J. BIOL. ENVIRON. SCI., 2019, 13(37), 39-47 Original Research Article Side Effects of Azadirachtin On Some Important Beneficial Insects in Laboratory* Mehmet Sadık Cura and Nimet Sema Gençer† Department of Plant Protection, Faculty of Agriculture, Bursa Uludag University, 16059, Bursa, TURKEY Received: 15.04.2019; Accepted: 03.04.2019; Published Online: 25.05.2019 ABSTRACT The toxic effect of azadirachtin (0.3 g/L) in recommended dose, its half and twice doses were tested on Encarsia formosa, Aphidius colemani, Orius laevigatus and Nesidiocoris tenuis under laboratory conditions. The mortalities of nymphs and adults were evaluated by the IOBC toxicity rating scale for pesticides. As a result of the study, the product was harmful to O. laevigatus and N. tenuis adults and E. formosa pupa. Slightly and moderately harmful on N. tenuis nymph and A. colemani at recommended and twice doses. In recommended dose it was harmless only on N.tenuis male and E. formosa pupa at 24 h. Twice dose of the product was very toxic to all of the beneficial insects. The adulticide effects on males and females of O. laevigatus and N. tenuis in half dose at 48 h were found to be 83.33% and 75.00% and 78.26% and 76.20%, respectively. Additionally, little toxic effect (28%) was detected for A. colemani at half dose after 48 h. Similarly, at this dose, the mortality rates indicated less toxic effects on N. tenuis nymphs. In conclusion, it was understood that the recommended and twice doses of azadirachtin had negative effects on natural enemies. It was suggested that azadirachtin should be used carefully in pest control programs. Keywords: Azadirachtin, Beneficial insects, Insecticidal effect, Toxicity, Side effect INTRODUCTION Pesticides and agrochemicals became an important component of worldwide agriculture systems during the last century, allowing for a noticeable increase in crop yields and food production (Carvalho 2017, Alexandratos and Bruinsma, 2012, Ngowi et al. 2007). Pesticides not only kill the pest organisms but also affect many natural enemy populations (Cloyd 2012). However, continuous use of pesticides may result in some potential ecological problems, including resistance and secondary pest epidemics (Ruberson et al.,1998). The adverse effects of pesticides against the human, animals and the environment cause alternative methods of pest control. In this case, one of the alternative pesticides used against pests is herbal compounds. Some studies have shown that the use of plant-based insecticides can keep insect pests below economic damage levels (Tepe 2010). Natural products having insecticidal effects have been used in the struggle of pests since ancient times (Isman 1997, Ujvary 2001). Neem is one of these plant-derived insecticides. Approximately 150 compounds have been detached from different parts of the neem tree Azadirachta indica A. Juss (Meliaceae) (Girish and Shankara 2008, Singh et al. 2010). Azadirachtin was a native compound derived from seeds and leaves of neem tree. It was known to act as a growth regulator (Mordue and Nisbet, 2000). Also it has mortality effect on insects, as well as negative impacts on the feeding development, behaviour and fertility (Hanning et al. 2009, Isman 2006, Waghmare et al. 2007). Azadirachtin was used against many pests found in greenhouses and open-field plant growing. It was believed that the IPM programs will have a selective effect on natural enemies (Schmutterer 1995, Simmonds et al. 2002, Santolamazza-Carbone and Fernández de Ana- Magán 2004). The effect of azadirachtin has been studied with many pest species (Schmutterer 1985, Lynn et al. 2012, Pavela et al. 2013, Tomé et al. 2013) and some natural enemies (Raguraman et al. 2004, Cordeiro et al. 2010, Celestino et al. 2014) and it has been found safer than other conventional insecticides. More than 550 harmful species can be controlled by the neem extract even if they are resistant to synthetic pesticides (Whalon et al. 2008, Ascher 1993). Among them, Western flower thrips, Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), and aphids were key pests of many agricultural plants that cause extensive economic losses in greenhouses and open-field area (Reitz et al. 2011, Minks and Harrewıjn 1987). The generalist predator, Orius spp. (Hemiptera: Anthocoridae), is the efficient predators of thrips, aphids, spider mites, whiteflies and moths (Blaeser et al. 2004, Silveira et al. 2004, Islam et al. 2010). Besides, parasitoid, Aphidius colemani * This article is part of the MSc thesis of the first author. † Corresponding author: nsgencer@uludag.edu.tr 39 J. BIOL. ENVIRON. SCI., 2019, 13(37), 39-47 Viereck (Hymenoptera: Braconidae), is used commercially for biological control of aphids in greenhouse crops. It has a host range of over 41 aphid species (Starý 1975). Side effects of azadirachtin were studied on some natural enemies, include larvae of Chrysoperla carnea Stephens (Neuroptera: Chrysopidae), eggs, nymphs and adults of Amphiareus constrictus (Stal) and Blaptostethus pallescens Poppius (Hemiptera: Anthocoridae), coccinellid predators Coccinella undecimpunctata L, Adonia variegata, egg parasitoid, Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) and aphid parasitoid, A. colemani (Medina et al. 2003, Gontijo et al. 2015, Swaminathan et al. 2010, Abudulai and Shepard 2003, Atalla et al. 2009, Schmutterer 1997, Stara et al. 2011). However, some toxicity effects of Neem Azal on some Tuta absoluta (Meyrick,1917) (Lep.: Gelechiidae) predators, Nesidiocoris tenuis Reuter and Macrolophus pygmaeus Rambur (Het.: Miridae) (Arno and Gabarra 2011). Additionally, behavioral and mortality effects of some botanical insecticides on the greenhouse whitefly Trialeurodes vaporariorum Westwood (Hem: Aleyrodidae) and its parasitoid Encarsia formosa Gahan (Hym: Aphelinidae) (Simmonds et al. 2002). Particularly, in recent years, there has been an increase in the use of biological control agents in IPM against pests in the greenhouses. Therefore, in this study, it was planned to study the side effect of different doses of a commercial formulation of azadirachtin on various biological stages of some natural enemies which can be found in natural environments or used in biological control methods. MATERIALS AND METHODS Host plant rearing In this study, common bean (Phaseolus vulgaris L.) (Fabaceae) (Magnum) were used as the test plant. To provide green leaves, bean plants were grown in plastic pots (12 x 11 cm) containing a mixture of vermiculite and soil. Natural enemies Commercial strains of predatory bugs, Orius laevigatus (Fieber) (Het.: Anthocoridae) and N. tenuis, Whitefly parasitoid, E. formosa and aphid parasitoid A. colemani were provided by Koppert Biological Systems (Antalya, Turkey). En-strip product has 3000 E. formosa pupae in one package. One-hundered ml bottle of Nesibug product had 500 adults and nymphs of N. tenuis. Tripor-L product was packed in bottles containing 500 O. laevigatus individuals dispersed in vermiculite. Aphipar product was mixed with wood-chips in one bottle containing 1,000 A. colemani mummies. Pesticide doses The commercial formulation of azadirachtin, nimbecidine (0.3 g/L azadirachtin) (Agrobest, Turkey), were tested at the recommended field dose (500 ml/100 L water) which is registrated for the western flower thrips in pepper, and two other doses (half and twice of the recomended doses) in the experiment. Contact or Residual method The method used in this study was adapted from the method stated by Simon (2014). In this method, formulated insecticide was diluted in a solvent (water) and the insecticide solution filled in water spray container. In the experiments, plastic Petri dishes (10 cm diameter) with 25 small air holes were used in the upper cover. Firstly, water-soaked paper towel were placed at the bottom of Petri dishes in order to prevent the bean leaves dry in a short time. Also, fully expanded leaves were selected to prevent leaf deterioration in a short time. Then the bean leaves approximately 4 cm diameter were placed on paper towel one by one. The azadirachtin solution was then sprayed on to the bean leaf in the Petri dishes and allowed to dry in room temperature at about 30-40 minutes. Mobile insects were released on the treated surface and thus got exposed to insecticide. But immobile stages (pupa) and first pupae were placed and then insecticide sprayed. 40 J. BIOL. ENVIRON. SCI., 2019, 13(37), 39-47 Experimental design We separated female, male and nymphes of predatoy bugs under stereomicroscobe. A total of two or three days old adults (10 female+10 male) and nymphes were selected and transferred using a fine paintbrush on to the threatened bean leaves. Exposure of E. formosa pupae to azadirachtin by direct contact+residues led to mortality in laboratory bioassays. Twenty E. formosa pupae and female were used for experiments. In the E. formosa pupae experiment first we put pupae on bean leaves then we treated with azadiracthin. E. formosa pupae on cards in glass tubes put into the climatic condition room (25±5°C, 65±5% R. H. and 16 L: 8 D hours) to provide adult emergence in a short time. They were released on to the bean leaves and the upper cover was closed. In A.colemani test, 10 females were taken into the glass tubes. Since parasitoids were very mobile, they were left in the refrigerator for 5 minutes in order to reduce movements. Thus, the release of parasitoids into the petri dishes was easier. After releasing 10 individual of parasitoid wasp into the dishes. In all experiments, we surrounded the dishes by parafilm in order to avoid the insects escape. The control bean leaves were treated with distilled water. There were three replications for each doses of pesticide. The results were controlled after 24, 48, 72 hours and the number of live and alive individuals were noted. Adult individuals were examined for mobility, however deformation of the pupae was examined. For the toxicity rating of pesticides (based in the total effect caused in the enemy), the IOBC classification for laboratory standard tests (residual exposure) (Hassan, 1994): 1 (harmless, <30%), 2 (slightly harmful, 30-79%), 3 (moderately harmful, 80-99%), 4 (harmful, >99%) has been used in the study. Statistical Analysis Toxic effects were analysed by two-way ANOVA. The means were compared with Tukey post-hoc test and the results were displayed in the form of letters. Variables were displayed as mean with 95% confidence interval (CI). Mortality rates were arranged according to Abbott’s formula (Abbott,1925). Statistical analysis were performed using JMP program. RESULTS AND DISCUSSION The toxicity of azadirachtin on Orius laevigatus The adulticide effects of half, recommended and twice doses of azadirachtin on adult females and males of O. laevigatus were shown in Fig.1. At the the half dose, mortality rate was 53.33% in female and male of O. laevigatus within 24 hour period (Fig.1). But mortality rate increased within 48 and 72 hours. Similarly, Bonsignore and Vacante (2012) found that the botanical insecticides rotenone and neem decreased number of adult O. laevigatus. In contrast to our results, Angeli et al. (2005) stated that azadirachtin had no significant effect on the predator O. laevigatus. However, other studies with botanical insecticide-acaricide (based on plant extracts of castor bean, chicalote and berberis) was evaluated on two pollinators-bees and bumblebees-and on the predators Chrysoperla carnea and Orius insidiosus was slightly toxic (>%25) (Luna-Cruz et al. 2018). The toxicity of azadirachtin on Nesidiocoris tenuis The effect of these different three doses of azadirachtin on adult females, males and last instars nymphes of N. tenuis were given in Fig. 2. Half dose of azadirachtin was only slightly toxic (20 %) on predatory bug N. tenuis male and female one day after treatment. At the recommended dose, mortality rate was 90.48% in female and 82.6% in male of N. tenuis within 48 hours. The significantly highest mortality rate (100 %) occured at the recommended and twice doses of the azadirachtin after 72 hours on predatory adult bugs. Also N. tenuis nymphal mortality was high in twice dose of azadirachtin within 72 hours. Also, the increase in doses causes an increase in death of natural enemies, as was observed by Momen et al. (1997). In agreement with Zanuncio et al. (2016) it was suggested that the mortality rates of different Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) nymph stages increased with increasing neem oil concentrations. Similarly, Arno and Gabarra (2011) found negative effect of azadirachtin on the Macrolophus pygmaeus Rambur (Hem.: Miridae) and N. tenuis. Also, Gontijo et al. (2015) found that azadirachtin affected the mortality 41 J. BIOL. ENVIRON. SCI., 2019, 13(37), 39-47 of adult, A. constrictus and B. pallescens important anthocorid predators of the tomato pinworm T. absoluta. Additionally, the same investigators reported that azadirachtin prevented the capacity of predator nymphs to reach the adult stage. 100 90 80 70 60 50 40 30 20 10 0 O.leavigatus(mal O.leavigatus(fem O.leavigatus(mal O.leavigatus(fem O.leavigatus(mal O.leavigatus(fem e)(24 hours) ale)(24 hours) e)(48 hours) ale)(48 hours) e)(72 hours) ale)(72 hours) 0,125 53,33 53,33 83,33 75 100 100 0,25 80 80 100 100 100 100 0,5 93,33 100 100 100 100 100 Figure 1. The mortality rates (%) (Mean ±SE) of half dose (250ml/l), recommended (500ml/l) and twice (1000ml/l) doses of azadirachtin on Orius laevigatus females, and males at different counting times (hour). 100 90 80 70 60 50 40 30 20 10 0 N.tenuis(ma N.tenuis(fe N.tenuis(ny N.tenuis(ma N.tenuis(fe N.tenuis(ny N.tenuis(ma N.tenuis(fe N.tenuis(ny le)(24 male)(24 mphe)(24 le)(48 male)(48 mphe)(48 le)(72 male)(72 mphe)(72 hours) hours) hours) hours) hours hours) hours) hours) hours) 0,125 20 20 28 78,26 76,2 43,48 95,45 95 77,78 0,25 28 32 36 82,6 90,48 52,17 100 100 83,33 0,5 60 56 52 100 100 60,87 100 100 88,89 Figure 2. The mortality rates (%) (Mean ±SE) of half dose ( 250ml/l), recommended (500ml/l) and twice (1000ml/l) doses of azadirachtin on Nesidiocoris tenuis females, males and nymphes at different counting times (hour). The toxicity of azadirachtin on Encarsia formosa The side effect of azadirachtin on the pupae and adults of Whitefly parasitoid E. formosa were shown in Fig.3. The high and significant mortality of E. formosa females were observed in Petri dishes treated by azadirachtin at twice doses within 48 h (97.92%) and 72 h (100%). Mortality was low (20.34%) at half doses in 24 h, but high (92.59%) in 72 h. According to Drobnjaković et al. (2018) the longevity of E.formosa adults exposed azadirachtin for 48 hour was shorter than that of control wasps. In contrast to us, Yankova et al. (2011) showed that azadirachtin was non-toxic to adult of the parasite E. formosa. Felthege and Schmutterer (1993) stated that the most suitable among the products combined E. formosa include azadirachtin. Mortality rates of E.formosa pupae were 80.64 % at half dose within 72 h. Güncan et al. (2005) stated that, neem was found to be more effective on 18 day E.formosa pupae (%53). Some other researchers have reported that products obtained from A. indica have a potential for use in IPM programs against white flies which are biologically challenged with E.formosa (Simmonds et al. 2002). It is known that botanicals have different mechanisms of action. Different doses of azadirachtin cause mortality, 42 Mortality rates(%) Mortality rates(%) J. BIOL. ENVIRON. SCI., 2019, 13(37), 39-47 inhibits adult emergence from the pupae and survival and results in anomalies on pupae. Like to see our work, Hossain et al. (2013) determined that some of them were biologically active chemical compounds. 120 100 80 60 40 20 0 Encarsia E.formosa(pupa) E.formosa(femal E.formosa(femal E.formosa(pupa) E.formosa(femal formosa(pupa)(4 (24 hours) e)(24 hours) e)(48 hours) (72 hours) e)(72 hours) 8 hours) 0,125 0 20,34 80,64 75,86 80,64 92,59 0,25 0 66,1 100 96,55 100 100 0,5 0 83,05 100 97,92 100 100 Figure 3. The mortality effects (Mean ±SE) of half dose (250ml/l), recommended (500ml/l) and twice (1000ml/l) doses of azadirachtin on Encarsia formosa pupae and females at different counting times (hour). The toxicity of azadirachtin on Aphidius colemani Neem extract at half doses was not high toxic effect at 24 and 48 hour (20.69% and 28%, respectively) in A.colemani (Fig. 4). In this study, A.colemani was relatively less susceptible compare to other species. However it was lethal to adult of A.colemani 80% and %100 at twice doses at 48 and 72 hours, respectively. Likewise, as also demonstrated by Stara et al. (2011) mortality rate was 100% after 48 h in adults of A. colemani exposed to azadirachtin. Some studies show that, after field treatment with insecticide, potential of recolonization by the parasitoid Aphidius ervi Haliday (Hym.: Braconidae) were reduced (Desneux et al. 2006 ab, Desneux et al. 2007, Joseph et al. 2011). 100 90 80 70 60 50 40 30 20 10 0 0,125 0,25 0,5 A.colemani(female)(24 hours) 20,69 41,38 65,52 A.colemani(female)(48 hours) 28 72 88 A.colemani(female)(72 hours) 66,67 95,83 100 Figure 4. The mortality effects (Mean ±SE) of half dose( 250ml/l), recommended (500ml/l) and twice (1000ml/l) doses of azadirachtin on Aphidius colemani females at different counting times (hour). The toxicity of azadirachtin depending on species, dose and exposure time The results of variance analysis in terms of species, dose and exposure time, and interactions between them were given in table 1. Main effect of azadirachtin were significant (P< 0.0001), while interaction between dose and 43 Mortality rates(%) Mortality rates(%) J. BIOL. ENVIRON. SCI., 2019, 13(37), 39-47 exposure time was significant (F=13,88; df=4,4; P<0,0001). All treatments were statistically different (F=22,56; df=71,144; P<0,0001). The effect of species, doses and time was found to be most significant in pupa of E. formosa and O.laevigatus adults (Table 1). One hundered percent mortality was observed at the 48 and 72 h at recommended and twice doses. But, E. formosa female was not significantly affected at half dose after 24 hour. In O. laevigatus females and males, 100% death was seen in 72 h. When we evaluated the effect of species and time, it was understood that N.tenuis male, female and nymphs was least affected by the 24th hour. When we evaluate the dose and species interaction, the females of A. colemani and N. tenuis nymphs were the least affected by the half dose of azadirachtin. Besides, we can say that N. tenuis nymphs are less affected by all doses. As the doses increased and time progressed, it was seen that the mortality rates increase in all species. According to the classification of toxicity of insecticides on natural enemies for laboratory tests, according to the IOBC/WPRS (Hassan 1994), most of the results of the present work are included within the second, third and fourth category of toxicity [ 2 (slightly harmful, 30-79%), 3 (moderately harmful, 80-99%), 4 (harmful, >99%)]. On the other hand, the product was highly harmful to the male and female of O. laevigatus and N.tenuis and E.formosa pupa. But, slightly and moderately harmful on N. tenuis (nymph) and A.colemani at half, recommended and twice doses, except twice dose at 72 h in A.colemani . Azadirachtin was harmless on N.tenuis male, female and nymph and E.formosa pupa at 24 h. However, in recommended doses it was harmless only on N.tenuis male and E. formosa pupa at 24 h. Table 1. Mortality effects of different azadirachtin doses on different stages of natural enemies in different exposure time. Species Hours Doses 0.125 0.25 0.5 Encarsia formosa (pupa) 24 0.00 l 0.00 l 33.33 ı-l 48 80.64 a-f 100.00 a 100.00 a 72 80.76 a-f 100.00 a 100.00 a Encarsia formosa (female) 24 20.33 kl 66.10 a-ı 83.05 a-f 48 75.86 a-g 96.55 a-c 98.27 ab 72 92.59 a-d 100.00 a 100.00 a Orius laevigatus (female) 24 53.33 e-k 80.00 a-g 100.00 a 48 75.00 a-g 100.00 a 100.00 a 72 100.00 a 100.00 a 100.00 a Orius laevigatus (male) 24 53.33 e-k 80.00 a-g 93.33 a-c 48 83.33 a-f 100.00 a 100.00 a 72 100.00 a 100.00 a 100.00 a Nesidiocoris tenuis (female) 24 19.99 kl 31.99 ı-l 55.99 d-k 48 76.19 a-g 90.47 a-d 100.00 a 72 95.00 a-c 100.00 a 100.00 a Nesidiocoris tenuis (male) 24 19.99 kl 27.99 j-l 59.99 c-j 48 78.25 a-g 82.60 a-f 100.00 a 72 95.45 a-c 100.00 a 100.00 a Nesidiocoris tenuis (nymph) 24 27.99 j-l 35.99 h-l 51.99 f-k 48 43.47 g-k 52.16 f-k 60.86 c-j 72 77.77 a-g 83.33 a-f 88.88a-e Aphidius colemani (female) 24 20.68 kl 38.04 h-k 65.51 a-ı 48 27.99 j-l 71.99 a-h 87.99 a-f 72 62.50 b-j 95.83 a-c 100.00 a CONCLUSIONS In conclusion, mortality rates of adult stage of O. laevigatus, N. tenius, E. formosa and A. colemani were high in twice doses compared with half and recommended doses. 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