Biocidal activity of polylactic acid-based nano-formulated abamectin on Acyrthosiphon pisum (Hemiptera: Aphididae) and the aphid predator Adalia bipunctata (Coleoptera: Coccinellidae)

Abamectin is a common biocide used to control agricultural insect pests. However, the water insolubility of abamectin may result in extra organic solvent introduced in the environment. To solve this issue, it is desirable to develop nanoformulations to encapsulate abamectin with environment-friendly polymers. In this study, two polylactic acid based abamectin nanoformulations were prepared. The average particle sizes, measured by dynamic light scattering and transmission electron microscope, were 240 nm and 150 nm, respectively. The insecticidal activity of these nano-formulated abamectin was examined in the laboratory on the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae). The acute toxicity of nano-formulated abamectin on non-target aphid predator Adalia bipunctata (Coleoptera: Coccinellidae) was also evaluated by topical, residual and oral exposure. The two nano-formulated abamectin had comparable insecticidal effect with commercial abamectin formulation against the pea aphid. Taking median lethal concentration (LC50) as the toxicological endpoint, nanoformulations had higher contact toxicity and lower oral toxicity to first-instar larvae of the predator A. bipunctata. These results are expected to contribute to the application of solvent-free nano-formulated pesticides that comply with the integrated pest management (IPM) strategies.


Introduction
One of the global challenges faced by the agriculture industry is the sustainable food production for the rapidly growing human population, reaching 9.7 billion of individuals by 2050 [1][2]. Therefore, plant protection products and fertilizers are required to maximize the agricultural productivity [3]. In order to avoid the well-documented deleterious effects of pesticides, the efforts of agrochemical industry are not only focused on looking for new active substances, but also in new pesticide formulations [4]. PLOS

Preparation of nano-formulated abamectin
PLA-based nano-formulated abamectin was synthesized according to a previously reported emulsion solvent evaporation method (O/W) [32]. PLA (40 mg/mL) and abamectin (40 mg/ mL) were dissolved in CH 2 Cl 2 by magnetic stirring to form an organic phase. PVA was dissolved in ultrapure water to form water phase (10 mg/mL). The organic phase was added dropwise over 10 min into the water phase under high shear emulsification (C25, ATS Engineering Ltd., Vancouver, Canada). CH 2 Cl 2 was eliminated from the nanosuspension by evaporation at room temperature with magnetic stirring (1,000 rpm) overnight. The abamectin PLA nanospheres (Abam-PLA-NS) were collected by centrifuging the nanosuspension at 15,000 rpm for 10 min at 4˚C and the deposition was redispersed in deionized water; this process was repeated three times to remove as much surfactant as possible. TA modified nanospheres were formulated by a self-assembly method. PEG (12 mg/mL) was added to the nanosuspension mentioned above, and followed by dropping TA (12 mg/mL). After stirring for 1h, the TA modified abamectin PLA nanospheres (Abam-PLA-Tannin-NS) were collected via centrifugation at 15,000 rpm for 10 min at 4˚C and was washed three times with deionized water.

Characterization of nano-formulated abamectin
The hydrodynamic particle size and polydispersity index (PDI) of nanospheres were investigated at 25˚C by dynamic light scattering (DLS; Zetasizer Nano-ZS90, Malvern, Worcestershire, UK). The morphology of the nano-formulated abamectin was observed via transmission electron microscope (TEM, HT7700, Hitachi Ltd., Tokyo, Japan). About 6 μl of the dispersed nanospheres was dropped on the surface of a cleaned copper grid. The TEM images were performed at 80 kV and 10 mA after the nanospheres were completely dried. The abamectin loading efficiency of nanospheres was investigated by high performance liquid chromatography (HPLC, 1260 Infinity, Agilent Company, California, USA). In brief, an appropriate aliquot of nanospheres was dispersed in CH 2 Cl 2 (5ml) and sonicated for 5min, followed by evaporation of the organic solvent at room temperature. Then abamectin was diluted to an appropriate volume with methanol. For the HPLC analysis, a C18 column (5 mm, 4.6 mm × 150 mm, Agilent Technologies; Santa Clara, CA, USA) was used to separate the target compound from others at room temperature. Methanol/water (90:10, v/v) was used as mobile phase at a flow rate of 1.0 mL/min. The wavelength of UV detector was 245 nm. Loading efficiency (%) = (weight of pesticide in nanospheres/weight of nanospheres) ×100%.

Biological materials
For aphid rearing, broad beans (Vicia faba L.) were used as host plants. The seeds were sown in 30 cm × 20 cm boxes, which contained a 1:1 mixture of vermiculite and perlite. The plants were infested with aphids at two-leaf stage. Aphids A. pisum were collected from Gembloux, Belgium, and they had been reared in the laboratory for several years. Aphids were kept under controlled conditions (22 ± 2˚C, 70 ± 10% R.H. and 16L:8D of photoperiod).
First instars of lady beetles A. bipunctata were purchased from Biobest Group NV, Belgium. Larvae were reared on a diet of frozen Ephestia kuehniella (Lepidoptera, Pyralidae) eggs and water until pupation or death. All studies were conducted at 22 ± 1˚C, 30 ± 5% R.H., and a photoperiod of 16L:8D of photoperiod.

Insecticidal effect of nano-formulated abamectin on the aphid A. pisum
An agar solution was prepared to perform the insecticidal assay on Petri dishes (diam. 3.5 cm) containing a broad bean leaf and aphids. Agar powder was mixed with distilled water (1%, w/w), heated until boiling and then allowed cooling while constantly mixing. After cooling for approximately 10 minutes, warm agar was poured into each Petri dish to a depth that was at least 3-4 mm. A round piece of leaf of 33 mm in diameter was cut using a sharpened metal tube, and put on the agar gel with abaxial surface facing skywards. Ten apterous aphid individuals were transferred onto each of the leaf discs using a fine brush.
The bioassay was conducted using Potter Precision Laboratory Spray Tower (Burkard Scientific, Uxbridge, UK) at a spray pressure 0.70 kg/cm 2 (69 kPa; 10 psi) [33]. The biocidal efficacies of Abam-PLA-NS, Abam-PLA-Tannin-NS and the commercial EC were evaluated. Water was used as the untreated control. Based on preliminary experiments to establish the range of concentrations to be tested, six concentrations, 3.125 mg/L, 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, and 100 mg/L were tested for each formulation. Each aphidcontaining Petri dish was sprayed with 1 mL of the tested solution, representing a deposit of 27.9 ± 2.1 mg on the leaf-disc. Then, all dishes were sealed with a close-fitting, ventilated lids. Two days after the application, the number of alive aphids was counted in each dish. An aphid was considered dead if it failed to react when touched by the brush. There were 3 replicates in each treatment (formulation × concentration) and for the water control.

Non-target effect of nano-formulated abamectin on coccinellid predator A. bipunctata
Abamectin was relatively safe to adult lady beetles [34][35], so first instars were selected as the objects in this study. Larvae were treated with Abam-PLA-NS, Abam-PLA-Tannin-NS and the commercial EC of abamectin, using the same six concentrations in the aphid bioassay. Controls were maintained using water alone. Abamectin acts on glutamate-gated chloride ion channels in arthropods to produce long-term, high-intensity inhibitory effects, causing insects to die. Abamectin has oral and contact toxicity. Therefore, three groups of insecticidal assays were conducted on lady beetles or aphids with Potter spray tower: (a) Topical exposure: Ten larvae were transferred to a Petri dish and then 1 mL of the tested solution was sprayed. Larvae were then individually transferred to clean plastic Petri dishes and checked for mortality daily.
(b) Residual exposure: Petri dishes were sprayed with 1mL of the tested solution (n = 10 for each concentration of a tested solution). A single larva was then individually transferred to a Petri dish. After 24 h of contact, they were transferred to clean plastic Petri dishes and checked for mortality daily.
(c) Oral exposure: 20 aphids (A. pisum) were sprayed with 1mL of the tested solution. After air dry, they were transferred to a clean plastic pot where a lady beetle larva was introduced (n = 10 for each concentration of a tested solution). Frozen Ephestia kuehniella eggs and water were offered after all the aphids died. Larvae were checked for mortality daily.

Statistical analysis
Insect mortality was corrected by Abbott's formula [36], taking into account the natural mortality observed on the control. The mortality of insect exposure to different formulations was analyzed by one-way ANOVA. Probit analysis was performed in order to estimate the LC 50 [37], in which, a Chi-square Goodness-of-fit test was used to analyze the mortality data. The dose-mortality relationships were considered valid when the observed data and the expected data did not diverge significantly (P<0.05). Data analysis was carried out on SPSS Statistics V.17 (IBM).

Characterization of nano-formulated abamectin
The hydrodynamic size of Abam-PLA-NS measured by DLS was 240.7±1.9 nm, and it increased to 243.6±1.2 nm for the Abam-PLA- Tannin

Insecticidal effect of nano-formulated abamectin on the aphid A. pisum
The bioassay results of nano-formulations and commercial EC on A. pisum were showed in Table 1  Nano-formulated pesticides are expected to improve the efficiency of pesticide and reduce environmental pollution [6,10]. These two solvent-free nano-formulated abamectin exhibited similar biocidal efficacy on aphids, because nano-sized formulations can improve the adhesivity and penetrability of pesticides on surface of organisms [20,38]. Abam-PLA-Tannin-NS had a lower LC 50 than Abam-PLA-NS, which attributed to the enhancing contact of abamectin on the epidermis of aphids, due to the adhesive properties of TA.
Abamectin is categorized as highly toxic with acute oral and dermal toxicity with low LD 50 concentrations for different organisms [39]. However, its popularity is growing due to the effective pest control. Usually, in the bioassay, the type, developmental stage and physiological condition of the target organism have a great influence on the efficacy of the pesticide. In addition, the environmental condition may also affect the response of the target to the agent. The recorded LC 50 of abamectin for mustard aphid Lipaphis erysimi Kalt (Hemiptera: Aphididae) was 0.63 mg/L [40]. The recorded LC 50 for peach aphid Myzus persicae Sulzer (Hemiptera: Aphididae) were 1.5 mg/L [41], and that was 5.5 mg/L for Anjum et al's bioassay [42]. But results of our study could get support from our recent bioassay on M. persicae (LC 50 of 17.38 mg/L and 10.68 mg/L for Abam-PLA-NS and Abam-PLA-Tanning-NS respctively) [32].

Non-target effect of nano-formulated abamectin on coccinellid predator A. bipunctata
Some studies have confirmed that nanoformulations were harmless to non-target organism, such as different cell lines and soil microorganisms [43], but only a few safety studies were carried out on natural predators [44].
The biocidal effects of all tested formulations against lady beetle larvae after 48 h and 120 h are presented in Tables 2, 3 and 4  Taking LC 50 as the toxicological endpoint, two nanoformulations showed higher contact toxicity than EC in both topical and residual applications 48 h and 120 h after the spray. It resulted in a similar trend as compared to aphids. This suggested that the nano-formulated abamectin had no selectivity for either insect group. For the commercial formulation, free abamectin on the surface of insects and Petri dishes were rapidly degraded, thereby reducing exposure to toxic residues. Nanoformualtions could enhance the stability of the pesticide and inhibit the degradation of abamectin [32,45], then, improved the toxicity to ladybeetles. While the oral exposure results showed totally opposite trends. Both nano-formulated abamectin showed higher LC 50 than abamectin EC after feeding. It could be accounted by the fact that PLA prevented abamectin from contacting with the lady beetles after entering the digestive tract [46]. The existence of TA also increased the toxicity of abamectin nanospheres due to the improved adhesion. When applied in crop protection, abamectin turns into a source of concern for non-targeted beneficial arthropods and evolving resistance in pests [47]. The effects of 13 agrochemicals used in grapevines on spider mite Panonychus ulmi (Acari: Tetranychidae) and the predatory mite Neoseiulus californicus (Acari: Phytoseiidae) were evaluated, and exposure to abamectin significantly reduced survival both of P. ulmi and of N. californicus [48]. A study proved that commercial abamectin was the most harmful substance (in term of lethal and sublethal effects) among 14 tested pesticides against Orius laevigatus Fieber (Hemiptera: Anthocoridae), a commonly used predator in biological control programs [49]. It was found that commercial abamectin EC at recommended concentration combined with 0.5% summer oil was highly toxic to Rhyzobius lophanthae Blaisdell (Coleoptera: Coccinellidae) adults and larvae in both direct applications and pesticide residue situations [50]. The recorded LC 50 of abamectin for the first instar and adult of multicolored Asian lady beetles, Harmonia axyridis (Coleoptera: Coccinellidae) were <0.09 mg/L and 4.88 mg/L, respectively, with a topical method in laboratory condition [51]. Whereas abamectin commercial formulation of 18 mg/L

Conclusions
In this study, two solvent-free nano-formulated abamectin were prepared, and their biocidal efficacy on aphids and on lady beetles was tested. Generally, there was no significant difference between nanoformulations and commercial EC in mortality of the acute toxicity. The environment-friendly compositions of nanoformulations make them more suitable as the plant protection products in IPM strategies. It is noticeable that the side effects on the non-target organisms at different sublethal concentrations, should also be considered for a complete understand of the nanoformulations [29,53]. Meanwhile, further studies on the field trial would be certainly worthwhile.