Effects of Imidacloprid on the Orientation Behavior and Parasitizing Capacity of Anagrus Nilaparvatae, an Egg Parasitoid of Nilaparvata Lugens

BioControl (2010) 55:473–483 DOI 10.1007/s10526-010-9276-z

Effects of imidacloprid on the orientation behavior and parasitizing capacity of Anagrus nilaparvatae, an egg parasitoid of Nilaparvata lugens

Fang Liu • Shan W. Bao • Ying Song • Hai Y. Lu • Jian X. Xu

Received: 17 June 2009 / Accepted: 8 March 2010 / Published online: 25 March 2010 Ó International Organization for Biological Control (IOBC) 2010

Abstract Anagrus nilaparvatae (Pang et Wang) imidacloprid treated rice plants by A. nilaparvatae (Hymenoptera: Mymaridae), is an egg parasitoid of decreased significantly. These effects involving dis- rice planthoppers, Nilaparvata lugens (Sta˚l) (Homop- turbed foraging ability and reduced parasitizing capac- tera: Delphacidae). This study evaluated effects of the ity of A. nilaparvatae indicated that imidacloprid could insecticide imidacloprid on orientation behavior and decrease the performance of this parasitoid. parasitizing capacity of A. nilaparvatae. Sub-lethal concentrations of imidacloprid (LC20 and LC10) dis- Keywords Mymaridae Anagrus nilaparvatae rupted the foraging ability of A. nilaparvatae exposed Imidacloprid Sub-lethal effect Indirect non-target to imidacloprid through contact or oral routes. Some effect Orientation behavior Parasitism survivors did not respond to volatiles from N. lugens- Rice infested plants. Responsive individuals were equally attracted to volatiles from N. lugens-infested and healthy plants. Volatiles emitted from rice plants Introduction treated with a low concentration of imidacloprid were more attractive to A. nilaparvatae than those from Anagrus nilaparvatae (Pang et Wang) (Hymenoptera: plants treated with a high concentration of imidaclo- Mymaridae), is a major egg parasitoid of such rice prid. Parasitism of N. lugens by A. nilaparvatae that planthoppers as Nilaparvata lugens (Sta˚l) (Homoptera: survived contact with sub-lethal concentrations of Delphacidae), Sogatella furcifera (Horvath) (Homop- imidacloprid did not decrease significantly. When tera: Delphacidae), and Laodelphax striatellus (Fallen) A. nilaparvatae were fed imidacloprid-honey mixture, (Homoptera: Delphacidae) (Cheng 1996). Biological parasitism rates were 1.49% and 0%, respectively, and ecological characters of A. nilaparvatae are well significantly lower than those of the control (9.58%). documented (Lou et al. 2002, 2005a, b; Lou and Cheng Parasitism of N. lugens eggs in high concentration of 1996, 2001; Luo and Zhuo 1981, 1986). In a field survey conducted in Fujian province, China, 40–60% of planthoppers eggs were parasitized by A. nilaparvatae Handling Editor: Dirk Babendreier. (Luo and Zhuo 1986). Parasitization rate of N. lugens eggs by A. nilaparvatae in Jiang-Huai Area ranged from F. Liu (&) S. W. Bao Y. Song H. Y. Lu J. X. Xu 10% to 60% (Cheng et al. 2003). The broad host range, College of Horticulture and Plant Protection, Yangzhou high fecundity, and short life cycle of A. nilaparvatae University, Yangzhou 225009, People’s Republic of China make it an excellent candidate for biological control of e-mail: [email protected] rice planthoppers (Lou and Cheng 2001). 123 474 F. Liu et al.

Chemical control remains a major method in directive 91/414/EEC. This directive lists active integrated pest management (IPM) because it is quick, ingredients allowed in the European Community efficient, easy to use, cost-effective, amendable to (Buffin 2003). In China, imidacloprid has been used practice, reliable and effective (Endo and Tsurumachi to control N. lugens for more than ten years. This 2001; Zhao 2000). Pesticides are designed to be toxic pesticide is also widely used in almost all N. lugens to target pests. However, even when used correctly, areas due to its high efficacy, long residual activity, and some pesticides can harm non-targeted organisms, environmental compatibility (Feng and Pu 2005; Liu including arthropod natural enemies particularly and Han 2006). However, wide use of imidacloprid important for crop pest control (Delaplane 2000). against N. lugens runs the risk of resistance develop- Pesticides exert a wide variety of direct and indirect ment in target insects (Liu and Han 2006; Wang et al. non-target effects on natural enemies. Lethal effects 2008b, 2009). Negative effects on non-target insects are often expressed as acute or chronic mortality have also resulted (Sun et al. 2008; Wang et al. 2008a). resulting from contact with or ingestion of pesticides Imidacloprid showed acute contact, oral and residual (Haseeb et al. 2004). Sub-lethal effects are defined as toxicity to adult A. nilaparvatae (Wang et al. 2008a). typically chronic, non-obvious effects (Haseeb et al. However, apart from a reduction of the parasitism of 2004) on individuals that survive pesticide exposure N. lugens (Xu et al. 2006), little is known about the sub- (Desneux et al. 2007). Sub-lethal effects include lethal and indirect non-target effects of imidacloprid changes in life-history traits, such as parasitism rate, on this parasitoid. longevity, egg viability, changes in consumption rates, This study investigated direct sub-lethal effect or behavioral change (Ruberson et al. 1998). Complete and indirect non-target effects of imidacloprid on analysis of pesticide impact must consider sub-lethal A. nilaparvatae. Sub-lethal effects of imidacloprid effects (Desneux et al. 2007). were assessed through direct contact and dietary In addition to direct lethal and sub-lethal effects, exposure. The study assessed indirect non-target indirect non-target effects of pesticides on natural effects that resulted from biochemical changes in rice enemies may occur, e.g., through feeding on plants due to imidacloprid stress. insecticide-intoxicated prey (Walker et al. 2007), contaminated nectar or honeydew (Longley and Materials and methods Jepson 1996; Stapel et al. 2000). Altered physiological and biochemical aspects of host plants may Rice plants also produce indirect non-target effects. For example, attraction of female parasitoid Microplitis croceipes Japonica rice variety Zhendao2 is widely planted in (Hymenoptera: Braconidae) to cotton decreased Jiangsu province, China and was used in this study. significantly after the cotton was sprayed with Plantings were made at successive 15 days intervals fenvalerate/chlordimeform mixture or with meth- so that 50–55 days old plants were always available omyl (Elzen et al. 1989). Neochrysocharis formosa for experiments. (Westwood) (Hymenoptera: Eulophidae) spent more time resting near or away from its hosts, but less time foraging for hosts if leaves of host plant, Insects kidney bean, were treated with imidacloprid (Tran et al. 2004). Few studies to date address indirect N. lugens were obtained from a stock population non-target effects on natural enemies via physio- maintained at China National Rice Research Institute logical and biochemical changes in rice plants due (Hangzhou, China). Prior to use in our experiments, to pesticide stress. N. lugens were reared on Shanyou63 (hybrid rice, Imidacloprid, the first of the neonicotinyl insecti- susceptible to N. lugens) for several generations in cides, acts on the insect nervous system as an agonist at covered cages in our laboratory at 28 ± 2°C, 12 h the nicotinic acetylcholine receptor (Zhang et al. photophase, and 70–80% R.H. 2000). Since its launch in 1991, products containing A. nilaparvatae were collected from an insecticide- imidacloprid have gained registrations in about 120 free rice field in Yangzhou, by using Shanyou63 rice countries and have been included in annex I of plants with N. lugens eggs as bait. The colony was 123 Effects of imidacloprid on the orientation behavior 475 propagated on N. lugens eggs in rice stems enclosed in Table 1 Combination of imidacloprid treatment variables in glass tubes (2.5 cm 9 20 cm), kept in a 26 ± 2°C each experiment room with 12 h photophase and 70–80% R.H. Each Experiments Side effects Parasitoids Rice day, newly emerged wasps were collected in clean investigated plants glass tubes and given access to water and honey Experiment 1 Orientation behavior ?- solution. Parasitoids were kept inside the tube for at Experiment 2 Orientation behavior -? least 2 h to ensure that all females mated. From the Experiment 3 Parasitizing capacity ?- second generation onwards, female parasitoids were Experiment 4 Parasitizing capacity -? used in experiments within 24 h after emergence. Note: ? indicated presence of imidacloprid, - indicated Insecticide absence of imidacloprid

Two formulations of imidacloprid were used. Techni- of imidacloprid for A. nilaparvatae via contact and cal grade imidacloprid (95.3%) (Jiangsu Changlong oral routes. In step 2 we exposed A. nilaparvatae to Chemical Co., Ltd., Jiangsu, China) was used to sub-lethal concentrations of imidacloprid as deter- determine sub-lethal effects via contact or oral routes mined by preliminary bioassay, using contact and oral to avoid inﬂuence of adjuvant added in commercial routes. In step 3 we observed orientation behavior of products. Imidacloprid wettable powder (WP) at a 10% A. nilaparvatae that had survived sub-lethal concen- concentration, a common formulation in the Chinese trations of imidacloprid to volatiles from N. lugens- market (Yangzhou Suling Pesticide Chemical Co., infested rice plants. Ltd., Jiangsu, China) was used in foliar spray treat- Step 1: Determination of sub-lethal concentrations ments to determine indirect non-target effects. The of imidacloprid using contact and oral routes. formulated imidacloprid was more applicable to foliar Imidacloprid concentrations sufﬁcient to cause 10– spray treatment because tension-active agents facili- 90% mortality of A. nilaparvatae were determined by tate penetration. a preliminary bioassay with the method described by Wang et al. (2008a). Adult parasitoids were exposed Experimental protocols to the serial concentrations to establish a concentration–mortality relationship. LC10 and LC20 were then Four separate sets of experiments were carried out to estimated from an appropriate regression (see below, investigate sub-lethal and indirect non-target effects of Statistical Analysis). imidacloprid. Experiment 1 investigated orientation Step 2: Exposure of A. nilaparvatae to sub-lethal behavior of surviving A. nilaparvatae after exposure to concentrations of imidacloprid via contact and oral sub-lethal concentrations of imidacloprid. Experiment routes. 2 studied A. nilaparvatae orientation to volatiles A. nilaparvatae females were exposed to two sub- from imidacloprid treated rice plants. Experiment 3 lethal concentrations of imidacloprid (LC10 and concerned parasitism of N. lugens by surviving LC20) via contact and oral routes. A. nilaparvatae A. nilaparvatae after exposure to sub-lethal concen- females exposed to 80% (v/v) acetone only were used trations of imidacloprid. Experiment 4 concerned as controls. After 1 h exposure, the survivors were parasitism of N. lugens in imidacloprid treated rice collected and placed individually in Petri dishes plants by A. nilaparvatae. Table 1 lists imidacloprid (5.3 cm in diameter) containing honey solution treatment variables for each experiment. (10%). Behavioral tests were performed within 2 h of the end of exposure. Experiment 1: Orientation behavior of surviving Step 3: Orientation behavior of surviving A. nila- A. nilaparvatae treated with sub-lethal parvatae treated with sub-lethal concentrations of imi- concentrations of imidacloprid to volatiles from dacloprid to volatiles from N. lugens-infested rice plants. N. lugens-infested rice plants Orientation behavior of surviving A. nilaparvatae females were tested using a Y-tube olfactometer In step 1 of this experiment (preliminary bioassay) we (Liu et al. 2002; Lou et al. 2005b), the standard determined sub-lethal concentrations (LC10 and LC20) apparatus for studying insect olfactory responses 123 476 F. Liu et al.

(e.g., parasitoids: Souissi, 1999; lacewings: Reddy, field rate (15 g a.i. ha-1). The higher concentration 2002; predatory mite: Boer et al. 2005). (46.7 mg a.i. l-1) was equal to the highest recom- A pair of odor sources was provided for each test: mended field rate (35 g a.i. ha-1). The desired amount plant-N. lugens gravid female complex (PF) and of imidacloprid was calculated based on the area unmanipulated plants (UP). Odor sources were occupied by potted rice plants. Imidacloprid diluted to prepared as follows. required concentrations with tap water, was sprayed using a Yantse 08 model sprayer. Tap water spray (1) Plant-N. lugens gravid female complex (PF). served as a control (CK). Plants infested by N. lugens, Potted plants were washed with running water then treated with low concentration of imidacloprid and thinned to yield ten plants per pot. Each plant (LI) or high concentration of imidacloprid (HI) was was then individually infested with ten gravid used as an odor source for behavior tests 1, 3, 5 and N. lugens females. After one day, ten plants (cut 7 days after imidacloprid treatment. off at soil level and the cut end wrapped with a Step 2: Orientation behavior of A. nilaparvatae to piece of moist cotton) with 100 females were volatiles from imidacloprid-treated rice plants. used as an odor source for bioassays. Lou et al. Behavioral responses of female A. nilaparvatae, (2005b) found that volatiles released from plant- within 24 h after emergence, to N. lugens-infested N. lugens complexes, in which the proportions rice plants treated with imidacloprid were examined among the volatile compounds were altered, were as in Experiment 1. In this experiment, A. nilaparva- more attractive to A. nilaparvatae than those tae were presented with three pairs of odor sources: from unmanipulated plants. Therefore, the plant- (1) HI vs. CK, (2) LI vs. CK, and (3) HI vs. LI. N. lugens gravid female complex was used to represent N. lugens-infested plants. Experiment 3: Parasitism of N. lugens (2) Unmanipulated plants (UP). Ten potted plants by surviving A. nilaparvatae treated were cut off at soil level to serve as an odor with sub-lethal concentrations of imidacloprid source. Unmanipulated plants were used to represent healthy rice plants. Mated A. nilaparvatae were exposed to LC10 and LC20 Female parasitoids were introduced individually of imidacloprid via contact and oral routes as described into the base tube of the Y-tube olfactometer and given in Experiment 1. Five surviving female parasitoids 10 min to walk toward the end of one of the arms. were introduced into a tube (9.5 9 4.5 cm) containing Choice of a preferred odor source was defined as a four rice stems, each stem containing approximately female crossing a line 7 cm beyond the division of the 40–50 N. lugens eggs. A. nilaparvatae exposed to 80% base tube and remaining there for at least 1 min. If a (v/v) acetone only were used as control. N. lugens eggs parasitoid did not make a choice within 10 min, this were renewed daily until the death of the wasp. Eight outcome was recorded as no response. At least 32 days after parasitizing, rice stems were dissected females were tested for each odor source pair. under a microscope to check the total and parasitized N. lugens eggs (Lou et al. 2005a). Parasitism rate was Experiment 2: Orientation behavior defined as the ratio of the number of parasitized eggs to of A. nilaparvatae to volatiles the total number of N. lugens eggs. Ten contact toxicity from imidacloprid- treated rice plants replicates and three oral toxicity replicates were carried out for each sub-lethal concentration. Step 1: Preparation of imidacloprid-treated rice plants. Experiment 4: Parasitism of N. lugens eggs Potted plants were washed with running water, then in imidacloprid-treated rice plants by thinned to ten plants per pot. Each plant was then A. nilaparvatae individually infested with ten gravid N. lugens females. Two days after infestation, plants infested Step 1: Potted plants were thinned to five plants per with N. lugens were sprayed with imidacloprid (WP) pot. 50 gravid N. lugens were introduced and plants at two concentrations. The lower concentration were covered with a transparent cylindrical plastic (20 mg a.i. l-1) was equal to the lowest recommended cage (6 cm 9 50 cm) for oviposition. Two days after 123 Effects of imidacloprid on the orientation behavior 477 oviposition, potted plants were treated with two y ¼ 0:9807 þ 1:3869x ð1Þ concentrations of imidacloprid (the low and the high concentration) as described in Experiment 2. Tap (x means logarithm of concentration, y means prob- water served as control. ability of mortality). From the linear regression, the Step 2: Gravid N. lugens were then removed and estimated concentrations causing 10% and 20% of mortality were 93.35 lg a.i. l-1 and 193.47 lg a.i. imidacloprid-treated plants along with the control -1 -1 were covered with a nylon cylindrical cage, attached l , respectively. 100 lg a.i. l , i.e. two thousandths of the highest recommended field rate, and 200 lg by a nylon thread to prevent oviposition by N. lugens. -1 Treated pots were then placed randomly in cement a.i. l , i.e. four thousandths of the highest recom- tanks containing rice plants (Variety: Shanyou63) in mended field rate were chosen to represent LC10 and the greenhouse that had not received pesticide LC20, respectively. In the oral toxicity bioassay, the treatment for [5 years. A natural population of regression line was A. nilaparvatae in the cement tanks parasitized y ¼ 2:5568 þ 1:2344x ð2Þ N. lugens eggs in imidacloprid-treated plants and control. From the linear regression, estimated concentrations causing 10% and 20% of mortality were 8.75 mg a.i. Step 3: Three days post-parasitization, potted -1 -1 -1 plants were brought back to the laboratory and l and 19.93 mg a.i. l , respectively. 10 mg a.i. l , i.e. one-fifth of the highest recommended field rate, placed at 28 ± 2°C, 12 h photophase, and 80% R.H. -1 Each pot was then placed in an 11 9 40 cm plastic and 20 mg a.i. l , i.e. two-fifths of the highest cage. After five days, potted plants were cut off at recommended field rate were chosen to represent soil level and dissected under a microscope to record LC10 and LC20, respectively. the total and parasitized N. lugens eggs (Lou et al. 2005a). One pot (five plants per pot) was considered a Experiment 1: Orientation behavior of surviving replication. Each treatment along with the control had A. nilaparvatae treated with sub-lethal three replications. concentration of imidacloprid to volatiles from N. lugens-infested rice plants Statistical analysis About 55% and 35% of the A. nilaparvatae that

Data were analyzed using Statistica (SAS Institute survived contact with sub-lethal concentrations (LC20 Inc., Cary, NC, USA). A linear regression model for and LC10) of imidacloprid had no response to mortality curves used logarithmic transformation of volatiles from rice plants infested by N. lugens. The concentrations and probit transformation of mortali- survivors that responded were equally attracted to ties (Log-probit model, Finney 1971). Differences in volatiles emitted from N. lugens-infested plants and behavioral responses of the parasitoid to pairs of those from healthy plants. However, all control odors were determined by v2 tests. Parasitism data A. nilaparvatae responded and were significantly were analyzed by ANOVA. When ANOVA analysis attracted by N. lugens-infested plant volatiles determined significance (P \ 0.05), Tukey’s honestly (Table 2). In the case of oral exposure, 42.5% significant difference tests were performed to detect surviving wasps having fed on LC20 honey-imidaclo- significant differences between groups. prid mixture showed no response to host-associated odors. A. nilaparvatae that survived two sub-lethal concentrations of imidacloprid had responses but Results could not distinguish volatiles from N. lugens infested plants and those from healthy plants. However, Determination of sub-lethal concentrations via control A. nilaparvatae were significantly attracted contact and oral routes to the volatiles from N. lugens infested plants (Table 2). Results indicated that imidacloprid treat- In the contact toxicity bioassay, the regression line ment via both contact and oral routes disrupted describing morality-concentration relationship was foraging ability of surviving A. nilaparvatae.

123 478 F. Liu et al.

Table 2 Behavioral responses of surviving A. nilaparvatae treated with sub-lethal concentrations of imidacloprid to volatiles from N. lugens-infested rice plants Exposure route Concentration No. of tested No. of no No. of the parasitoid to the odor v2 value of imidacloprid parasitoids response parasitoids Odor 1 Odor 2

Contact Control 32 0 25 7 9.03**

LC10 40 14 13 13 0.04

LC20 40 22 13 5 2.72 Oral ingestion Control 40 0 31 9 11.03**

LC10 40 0 20 20 0.03

LC20 40 17 15 8 1.57 Note: The degree of freedom for v2 test was uniformly equal to one. Control means A. nilaparvatae exposed to 80% (v/v) acetone -1 -1 only. LC10 and LC20 were 100 and 200 lg a.i. l in the contact toxicity, and were 10 and 20 mg a.i. l in the oral toxicity, respectively. Odor 1 means rice plants infested by N. lugens, and Odor 2 means healthy rice plants 2 ** indicated signiﬁcant difference at a = 0.01 level, df = 1, v0.01 = 6.63

Experiment 2: Orientation behavior of Experiment 4: Parasitism of N. lugens eggs A. nilaparvatae to volatile from imidacloprid- in imidacloprid-treated rice plants treated rice plants by A. nilaparvatae

Parasitoids offered pairs of odors from imidacloprid High imidacloprid concentration affected parasitism of treated plants and from untreated plants showed no N. lugens by A. nilaparvatae. Parasitism of N. lugens in odor preference (Fig. 1A, B). However, A. nila- plants treated with a high concentration was parvatae preferred volatiles from low concentration 4.83 ± 2.07%, signiﬁcantly lower than control treated plants to volatiles from high concentration (11.62 ± 2.25%) (F = 8.43, df = 2, 6, P = 0.0181). treated plants (Fig. 1C). These results showed that However, treatment with a low concentration of volatiles released from rice plants treated with a imidacloprid had little impact on parasitism rate high concentration of imidacloprid disturbed the (12.55 ± 3.11%) (Fig. 3). orientation behavior of A. nilaparvatae.

Discussion Experiment 3: Parasitism of N. lugens by surviving A. nilaparvatae treated with sub-lethal Our experiments sought to simulate effects of field concentrations of imidacloprid spraying of imidacloprid and served to assess sub- lethal effects of imidacloprid on a parasitoid, Parasitism of N. lugens by surviving A. nilaparvatae A. nilaparvatae. Generally, parasitoids spend a after contact with LC20 and LC10 imidacloprid did not significant proportion of their adulthood searching differ significantly from the control (F = 2.54, df = 2, for places where their hosts can potentially be found 27, P = 0.0977) (Fig. 2A). A significant difference (Vinson 1998). When foraging for hosts, A. nila- was observed for oral exposure (F = 49.89, df = 2, 6, parvatae adults can potentially come in direct contact P = 0.0002). Parasitism of N. lugens by surviving A. with pesticides. They may feed on pesticide contam- nilaparvatae that had ingested LC10 and LC20 imida- inated honeydews secreted by rice planthoppers cloprid-honey mixture were 1.49 ± 1.42% and 0%, (Wang et al. 2008a). Subsequently, parasitoids may respectively, significantly less than control (9.58 ± die or their performance may be adversely influ- 1.67%) (Fig. 2B). enced (Haseeb and Amano 2002). Experiments were

123 Effects of imidacloprid on the orientation behavior 479

(A) 40 (A) 2 35 Contact toxicity DAT HI vs. CK value HI a 30

CK (%) 1d 0.03 25 a a 20 3d 0.78 15 0.03 5d 10

7d 0.28 A. nilaparvatae 5 0 20 10 0 10 20 30 by 14 (B) (B) 12 Oral toxicity 2 a DAT LI vs. CK value LI N. lugens 10 CK 1d 0.03 8 6 3d 0.78 4

Parasitism of b 5d 0.03 2 b 7d 0.28 0 20 10 0 10 20 30 Control LC10 LC20 Imidacloprid treatment (C) 2 DAT HI vs. LI value HI Fig. 2 Parasitism (mean ? SD) of N. lugens by surviving LI A. nilaparvatae treated with imidacloprid. A Parasitism of N. lugens by surviving A. nilaparvatae after contact with 1d 4.65* imidacloprid; B Parasitism of N. lugens by surviving 3d 2.70 A. nilaparvatae having ingested imidacloprid. Control means A. nilaparvatae exposed to 80% (v/v) acetone only. LC10 and 5d 9.03** -1 LC20 were 100 and 200 lg a.i. l in the contact toxicity, and -1 7d 4.65* were 10 and 20 mg a.i. l in the oral toxicity, respectively. Different letters indicated signiﬁcant differences among treat- 20 10 0 10 20 30 ments (P \ 0.05, Tukey’s honestly signiﬁcant difference tests) No. of parasitoid attracted

Fig. 1 Number of A. nilaparvatae adult females attracted by volatiles released from pairs of odors. A Rice plants treated 18 with high concentration of imidacloprid (HI) vs. CK (Rice 16 a

(%) a plants treated with tap water); B Rice plants treated with low 14 concentration of imidacloprid (LI) vs. CK (Rice plants treated 12 with tap water); C Rice plants treated with high concentration N. lugens by 10 (HI) vs. low concentration of imidacloprid (LI). DAT is days after imidacloprid treatment. The degree of freedom for v2 test 8 b 6 was uniformly equal to one. * indicated signiﬁcant difference A. nilaparvatae 2 at a = 0.05 level, df = 1, v0.05 = 3.84. ** indicated signiﬁ- 4 2 Parasitism of cant difference at a = 0.01 level, df = 1, v0.01 = 6.63 2 0 designed to investigate sub-lethal effects of imida- Control Low concentration High concentration cloprid on A. nilaparvatae via contact and oral Imidacloprid treatment routes because lethal effects of the insecticide on Fig. 3 Parasitism (mean ± SD) of N. lugens eggs in rice A. nilaparvatae have been reported (Wang et al. plants treated with imidacloprid by A. nilaparvatae. Control 2008a). Adult A. nilaparvatae were highly sensitive to means rice plants treated with tap water insecticides and exhibited toxic symptoms after a few minutes of exposure (Luo et al. 1981b). Moreover, the rather than a 24-h exposure period (e.g., Desneux et al. adulthood of A. nilaparvatae, generally ranging from 2004; Delpuech et al. 1998b) was selected to assess one to three days, is very short (Luo and Zhuo 1980; sub-lethal effects of imidacloprid on A. nilaparvatae Luo et al. 1981). Therefore, a 1-h exposure period in our study, as in Wang et al. (2008a). 123 480 F. Liu et al.

Parasitoid host search involves orientation to host contact with imidacloprid was found. This result and to host-plant odors (Vinson 1998). In this paper indicated that the sub-lethal effect of imidacloprid on we focused on volatile cues from the host plants. parasitization by A. nilaparvatae differed depending Such cues seem particularly important for long range on the exposure route. Desneux et al. (2006) reported detection of infested plants by parasitoids. Previous that longevity of Aphidius ervi (Hymenoptera: Aphi- studies found that A. nilaparvatae was attracted by diidae) decreased significantly (approximately 21%) volatiles released from N. lugens-infested rice plants when exposed to deltamethrin spray application but did (Liu et al. 2002; Lou and Cheng 1996; Lou et al. not decrease after topical application. Their results 2002; 2005a, b; 2006). indicated that sub-lethal effects of pesticides on We found that imidacloprid decreased the ability parasitoids may be different when parasitoids are of A. nilaparvatae to perceive host-plant odor cues exposed to pesticides by different exposure methods, when A. nilaparvatae were treated with sub-lethal but via the same exposure route. For oral exposure, the concentrations of imidacloprid. After exposure to reduction in parasitization possibly resulted from sub-lethal concentrations of imidacloprid via contact altered oviposition behavior, reduced longevity and or oral routes, some surviving A. nilaparvatae had no fecundity of A. nilaparvatae after exposure. However, response to volatiles from N. lugens-infested plants. these altered traits may not be observed in our contact Survivors that responded were equally attracted to exposure experiments. More studies are needed to volatiles from N. lugens-infested and healthy plants. verify these suggestions. Stapel et al. (2000) reported that imidacloprid could Previous studies indicated that foliar sprays of disturb host searching behavior in M. croceipes. After imidacloprid, buprofezin, and triazophos reduced the feeding on extrafloral nectar of imidacloprid-treated photosynthetic rate of rice leaves and also produced cotton, foraging ability of M. croceipes decreased several other physiological and biochemical changes drastically. From odor detection to associated behav- in rice plants (Luo et al. 2002; Qiu et al. 2004; Wu et al. iors, olfaction depends entirely on nervous transmis- 2001, 2003). These alterations in rice plants resulting sions, precisely the targeted pathway of neurotoxic from pesticide applications indirectly induced changes insecticides (Desneux et al. 2004). Therefore, the in reproductive rates of N. lugens and Tryporyza possible cause for reduced foraging ability of surviv- incertulas (Walker) (Lepidoptera: Pyralidae) (Wang ing parasitoids exposed to imidacloprid could involve et al. 2005; Wu et al. 2001). Triazophos sprays altered alteration in those functions necessary for olfactory proportions of the volatile compounds in rice plants responses in the olfactometer. Olfactory responses of (Lu et al. 2008). Our experiments sought to investi- beneficial insects impaired by other neurotoxic gate indirect non-target effects of imidacloprid on insecticides, such as chlorpyriphos (organophos- A. nilaparvatae via biochemical changes in rice plants. phate) (Delpuech et al. 1998a, b), lambda-cyhalothrin Attraction of female A. nilaparvatae to rice plants (pyrethroids) (Desneux et al. 2003), and triazamate sprayed with a low concentration of imidacloprid was (carbamyltriazol) (Desneux et al. 2004) have been significantly higher than attraction to plants sprayed previously demonstrated. with a high concentration of imidacloprid (Fig. 1C). Effects of imidacloprid on parasitizing capacity of Preference of A. nilaparvatae for rice plants treated parasitoids were examined with varying results. with a low concentration of imidacloprid was possibly Ozawa et al. (1998) found that imidacloprid had a due to altered volatiles released from these plants. harmful effect on parasitism of the Liriomyza trifolii Preference of A. nilaparvatae for rice plants treated (Diptera: Agromyzidae) by a parasitoid Diglyphus with a low concentration of imidacloprid might result isaea (Hymenoptera: Eulophidae). However, parasit- from evolved tolerance to imidacloprid in view of a ism of Bemisia argentifolii (Homoptera: Aleyrodidae) long history of exposure to imidacloprid. Resistance by Encarsia formosa (Gahan) (Hymenoptera: Aphe- surveys in 2005 indicated that four field popula- linidae) was not affected by imidacloprid (Bethke and tions (Nanning, Nanjing, Haiyan and Tongzhou) of Redak 1997). In our study, parasitizing capacity of N. lugens developed extremely high resistance levels surviving A. nilaparvatae decreased significantly after to imidacloprid with resistance ratio ranging from 200 feeding on an imidacloprid-honey mixture. However, to 799 for up to 13 years imidacloprid application little impact on parasitization by the survivors after (Wang et al. 2008b). After temporary suspension of 123 Effects of imidacloprid on the orientation behavior 481 imidacloprid control for N. lugens, resistance ratios in information for understanding the compatibility of four populations ranged from 107 to 625 in 2006 and imidacloprid with A. nilaparvatae. However, the from 135 to 233 in 2007, respectively. Resistance actual mechanism causing these adverse effects still level of N. lugens to imidacloprid remained high to remains unknown. Further studies on physiology and extremely high (Wang et al. 2008b, 2009). We neurotoxicology may be necessary for full understand- speculated that A. nilaparvatae has developed some ing of the impact of imidacloprid on A. nilaparvatae. tolerance to imidacloprid due to its long application history, although these parasitoids were collected Acknowledgements This research was financially supported from an insecticide-free rice field. by National Natural Science Foundation of China (No. 30500329) and National Basic Research Program of China (973 Program) A. nilaparvatae exhibit a preference for plants (No. 2010CB126200). We thank Dr. Y Hu of China National Rice treated with a low concentration of imidacloprid Research Institute for revising the manuscript. We also thank compared to plants treated with a high concentration. Professor SL Gu for help with statistics. We are very grateful However, attraction of this parasitoid to volatiles from to two anonymous reviewers for most valubale comments improving the manuscript considerably. imidacloprid treated plants and to those from the control (plants treated with water) did not differ detectably (Fig. 1A, B). For a foraging parasitoid, References insecticide treated plants may be associated with more hosts (benefit) because the insecticide is normally used Bethke JA, Redak RA (1997) Effect of imidacloprid on the silverleaf whitefly, Bemisia argentifolii Bellows and when pest density is high. At the same time, insecticide Perring (Homoptera: Aleyrodidae), and whitefly parasit- treated plants are also associated with higher risk (cost) ism. Ann Appl Biol 130:397–407 because insecticides are mostly toxic to parasitoids. 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biochemical changes in Tryporyza incertulas (Lepidep- Nilaparvata lugens by Anagrus nilaparvatae. Entomol tera: Pyralidae). J Econ Entomol 98:1144–1149 Knowledge 43:789–793 Wang HY, Yang Y, Su JY, Shen JL, Gao CF, Zhu YC (2008a) Zhang A, Kayser H, Maienfisch P, Casida JF (2000) Insect Assessment of the impact of insecticides on Anagrus acetylcholine receptor: conserved neonicotinoid specific- nilaparvatae (Pang et Wang) (Hymenoptera: Mymani- ity of (3H) imidacloprid binding site. J Neurochem dae), an egg parasitoid of the rice planthopper, Nila- 75:1294–1303 parvata lugens (Hemiptera: Delphacidae). Crop Prot Zhao SH (2000) Plant chemical protection. Agriculture Press 27:514–522 of China, Beijing, China Wang YH, Gao CF, Zhu YC, Chen J, Li WH, Zhuang YL, Dai DJ, Zhou WJ, Ma CY, Shen JL (2008b) Imidacloprid Author Biographies susceptibility survey and selection risk assessment in field populations of Nilaparvata lugens (Homoptera: Delpha- cidae). J Econ Entomol 101(2):515–522 Fang Liu is involved in studies related with compatibility of Wang YH, Wu SG, Zhu YC, Chen J, Liu FY, Zhao XP, Wang pesticide with natural enemy, potential applications of semio- Q, Li Z, Bo XP, Shen JL (2009) Dynamics of imidaclo- chemicals and varietal resistances. prid resistance and cross-resistance in the brown planthopper, Nilaparvata lugens. Entomol Exp Appl Shan W. Bao is studying side effects of insecticides on natural 131(10):20–29 enemies of rice pests. Wu JC, Xu JX, Yuan SZ, Liu JL, Jiang YH, Xu JF (2001) Pesticide-induced susceptibility of rice to brown plant- Ying Song is studying biological control of rice pests. hopper Nilaparvata lugens. Entomol Exp Appl 100:119–126 Hai Y. Lu is studying indirect non-target effects on natural Wu JC, Xu JF, Feng XM, Liu JL, Qiu HM, Luo SS (2003) enemies via physiological and biochemical changes in rice Impacts of pesticides on physiology and biochemistry of plants due to pesticide stress. rice. Sci Agric Sin 36:536–541 Xu ZY, Liu F, Song Y, Bao SW, Zhang J, Wang KP (2006) Jian X. Xu is a researcher in entomology devoted to study Effects of buprofezin and imidacloprid on parasitism of ecology of rice planthoppers and some vegetable pests.

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