Insecticidal and P450 mediate metabolism of fluralaner against red imported fire ant, Solenopsis invicta (Hymenoptera: Formicidae).

The red imported fire ant (Solenopsis invicta), a worldwide invasive and polyphagous pest, and often nests in residential areas. Finding an alternative pesticide that is both effective on S. invicta and environmentally friendly is urgent and crucial. Fluralaner, a novel isoxazoline insecticide, has been proven to possess selective toxicity for insects versus mammals and has been safe for mammals and non-target organisms, suggesting its potential in pest management. However, little toxicity information is available for the controlment of S. invicta. In this article, we studied the toxicity of fluralaner against S. invicta systematically, and the roles of metabolism-related enzymes in the metabolism process of fluralaner. The toxicity results showed that the topical application and feeding application were all effective for S. invicta. Moreover, fluralaner can be transmitted among workers by contacting and feeding which leads to a toxic reaction among nestmates. By exploring the biochemistry change, we found cytochrome P450 monooxygenase (P450) may be involved in the detoxification of fluralaner as well as carboxylesterase (CarE), but not glutathione S-transferase (GST). Synergism assays gave solid evidence in which piperonyl butoxide, an activity inhibitor of P450, increased the toxicity of fluralaner to S. invicta. Importantly, with the RNAi treatment, four of S.invicta P450 genes were significantly inhibited and showed more sensitivity to fluralaner at LC50 concentration. Our result indicated that fluralaner could be a potential alternative pesticide in S. invicta control. And CYP9AS16, CYP6AS161, CYP6SQ20, and CYP336A45 genes were closely associated with the metabolism process of fluralaner.


Introduction
In modern agriculture, the use of pesticides is still an effective tool on crop production because it provides much faster, more targeted, and effective control of pests and diseases. In the meantime, the increasing consumption of pesticides caused severe problems to environment and human health due to the residual or bioaccumulation, even to pest control due to pest resistance or negative impact on non-target arthropods [1][2][3] . Developing a new pesticide which is safety to non-target organisms and easy to degrade might weaken the effect caused by the problems mentioned above. However, the research of new pesticide is time-consuming process costly 4 . Digging out an alternative chemical in the exist chemistries is more helpful to face the challenge in pest management.
Fluralaner (CAS 864731-61-3), a novel isoxazoline insecticide, inhibitors of γaminobutyric acid (GABA)-gated chloride channels (GABACls) and L-glutamategated chloride channels (GluCls). It was firstly synthesized by Nissan Chemical Industries 5 , and it has been marketed as BRAVECTO® in Europe, America and East Asia for its excellent activities on parasitic and sanitary pest. In previous studies, Fluralaner has been used orally to dogs, cat, sheep and other production animals in the management of stomoxys calcitrans, Aedes aegypti and Siphonaptera etc. 6,7 . And the vitro contact activity of fluralaner is better than spinosad, phoxim, propoxur against Ornithonyssus sylviarum 8 . High activities also have been found under lab conditions against multiple agricultural pests such as Tetranychus urticae, Chilo suppressalis, Spodoptera litura, Frankliniella occidentalis 9,10 , urban pests as Blattella germanica, and stored product insect like Tribolium castaneum 11 . Besides, fluralaner and its formulated product are of low toxicity to non-target organisms, such as zebrafish 12 .
Mechanism research indicated this chemical has different action site between mammals and insect, it was more active and toxic toward insects than animal 13 , this make it a promising green agrochemical.
Red imported fire ant (Solenopsis invicta) is a dangerous invasive insect worldwide, which caused huge damage in invaded place, including reduction of native arthropods amount, losses of agricultural production, as well as threat on human health 14-16 . S.invicta prefers to build their nest in disturbed neighborhoods, this means more vulnerable environment, which is extremely sensitive to pesticides 17 . The twostep method is an alternative control strategies used for the whole colony management of S.invicta 18 . Low toxic bait is the key to ensure the contact of most colony members with insecticide. With its underground nest habit and intensive contact with living activities, the chemical choice against S.invicta is limited. Fipronil is the most popular bait used in S.invicta management, but it has been restricted or totally forbidden in many countries for its high toxicity to non-target pest especially for aquatic organisms 19,20 . Thus, novel, low-toxic, and environmentally friendly pesticide is substantially needed for the controlment of this complicated pest.
Although studies have demonstrated fluralaner has significant activity against wide range of insects, the effects of fluralaner on social insects are still unknown. In our present study, fluralaner insecticidal activity through both contact and feeding exposure against S.invicta were taken on workers. Moreover, to evaluate the potential of this new chemical as an active bait ingredient, trophallaxis effects among S.invicta colony after taking fluralaner was conducted, using rubidium as the tracer. Synergism assays using three main detoxification enzyme synergists, including piperonyl butoxide (PBO), diethyl maleate (DEM), and triphenyl phosphate (TPP) together with enzyme activities comparison before and after exposure to fluralaner at sublethal does were conducted to investigate the possible metabolism mechanism. and qRT-PCR were assessed aim to know the P450 gene induction of this novel chemical in S.invicta.

Results
Topical toxicity of fluralaner against S.incivta. The contact toxicity of fluralaner against S. invicta was determined by a topical application method. The mortality of S. invicta increased with dose-and time-dependent effects, with the 24-h and 48-h LD50 values were 7.99 and 2.21 ng/insect, respectively (Table 2). In comparison, the LD50 values for fipronil were 1.11 ng/insect at 24h and 0.44 ng / insect at 48h, respectively.  (Table 3). Synergism effects of PBO, DEM, and TPP were topical pretreat 4h before feeding assay. PBO and DEM displayed statistically significant synergism on fluralaner toxicity, with the lower LC50 value than the no synergist treatment group. And the synergist ratios of PBO and DEM was 1.53, 1.36 at 24-h, 1.62, 1.59 at 48-h, respectively. In contrast, TPP displayed no synergist effect on fluralaner toxicity, as the 95% confidence intervals of fluralaner treatment and TPP pre-treat group overlapped ( Table 3).
Effect of fluralaner on toxicity transmit. Horizontal toxicity transmit effects were conducted to determine whether fluralaner toxicity can be horizontally transferred among workers after topical expose. Both the cumulative mortality of donors and recipients was increased within 48h, and exerted dose-dependent effects, but the transmit rates between donors and recipient were not different (P>0.05, ANOVA) ( Table 4).
Effect of fluralaner on food exchange. Donor workers were fed with Sublethal does fluralaner to know the effect on reciprocal food exchange and nutrition transmission among colony use rubidium as the food tracer. After feeding for 24h, food consumption was unaffected by fluralaner compared with controls (Fig.1A). For recipient ants, worker recipients exhibited no diffidence from control. Female and larvae recipients get more food from donors in the presence of sublethal does of fluralaner (Fig. 1B).
Besides, worker donor and larvae recipient showed notable difference in food transmit ratio compared with control (Fig.1C).
Enzyme activity comparisons after feeding with sublethal does of fluralaner. The enzyme activities results showed that the P450 activity but not GST and CarE were significant different from controls ( Fig. 2A,B). The activity of P450 monooxygen activity was 2.69-fold higher after sublethal does fluralaner treatment (2502.67 nmol / min/mg  prot) compared with control (803.75 nmol/min/mg  prot), Meanwhile, PBO pre-treat inhibits P450 enzyme activity notably (Fig.2B).
Fluralaner-induced expression of P450 gene of S.invicta. Based on qRT-PCR analysis, the expression of twelve P450 genes were significantly upregulated after LC50 concentrate fluralaner feeding for 24hours compared with the solvent treated group ( Figure 3). The relative expression of CYP4G323, CYP336A44, CYP301A1, CYP305D1v1 and CYP306A1 were more than 4-fold higher than control treatment.
CYP4SJ1 and CYP336A45 were significantly downregulated after fluralaner treated.

Discussion
Fluralaner, a potent isoxazoline insecticide, has been confirmed with high activity towards wide spectrum of pest both agriculture and sanitary pest 10,11,21 . In present study, topical toxicity and feeding toxicity assays all showed a lower toxicity against S.invicta, than fipronil (Table 2), and the LC50 value of feeding toxicity was 15.61, 8.63 mg / L( Table 3). Fipronil was a universal active ingredient used in S.invicta controlment, and its LD50 value was 1.11, 0.44 ng/insect after treated for 24, 48h ( Table 2). Compared with fipronil, fluralaner showed a delayed toxicity and is more safe on non-target organisms 20,22 . In social insects management, relatively delayed toxicity is needed for sufficient amounts of toxicant to delivered among the colony 23,24 . In studies of Zhao et al 11 , fluralaner showed higher contact activities toward many agricultural insects compare with fipronil, with LD50 value varies from 22.3 to 107.0 ng/insect at 24h, but for the German cockroach fluralaner showed lowed contact activity, this was consistent with our findings, and we speculate that this might be because the thicker cuticles than other insects. In other studies, Fluralaner showed high toxicity to fipronil resistance strains, with LC50 values of 7.6 and 10.0 mg/L to Laodelphax striatellus susceptible and resistance strains, respectively 25 , and the toxicity of fluralaner to susceptible and resistant strains of D.melanogaster strains was equivalent 6 . So, fluralaner may be a good alternative of fipronil, owning to the differential actions of fluralaner and fipronil on the GABAR 25  g/ant 26 , much higher than fluralaner in present study, this indicated fluralaner is a more promising isoxazoline insecticide.
Toxicity transfer of insecticides occurs after insects contacting or ingesting an insecticide, and transferd it to other conspecific insects through trophallxis, necrophoric, contact and mutual grooming [27][28][29] . This is a common phenomenon in many social insects. In our present study, fluralaner was added in food, and Rb content in doners and recipient did not show much difference, this may indicate that this chemical do not affect the food sharing in S.invicta colony (Fig 1), which may be beneficial for toxicity transmit along with food consumption. The topical horizontal toxicity results confirmed that after fluralaner topical treatment, toxicity could be transferred from donors to recipients, and the transmit ratio between donors and recipient did not show any does dependent differences (Table 4). Horizontally transfer is an important index to evaluate whether this insecticide is a good candidate for ant control. For example, after exposed to contact insecticides, fipronil was readily horizontally transferable than bifenthrin, cyfluthrin, and chlorfenapyr. When ants contacted contaminated crops and the dead ants, the mortality occurred, this may resulted from necrophoresis behavior 28,30 . Similar study results also been reported by Wiltz et al 27 , and they think after treated, bifenthrin may has a reduction in recruitment effect among nestmates, this may hamper the effectiveness of pesticide. Trophallaxis, the main food sharing behaviors in social insects, also is an important factor for cohesion and crucial communication process 31,32 .
In trophallaxis process ants ingest and store a liquid toxic bait in food and then regurgitate it to other nestmates 33 . In Furman's study, RFIA can transmitted indoxacarb toxicity between RFIA workers and brood by trophallactic behaviors 34 . Neoh et al, found after exposed to chlorantraniliprole termitea were likely to cease feeding and undergo starvation, which made the pesticide low efficiency 35 . Because in the management of social insect, repellent or fast-acting toxicant may provide effective barriers 36 . Non-radiolable rubidium (Rb) have been used as a good trace in termites' trophallaxis process [37][38][39] , but had not yet been reported in S.invicta. In our study, Rb can be a good indicator of food allocation. Our study results indicate fluralaner can be a good candidate for S.invicta controlment.
The information about the metabolism of fluralaner in insects is limited, especially in insect. In present synergetic assay indicated that PBO and DEM displayed significant synergism on fluralaner toxicity during all the observation periods (Table 3). And enzyme measurement result confirmed that P450 monooxygen showed 2.69-fold higher activity was founded after LC30 fluralaner treatment compared with control (Fig.2).
Quantitative analysis showed twelve P450s genes were significantly up regulated by fluralaner (Fig.3). PBO is a potent P450 monooxygen and esterase inhibitor and can synergise insecticide through phaseⅠ metabolic enzyme systems 40,41 . And synergists could be used to know the enzyme detoxification mechanisms of the chemicals roughly. Three groups of experiment were conducted using a simple donor-recipient model 39 . In worker-worker group, innocuity paint was used to distinguish donors from recipients. Fire ants from three different colonies were chose as the duplication. In each experiment 50 donor worker fire ant were starved for 24 hours, then fed with 1ml 10% honey water contain LC30 (9.17mg/L) fluralaner together with 8000 mg/L Rubidium fluoride for 24 hours. Control group were fed with 1mL 10% honey water contain 8000 mg/L rubidium fluoride only. After that, the test tube was removed and another 50 starveling pre-treated workers or 30 starveling pre-treated larvae or 15 starveling pre-treated female were transplanted into the box as the recipients, 24h later, the donors and recipients were frozen at -20°C and then placed in silica beads test tube, and dried at 65°C for 24h respectively. The dried S.invicta were digested with 1.0 mL of 65% nitric acid for 24h. And after that 0.5 mL of 60% perchloric acid was added to each tube and heated in a 95°C water bath for 30min, cooling in room temperature, thereafter, 20mL of deionized water were added to each sample. The Rb content of each sample were determined using inductively coupled plasma optical emission spectrometry (ICP-MS) (VARIAN, 710-ES, USA).
Enzyme extraction and analysis. Thirty adult workers were homogenized, 24h after feeding LC30 does fluralaner using a vitreous homogenizer (diameter = 0.8 cm) in 300L of ice-cold phosphate buffer (0.1 M, pH 7.5, containing 0.1% Triton X-100) in a 1.5mL centrifuge tube. The homogenate was centrifuged at 8,000 g at 4°C for 10 min. Thereafter, the supernatant was collected and diluted appropriately with addition of homogenization buffer without Triton X-100 and used as the sources of enzymes. Protein concentration was determined after the method of Bradford 49 .
After incubating at 37°C for 30 min, 45L mixture solution of 1.0% fast blue B-salt in 5.0% SDS (2:5) was added in each microplate. After another 15 min incubation period at room temperature, absorbance was measured at 600 and 560 nm. The activity of CarE activity was reported as nanomoles of product formed / min / mg  protein via comparing to the -naphthol or β-naphthol standard curve.
The activity of Glutathione S-transferase (GST) was determined using 1-chloro-2,4dinitrobenzene (CDNB) and reduced glutathione (GSH) following the method of Salman et al 50  L of nuclease-free water according to the manufacturers protocol(Takara, Dalian, China).
The relative gene expression was analyzed using the 2 -ΔΔ Ct method as described by Livak and Schmittgen 53 . The expression level of solvent treated S.invicta was used as the calibrator. The significance of differences between two treatment was evaluated using a one-way ANOVA (SPSS version 17.0) followed by the Duncan's multiple range test at 0.05 level. Three biological replicates and three technical replicates were performed in each qRT-PCR reaction.
One-way ANOVA with means separated using Tukey's honestly significant difference (HSD) test were used to analyze the differences of detoxifying enzymes among castes and development stages of S.invicta at p = 0.05 level.

Author Contributions
T, Xiong., X. N., Zeng conceived and designed the experimental plan. T, Xiong. Wrote the main manuscript text and conducted the experiments and analysed the results. S Q., Ling and J L., Liu provided some suggestions for revision and corrected the grammatical mistakes. X. N., Zeng supervised the study. All authors reviewed the manuscript.

Additional Information
Competing Interests: The authors declare no competing interests.  Table 2. Contact toxicity of fluralaner on workers of S.invicta Table 3. Feeding toxicity and synergism effects of fluralaner against S.invicta Table 4. Horizontal toxicity transmit of Fluralaner between workers of S.invicta