375 ISJ 14: 375-378, 2017 ISSN 1824-307X SHORT COMMUNICATION Evaluation of the effects of some insecticides based on neonicotinoids on entomopathogenic nematodes, Steinernema feltiae and S. carpocapsae V Kwizera, IA Susurluk Uludag University, Faculty of Agriculture, Department of Plant Protection, 16059 Nilufer-Bursa, Turkey Accepted September 21, 2017 Abstract Used of entomopathogenic nematodes (EPNs) are one biological control way against some insect pests, which especially pass one stage of their life in the soil. There have been a lot of developments and new discoveries in the field of entomopathogenic nematology. In spite of developments in the field of biological control, pesticides are still widely used for plant protection. These pesticides have side effects on soil biotop such as EPN species. In this study, we evaluated the effects of two insecticides (acetamiprid and imidacloprid) belong to neonicotinoid group on two EPNs (Steinernema carpocapsae and Steinernema feltiae). The present experiments were carried out in the laboratory. Mortality rate of the EPN species was performed at 24, 48 and 96 h, after treatment. Inspired by the compatibility observed between the neonicotinoid insecticides and EPNs against insect pest larvae, it was evaluated the direct effects of those insecticides on EPNs. Our results showed less mortality by imidacloprid and mortality rates are a little higher by acetamiprid. These results confirm the high compatibility of imidacloprid with EPNs and the low synergism with acetamiprid. Despite their effects on pollinators and other useful organisms of the biotop, these insecticides can be used effectively against soil dwelling insect pests without high risk on EPNs. Key Words: entomopathogenic nematodes; neonicotinoid; Steinernema feltiae; Steinernema carpocapsae Introduction Entomopathogenic nematodes (EPNs) are natural agents that control some pest insects (Gaugler and Kaya, 1990). It was first discovered in 1923 and since then the domain of EPN has made great progress (Thomas and Poinar, 1979; Poinar and Grewal, 2012). EPNs in the future may have great potential for farmers to control many harmful insects. EPNs are a group of nematodes used to control insect pests (Tomalak, 2004, 2005; Jacobson and Martin, 2011). EPNs of the families Steinernematidae and Heterorhabditidae are symbiotically associated with bacteria in the genera Xenorhabdus and Photorhabdus, respectively (Griffin et al., 2005). When an infective juvenile (IJ) of EPNs enters the body cavity of a susceptible host, the bacteria are released, multiply and host death occurs within two days, hence the term, entomopathogenic (Webster et al., 2002). The nematodes develop and reproduce within the insect ___________________________________________________________________________ Corresponding author: I. Alper Susurluk Uludag University Faculty of Agriculture Department of Plant Protection E-mail: susurluk@uludag.edu.tr cadaver, feeding on the symbiotic bacteria and degraded host tissues (Forst et al., 2002; Poinar and Grewal, 2012). EPNs are a potential alternative to the use of insecticides, which have side effects on the soil biotope. Since their discovery in 1923, researches on EPNs have been intense and a lot of progress have been made like mass production and shipment of IJs (Ehlers and Shapiro-Ilan, 2005; Poinar and Grewal, 2012). Despite the promising sustainability of biological control, insecticides are largely used to control insect pests. Beside pest control, these insecticides have side effects on useful insects. This is the case for the bees, which populations are dangerously reduced by nicotinoid insecticides (Woodcock et al., 2016). Altought there are many studied on effects of pesticides that largely used in agriculture on EPNs, the studies on neonicotinoids is very limited (Ulu et al., 2016). The 20 th century was going to be a century of neonicotinoids, this is after the discover of nicotinoids, their wide range use and their substitution to many insecticides as they are successfully applied to control pests in a variety of agricultural crops (Yamamoto and Casida, 1999). The first nicotinoid to be on the market was imidacloprid (Thyssen and Machemer, 1999). Neonicotinoids are a class of insecticides chemically http://en.wikipedia.org/wiki/Neonicotinoid 376 Fig. 1 Effects of acetamiprid on Steinernema carpocapsae (Sc) and Steinernema feltiae (Sf) (F = 37,4948; df = 5, 18; p = < 0,0001). related to nicotine. The name literally means “new nicotine-like insecticides”. Initially neonicotinoids were praised for their low-toxicity to many beneficial insects, including bees; however this claim has come into question. Researches point to potential toxicity to bees and other beneficial insects (Penelope et al., 2012; Tjeerd Blacquière et al., 2012; Gennaro Di Prisco et al., 2013). Neonicotinoids ere today recognized as the first bee killer (Jennifer et al., 2012). Despite their side effects on bees and other beneficial insects, there are some researches showing synergism of neonicotinoid insecticides with EPNs (Pisa et al, 2014). But no previous research has evaluated the direct effects of neonicotinoids on EPNs. This paper will evaluate the direct effects of two neonicotinoids (imidacloprid and acetamiprid) on IJs of two EPNs (Steinernema carpocapsae and Steinernema feltiae). Materials and Methods Imidacloprid and acetamiprid (neonicotinoid insecticides) and two EPNs Steinernema carpocapsae and Steinernema feltiae were used. The first step was to multiply the EPN samples on Galleria mellonella. Multiplication of EPNs Twenty-four well cell culture dishes were used for the multiplication of EPNs. A larva was placed in each well and 10 % moistened sand was added. After filling the sand, 2 μl of IJ nematodes were given. It was then incubated at 27 °C. The infected larvae are removed after 48 h and placed in larger wells. Wells were left at room temperature and new IJs were obtained after 10 days (Kaya and Stock, 1997). Infection Twenty-four well cell culture vessels which we used for the replication of EPNs were also used here. 70-100 IJ micropipettes were placed in each well and the drug was applied. Four replications were used for each drug. In addition to these, 4 controlled recurrences were applied. Doses Field dose of each insecticide was applied. In the fields, doses are normally diluted in 100 liters. In this application, the dose is calculated and diluted in 20 ml: 1.2 mg acetamiprid and 3 µl imidacloprid in 1 ml, respectively. Tap water is used in control wells. Dead EPNs counting Counts were made at 24, 48 and 96 h. Live IJs are moving. Dead IJs are still and flat (Ulu et al., 2016). Two watches were used to count the dead- living distinction. After counting, the IJs in each well were recorded. Statistical analysis The ANOVA test was used to compare the mortality rates of the species with those of each other and themselves in drug efficacy trials. ANOVA test and LSD were performed in the JMP® 7.0 program. Results and Discussion Effects of acetamiprid According to the results, the mortality rate increases with time (24, 48 and 96 h) (Fig. 1). The lowest mortality rate was significantly detected at 24 hours. Time-dependent differences were observed. http://www.sciencemag.org/content/335/6076/1555.short http://www.sciencemag.org/content/335/6076/1555.short 377 Fig. 2 Effects of imidacloprid on Steinernema carpocapsae (Sc) and Steinernema feltiae (Sf) (F= 2,6041; df = 5, 18; p = 0,0610) Effects of imidacloprid According to the results obtained, the mortality rate increases with time and the lowest mortality was observed at 24 h (Fig. 2). It was determined that the mortality rate changed with time and dose. The lowest mortality rates were observed at 24 h, high mortality at 96 h. There is no direct numerical correlation between doses and mortality rates (Fig. 2). Mortality increases with time without direct numerical correlation with dose variation. Since there were no previous studies evaluating the direct effects of insecticides on EPNs, this study focuses on the effects of the two insecticides on EPNs. Comparing these results, it is observed that the acetamiprid has a higher mortality than imidacloprid. This confirms existing research evaluating the compatibility of EPNs and imidacloprid and acetamiprid to control of pests. According to those researchers, compatibility with imidacloprid is high. Acetamiprid is less compatible. These results were reported by Koppenhöfer et al. (2003). In previous green houses and field studies on control of five scarab larvae, neonicotinoid insecticide imidacloprid has been shown to interact synergistically with five entomopathogenic nematode species. Two other neonicotinoids, thiamethoxamine and acetamiprid showed weaker interaction with nematodes in the scarab larvae. Imidacloprid and EPNs have shown good synergy against harmful insects (Farkhanda, 2012; Sheykhnejad et al., 2024), and are consistent with this research; imidacloprid was found to have a mortality rate of 0,122 % to 14,25 % in this study. 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