- pared using the oral application. Materials and Methods IGRs Both IGRs were of technical grade: cyromaz- ine (99.9% a.i., Novartis) and triflumuron (99.6% a.i., Bayer). Insects House-fly larvae were of two strains: Rento- kil, a non-resistant laboratory strain, and Chicken house, a wild strain. The house-flies were reared at Insect Investigations Ltd. (Cardiff University of Wales) in constant environmental chambers (25 °C, 55%-60% RH and 16hL: 8hD). The rearing method was that of Cetin et al. (2006) and Kristensen and Jespersen (2003) with some modifications. The larvae were reared in a medium made by mixing the following ingredi- ents: 170g dog food pellets, 20g-yeast extract (Sigma) and 30g milk powder. The pellets were placed into a plastic container, and sufficient water added to soften them. The pellets were then soaked in the water for at least one hour. The pellets, milk powder and yeast extract were together placed into a food mixer, and blended thoroughly. The resultant mixture was left for another hour, after which its time consistency was checked, and more water added if necessary. The mixture should be not much watery or thick. Oral Application (OA) To examine the effects of OA, second-instar larvae were used, because of their higher food consumption rate. The larvae were provided with: 0.125, 0.25, 0.5 and 1 mg/kg larval medium (Kelly et al. 1987). Distilled water was used as the solvent for cyromazine and acetone was used for triflumuron. Twenty-five of the second-instar larvae were placed in a 250cm glass-jar containing 70gr of IGR-contaminated larval medium. The latter was treated with the highest volume of acetone, 1000µl/Kg diet, as this was the volume used in Iranian J Arthropod-Borne Dis, 2007, 1(2): 7-13 B Vazirianzadeh et al.: The Effects of Oral Application … 9 the treated control (see above). A non-treated control did not receive any IGR or acetone. In all cases three replicates were used for both strains. The jars containing larvae were kept in a constant environmental chamber and monitored for two weeks. Emergence of the adults usually took< 10 days. The numbers of emerged house- flies at the end of experiment was counted to record the rate of survival which was then con- verted to the rate of mortality. All the experiments were conducted in School of Biosciences, Cardiff University of Wales, UK. Data analysis All percentage mortality data were arcsine square root-transformed. They were checked for the homogeneity of their variances using Bartlett or Levene tests and for the normality of their residuals using the Ryan-Joyner test, which is similar to the Shapiro-Wilks test. One- way ANOVAs were then performed on the data followed by the Least Significant Difference (Fisher’s LSD and Bonfferoni correction) method for distinguishing between the means to find any significant differences in mortality between the different concentrations and control, also amongst the concentrations. If the ANOVA table indicates a significant difference between the means, then pairs of means were compared, using LSD method to determine if they are signifi- cantly different from each other. Then concentra- tion-mortality regression was performed by probit analysis (Chi package, 2000) to determine the LC 50 and LC 90 for each treatment. The Chi pack- age is based on Finney’s probit analysis (Finney, 1978) and takes Abbott’s formula (Abott 1925) as a common correction. Differences in toxicity at LC50 and LC90 were considered to be significant when fiducial limits did not overlap (Sheppard et al. 1992, Stark et al. 1991). Results Overall one way-ANOVAs revealed significant differences in mortality among the treatments and controls (P< 0.001). Moreover both IGRs had a significant effect on mortality of larval mortality compared with their controls among the concentrations (P< 0.01, Fisher’s LSD with Bonfferoni correction). The results are summa- rised in Table 1 and 2. There was a dose- dependent relationship in the rate of mortality of larvae with both IGRs. This was confirmed using probit analysis. Comparisons among LC50 and LC90 values, using fiducial limits, showed that cyromazine was significantly more toxic to the larvae of the two strains than triflumuron. Besides, there was no significant difference in the suscep- tibility of the two strains to either cyromazine or triflumuron. There was no difference in mortality between treated and un-treated controls. Acetone had no side effects on the larval mortality. Table1. Probit analysis, using oral application of 2nd instar, for both IGRs against both strains of house-fly larvae, mortality of larvae to adults Treatment LC50 mg/kg diet FL* of LC50 LC90 mg/kg diet FL* of LC90 chi-squared values df SD of slope cy/Re 0.207 0.176-0.238 0.566 0.473-0.727 0.362 2 0.342 cy/Ch 0.216 0.190-0.241 0.566 0.473-0.727 0.249 2 0.349 tr/Re 0.531 0.475-0.602 3.455 2.567-5.132 0.085 2 0.267 tr/Ch 0.594 0.541-0.659 3.905 3.024-5.390 0.005 2 0.272 cy/Re= cyromazine # Rentokil, cy/Ch= cyromazine # Chicken, tr/Re= triflumuron # Rentokil, tr/Ch= triflumuron # Chicken, FL*= Fiducial Limits Iranian J Arthropod-Borne Dis, 2007, 1(2): 7-13 B Vazirianzadeh et al.: The Effects of Oral Application … 10 Table 2. Toxicity of two IGRs against house-fly larvae, using Fiducial Limits (mortality of larvae to adults), topical application of 2nd instar Comparisons Ratio of toxicity (LC50) Significant Ratio of toxicity (LC90) Significant trCh=trRe 1.12 ns 1.10 ns cyCh=cyRe 1.04 ns 1.00 ns trRe