Impact of the neonicotinoid acetamiprid on immature stages of the predator Eriopis connexa (Coleoptera: Coccinellidae)

Eriopis connexa is a native coccinelid predator in the Neotropical Region. In Argentina it is commonly found associated to sucking pests in several crops and among them aphids and whiteflies. These pests are usually controlled with newly developed systemic insecticides, such as the neonicotinoids. However, the compatibility between selective pesticides and natural enemies is required before incorporating them in integrated pest management (IPM) packages. Within this frame, the objective of this study was to evaluate the side effect of various concentrations/doses of one commonly used neonicotinoid in vegetal crops, acetamiprid, on immature stages of E. connexa by dipping or topical exposure for eggs and larvae, respectively. Acetamiprid reduced egg hatching from 34 to 100 %. Moreover, the embryogenesis was disrupted by insecticide at early embryo stage at all tested concentrations. Second larval instar was more susceptible to acetamiprid than the fourth one and this susceptibility was positively related with the tested concentrations. On the other hand, the survival reduction at larval stage reached 100 % from 20 mg a.i./L (10 % of maximum field concentration). Besides, the reproduction of the females developed from topical bioassays on fourth instar larvae was strongly affected, with reduction in fecundity and fertility from 22 to 44 % and from 37 to 45 %, respectively. Overall the results showed a high toxicity of acetamiprid on immature stages of E. connexa, demonstrating that this broadly used insecticide could reduce biocontrol services provided by this predator and could also likely disturb IPM programs.


Introduction
Neonicotinoid-based agrochemicals are widely used to control sucking pests, such as aphids (Hemiptera: Aphididae), mirid bugs (Hemiptera: Miridae) and whiteflies (Hemiptera: Aleyrodidae) (Palumbo et al. 2001;Ishaaya et al. 2007;Liang et al. 2012;Seagraves and Lundgren 2012;Tan et al. 2012). They are very popular insecticides owing to their high efficacy in pest control (namely ovicidal and larvicidal activity), systemic action as well as long lasting effects and environmentally-friendly profiles (Ghanim and Ishaaya 2010). Due to their high specificity, high efficacy and relatively low toxicity to mammals and the environment, these insecticides have been considered a good alternative for the organophosphate insecticides (Tomizawa and Casida 2005;EPA 2012). Indeed, the US Environmental Protection Agency (US EPA) categorizes neonicotinoids as biorational insecticides being compatible with arthropod natural enemies and adequate compounds within Integrated Pest Management (IPM) programs (Ishaaya et al. 2007).
One of the major purposes of IPM strategies is the combination of selective pesticides with biological control agents, i.e. predators and parasitoids (Desneux et al. 2006a). Therefore, the evaluation of side effects of pesticides on natural enemies, both lethal and sublethal effects, is essential prior to IPM programs implementation Stark et al. 2007). The Horticultural Green Belt (Gran La Plata Region, Province of Buenos Aires, Argentina) is one of the main producing areas of Argentina, with 7,538 ha of cultivated area (CFHB (Censo flori-hortícola bonaerense) 2005). Pest control is mostly conducted by newly developed synthetic pesticides, being neonicotinoids between the most extensively used for the control of sucking pests, such as aphids and whiteflies, mites, scales and mealybugs in both, field and greenhouse crops (Cappello and Fortunato 2008).
Generalist arthropod predators are known worldwide as regulators of insect herbivore populations in agricultural and forest ecosystems (Symondson et al. 2002;Desneux et al. 2006b;Lu et al. 2012); moreover, they are able to establish populations in highly disturbed ecosystems, such as annual cropping systems, by exploiting alternative preys (Harwood et al. 2007;Desneux and O'Neil 2008;Juen et al. 2012). Eriopis connexa (Gemar) (Coleptera: Coccinellidae) is an indigenous generalist predator in Argentina. It is widely distributed in the Neotropical Region and it is considered a potential control agent of various pests on several crops (Almeida-Sarmento et al. 2007;Duarte Gómez and Zenner de Polanía 2009). Both larval and adult stages of all coccinellid predator species provide important biocontrol services feeding on different soft-body pests, such as aphids, whiteflies, mites, and lepidopteran eggs and larvae (Obrycki and Kring 1998).
The synthetic neonicotinoid insecticide acetamiprid, (E)-N1-[(6-chloro-3-pyridyl)methyl]-N2-cyano-N1-methylacetamidine, belongs to a relatively new group of active ingredients and it is characterized by a novel mode of action compared to conventional neurotoxic insecticides (Tomizawa and Casida 2005). This compound acts on the central and peripheral nervous system of insects, specifically interacts with nicotinic acetylcholine receptors (nAChR), resulting in excitation and paralysis, followed by death (Ghanim and Ishaaya 2010). Mindful of this context, the objective of the present study was to assess the lethal and sublethal effects of various concentrations/doses of acetamiprid under controlled laboratory conditions on immature stages (egg, second and fourth larval instars) of the Neotropical generalist predator E. connexa.

Insects
The E. connexa colony was established from samplings collected in infested vegetable crops in La Plata region, Argentina (348 57 0 17 00 S, 578 53 0 26 00 W) in 2008. Adults were collected manually using plastic tubes (10 cm length 9 1.5 cm diameter) conditioned and isolated to avoid potential field diseases (fungal, bacterial or viral infections) and/or parasitism. Then, their healthy progeny were used to initiate the predator rearing. Annually, the colony was infused with wild stock (between 50 and 80 adults each) collected from the same geographical area, to maintain its genetic variability.
Rhopalosiphum padi L. (Hemiptera: Aphididae) was used as prey. The aphid colony was initiated from clones obtained from the Faculty of Agricultural and Forestry Sciences (National University of La Plata) and it was reared on pesticide-free wheat seedlings (Triticum aestivum L.) (cultivar ACA 901). An artificial diet based on beef liver (Martos and Niemeyer 1990) was offered ad libitum as nutritional supplement for larvae and adults of the predator. Insect colonies and all the bioassays were carried out in a growth chamber with controlled environmental conditions (25 ± 2°C, 70 ± 5 % HR and 16:8 h L:D).

Effects on eggs: embryogenesis and development
A commercial formulation of acetamiprid (Mospilan Ò 10 % p/p, Summit-Agro S.A., Argentina) was used for this experiment. It was tested at its maximum field concentration (200 mg a.i./L) and its half (100 mg a.i./L) The insecticide solutions were diluted in distilled water and, to facilitate adhesion of the insecticide to the egg chorion, a commercial tensioactive (Tween 80 Ò , Merck, Darmstadt, Germany) was added at its label concentrations, i.e. 0.1 ml/ L. The untreated control was sprayed only with distilled water plus the tensioactive.
Coetaneous eggs (B48 hold) were treated by dipping in the solutions for 15 s according to Schneider et al. (2009), left to dry in a fume hood and then maintained in plastic Petri dishes (9 cm diameter and 1.5 cm high). Each treatment consisted of 3 to 4 replicates with 20-30 eggs per replicate. Embryogenesis and egg hatching were checked daily by a stereomicroscope 24 h after treatment and for seven consecutive days. Hatching generally occurs between 3-4 days after oviposition (Fogel MN, pers. obs.). Therefore, treated and untreated eggs that did not hatch during the 7 days of observations were considered dead. The percentage of eggs hatched was evaluated from the following formula [(Number of eggs hatched/N8 initial eggs) 9 100]. To assess the effects on the embryos development, from 20 to 30 unhatched eggs were randomly selected from each treatments and were placed in Bouin Ò solution, then dehydrated in ethanol series of analytical grade (70, 90 and 100 %) and mounted between slide and cover glass in Hoyer's medium and dried in stove at 40°C.

Effects on larval survival and development
Second and fourth instar larvae were treated topically with seven concentrations, including the maximum field concentration (see Table 1 for details), of acetamiprid. The weight of the tested larvae averaged 2.66 ± 0.7 mg and 5.7 ± 0.4 mg for L 2 and L 4 , respectively. To improve the insecticide dissolution, the solutions were prepared with acetone (ACS analytical grade) diluted in distilled water (80:20 v/v) and used as solvent following the methodology by Youn et al. (2003). The treatments were performed with a manual micro-applicator (Burkard, Rickmansworth, UK) applying 0.5 and 1 lL of insecticide solution to the first abdominal segment of each tested individual. Acetone 80 % was used for untreated controls. Treated larvae were placed into plastic Petri dishes and then were transferred into a growth chamber until adult emergence. They were fed R. padi specimens and beef liver-based artificial diet ad libitum (Martos and Niemeyer 1990). Each treatment consisted of three replicates of ten larvae.
Survival and development time of immature stages (from larva to pupa) were checked, every 24 h until adult emergence. Survival data were used to obtain the reduction of this parameter according to the following formula: where SC is proportion of survivors in the control and ST refers to the proportion of survivors in the treatments.

Effects on reproduction
Sublethal effects on fecundity and fertility were assessed on the adults developed from treated larvae. Because the survival of the treated larvae was very low (see ''Effects on larval survival and development'' section), the effects on reproduction were assessed on the adults developed from fourth instar larvae treated with the three lower acetamiprid doses, i.e. 0.0001, 0.0008 and 0.0017 lg a.i./g. Newly emerged adults, both males and females, were placed in plastic cylindrical containers (18 cm diameter and 15.5 cm height) and, to help the female ovary development, R. padi specimens and artificial diet were offered ad libitum. After 5 days, ten mated females having a large abdomen (i.e. with developed ovaries) per treatment were randomly selected for the reproduction assessment. Females were placed individually in plastic glasses (5 cm diameter and 10 cm height) with a fine mesh net fixed on the upper opening to allow ventilation. The inner walls of the containers were previously covered with untreated paper as substrate for the oviposition, whereas aphids and artificial diet were provided ad libitum. During the following 5 days they were checked for the presence of egg batches and newly laid eggs were collected and placed in Petri dishes. Therefore, fecundity (number of laid eggs) and fertility (number of hatched eggs) were registered for each egg clutch and female.
Unhatched eggs became black 48 h after treatment and some of them got dehydrated. For acetamiprid treatment at 50 % of the maximum field concentration (100 mg a.i./L), the eggs hatching rate was significantly lower than in the control (F = 52.9; df = 2,3 p = \ 0.012; Fig. 1a). However, the neonate larvae that were able to emerge showed a significant reduction of survivorship compared to the control (F = 47.1; df = 2,3; p = 0.0001), with values of 10.0 ± 8.6 and 90.0 ± 3.4 %, for acetamiprid (100 mg a.i./L) and control, respectively. Development time of the eggs (embryos development) was significantly longer in the acetamiprid treatment (100 mg a.i./L) compared to the control (K = 3.84, p = 0.034); reaching 2.5 ± 0.28 days from treatment (0.5 days more than control) (Fig. 1b). In those where no larvae emerged, the embryos mortality was corroborated by observation under the stereo microscope preparations (Fig. 2). Embryos of the untreated control ( Fig. 2a, b) were fully developed at 48 h post-treatment (72-96 h from oviposition), being observed abdominal segments, legs, setae and mandibles. However in treated ones, the vitelline membrane is the only detectable and the developed embryos showed just mandibles (Fig. 2d, Fig. 2e, f).

Effects on larval survival and development
The effects of acetamiprid on E. connexa larvae survival were significantly different according to the larval instar being exposed (instar factor) and to the insecticide concentration applied (dose factor). Moreover, the interaction between these two factors was also significant (Table 2). Indeed, second instar larvae were significantly more susceptible to acetamiprid than the fourth instar ones at all the tested concentrations (Fig. 3a). The reduction of larval survival was more evident at higher concentrations of acetamiprid (200, 100, 50 and 20 mg a.i./L) than at lower ones (10, 5 and 1 mg a.i./L) (Fig. 3b). The development time of fourth instar larvae to adults in acetamiprid treatments did not differ significantly from the control ones reaching values of 7.3 ± 0.21; 7.0 ± 0.19; 6.9 ± 0.34; 7.15 ± 0.22 days for control and acetamiprid doses of 0.0001, 0.0008 and 0.0017 lg/g, respectively (K = 7.81, p = 0.504). At higher doses this parameter was not evaluated due to high mortality recorded at second day from treatment. On the other hand, the development time to adults could not be recorded for the treated second instar larvae, since all the specimens died few hours after treatment regardless of the insecticide concentration considered.

Effects on reproduction
Reproduction of E. connexa just was evaluated at lower doses of acetamiprid and in adults emerged from fourth instar larvae because at second instar larvae and higher doses no survivors were obtained. Acetamiprid effects on fecundity and fertility were evaluated taking into account the time (time factor) and the pesticide dose (doses factor) ( Table 3). Acetamiprid at 0.0017 lg a.i./g significantly reduced the daily fecundity of E. connexa females emerged from treated fourth instar larvae (p = 0.001) (Fig. 4a). Analyzing data according to time factor, a tendency in the reduction of the fecundity after the first oviposition day was detected. The fecundity was significantly affected at day five compared to first day, regardless of the concentration considered (p = 0.007) (Fig 4b). The acetamiprid doses of 0.0017, 0.0008 and 0.0001 lg a.i./g significantly reduced the number of daily hatched eggs (fertility) compared to controls (p \ 0.0001) (Fig. 4c). By contrast, no significant differences were observed among the five oviposition days in the portion of the hatched eggs (Fig 4d).

Discussion
Exposure of beneficial arthropods to pesticides can result in a wide range of effects, including the simultaneous occurrence of multiple sublethal effects Stark et al. 2007;2012b). Besides the short term effects (acute toxicity), the long-term ones could also strongly impact on natural enemies reproduction, leading to a reduction in offspring, affecting the population growth rates (Stark et al. 2004). In the present study we observed both lethal and sublethal effects of various concentrations/doses of acetamiprid on immature stages of E. connexa.
The observed deleterious effects were highlighted by the significant reduction of survival, both on egg and larval stages exposed to the recommended field and to lower concentrations. Moreover, the lower tested concentrations not only affected the survival, but also decreased the fecundity, fertility and embryogenesis of the developed adults. Indeed, high mortality of embryos and of the newly hatched larvae after the direct exposure of eggs to acetamiprid was observed. Although the chorion surface layer is a sclerotized protein membrane providing mechanic resistance with non-permeable properties (Nation 2008), some chemicals can pass through it affecting (i) the embryo development (ii) or, in the case of successful hatching, the individual may die when feeding the insecticide-contemned  chorion (Trisyono et al. 2000;Consoli et al. 2001;Galvan et al. 2005;Rimoldi et al. 2008). Coccinellidae eggs have a ring of micropyles useful for the fecundation process and for oxygen diffusion inside the eggs (Nedvȇd and Honȇk 2012). Furthermore, the treatment with 50 % of acetamiprid maximum field recommended concentration induced a lengthening of the embryogenesis of the predator eggs. According to our studies it could be hypothesized that acetamirprid was able to trespass the chorion of E. connexa eggs or to penetrate through the ring of mycropiles, blocking the embryo development.
Even though neonicotinoids exhibit the same mode of action targeting the nicotinic acetylcholine receptor, ovicidal activity and effects on embryos is variable between compounds of this chemical group (Hoffmann et al. 2008). Our results match with those reported by Youn et al. (2003), which observed no larvae emergence when dipping the eggs of Harmonia axyridis Pallas (Coleoptera: Coccinellidae) in the two neonicotinoids, acetamiprid and imidacloprid. While another insecticide belonging to this family, thiametoxam, did not cause any deleterious effect (Youn et al. 2003). Additionally, Kim et al. (2006) observed that, although acetamiprid-treated eggs of the predator Deraeocoris brevis Uhler (Hemiptera: Miridae) did not evidence effects on hatching; a significant reduction of survival of emergent nymphs was observed. Furthermore, studies on the exposure of egg of the predator Podisus maculiventris (Heteroptera: Pentatomidae) to imidacloprid did not cause any egg mortality, although a significant reduction of emergent larvae was recorded (Cutler et al. 2006). Whereas, imidacloprid caused lengthening in the embryogenesis of Apolygus lucorum Meyer-Dur (Hemiptera: Miridae) in eggs laid by treated females (Tan et al. 2012).
It is well known that susceptibility of natural enemies toward pesticides varies with the development stage of  tested organisms, being generally the immature stages more susceptible than adults (Delbeke et al. 1997;Michaud 2002;Grafton-Cardwell and Gu 2003;Schneider et al. 2004;Galvan et al. 2005;Cutler et al. 2006). The difference could be associated to a thinner and more permeable cuticle of immature stages with lower chitin content added of less active enzymatic detoxifying processes in comparison to adult stage (Stark et al. 2004). According to the results of the toxicity bioassays with second and fourth instar larvae of E. connexa, it is evident that both instars are highly susceptible to acetamiprid but the effect was even clearer on second instar larvae bioassays. However, the survival reduction in fourth instar larvae was observed at concentrations between 20 and 200 mg a.i./L. Other authors have reported similar results for neonicotinoids on various beneficial insects. Acetamiprid caused 100 % mortality of H. axydiris fourth instar larvae and imidacloprid was more toxic to the second instar larvae than the fourth instar one (Youn et al. 2003 (Kim et al. 2006) and Picromerus bidens L. (Heteroptera: Pentatomidae) (Mahdian et al. 2007). Reproductive capacity of females emerged from fourth instar larvae survivors was significantly affected by very low doses of acetamiprid. These results agree with those reported by Sohrabi et al. (2012), where the fecundity and fertility of Encarsia inaron (Walker) (Hymenoptera: Aphelinidae) adults developed from imidacloprid-treated larvae were significantly affected. Likewise, Grafton-Cardwell and Gu (2003) observed a fertility reduction of R. cardinalis females exposed to acetamiprid, imidacloprid and thiametoxan. Similarly, fecundity reduction of H. undecimnotata females was observed when this predator was fed with aphid treated with sublethal concentrations of imidacloprid and carbofuran (Papachristos and Milonas 2008). On the contrary, no adverse effects on fecundity and fertility were observed on treated females of D. brevis under topic exposure of predator's nymphs (Kim et al. Fig. 4 Means (±SE) of sublethal effects of acetamiprid at 0.0017, 0.0008, and 0.0001 (lg a.i./g) on fecundity (number of eggs laid daily/ female) and fertility (number of eggs hatching/female) of Eriopis connexa emerged from treated fourth instar larvae. a and b Effects on females' fecundity respect to dose and time as main factors, respectively. c and d Effects on females' fertility respect to dose and time as factors, respectively. Treatments with different letters are significantly different. Repeated-measures ANOVA (P \ 0.05) 2006). Likewise, this insecticide did not induce any side effects on reproductive parameters of the predator P. bidens (Mahdian et al. 2007).
Taken as whole, although they should be confirmed with field studies, the results from our laboratory experiments demonstrated that acetamiprid would strongly reduce the E. connexa population development throughout the impairment of crucial physiological processes in the juvenile development. This could result in limiting the predator biocontrol services in the crops where this insecticide is broadly used, with negative implications for the Integrated Pest Management programs. Finally, this research provides new insights into side-effects of acetamiprid in the embryogenesis and highlights the importance of incorporating juvenile stages in pesticide risk assessments.