**1. Introduction**

The white-back planthopper (WBPH), *Sogatella furcifera* (Horváth) (Homoptera: Delphacidae), is an important insect pest in rice-growing countries in Asia [1,2] that seriously affects rice yields by sucking juice from the rice stem, resulting in slow growth, yellowing, and even lodging and a phenomenon known as'hopper burn', leading to the death of rice plants in severe cases and crop failure. In addition, recent studies have shown that the southern rice black-streaked dwarf virus (SRBSDV) can be transmitted in the main rice-growing areas of Asia, including China, Vietnam, and Japan, through the stylets of *S. furcifera* when injecting juice containing the SRBSDV into the stems of healthy rice, causing great rice yield losses. *S. furcifera* is a typical *r*-strategy pest and can reproduce rapidly when conditions are

suitable [3–5]. It can also migrate long distances to adapt to environmental changes [6]. Over the past few decades, chemical pesticides have been the main strategy for controlling this pest [7]. The long-term use of insecticides against the rice planthopper has resulted in the development of resistance and population increases [8]. *S. furcifera* has now developed different levels of resistance to organophosphorus, carbamate, phenylpyrazole, neonicotinoid, pyrethroid, and insect growth regulator insecticides [9–13]. When insects are successively exposed chemicals, insecticide resistance is a natural adaptability feature and one of the most important factors is the enhanced detoxification metabolic ability by enzymes to insecticides, such as mixed-function oxidase (MFO), carboxylesterase (CarEs) and glutathione *S*-transferase (GSTs) [14], especially those for cytochrome P450 monooxygenases (P450s) activity, could play a major role in the detoxification of insecticides in a number of insect pests [15,16].

Sulfoxaflor is an insecticide produced by Dow AgroSciences (DAS) from a new chemical class of sulfoximines, which act on nicotinic acetylcholine receptors (nAChRs) in the insect nervous system [17,18], and is the first commercial agrochemical to be used for the control of a broad range of sap-feeding insect pests [19]. Although several other chemically distinct classes of insecticides (spinosyns, neonicotinoids, nereistoxin analogs) also act on nAChRs, sulfoxaflor presents some special biological characteristics, such as an absence of cross-resistance between sulfoxaflor and the other nAChR-acting insecticides, which make it highly effective against a wide range of sap-feeding insects, especially against aphids such as the green peach aphid [20]. Sulfoxaflor is also effective against insect pests that are resistant to other classes of insecticides, including many insects that are resistant to neonicotinoids [21]. In 2012, sulfoxaflor was first registered for the control of Miridae pests in cotton cultivation in Arkansas, Louisiana, and Mississippi in the United States, and in 2013, Dow AgroSciences introduced sulfoxaflor to China for the control of cotton aphids, wheat aphids, scale insect pests in citrus, and Delphacidae insect pests in rice [22]. As a relatively recently developed insecticide, there is still a risk that the efficiency of sulfoxaflor may become compromised through various mechanisms, e.g., through modification of the target site of neonicotinoid insecticides or positive sublethal effects (hormesis) in exposed individuals [23]. Thus, it is crucial to understand the resistance mechanisms of insects related to this insecticide before its widespread use in integrated pest management (IPM).

In this study, we examined toxicity and transcript profiles in a susceptible laboratory (Sus-Lab) strain of *S. furcifera* and the Sus-Lab strain in which resistance was continuously induced by treatment with the sublethal LC25 dose of sulfoxaflor for six generations (SF-Sel). We analyzed the genes showing upregulated expression and verified the results using qRT-PCR. Furthermore, the functions of two P450 genes, *CYP6FD1* and *CYP4FD2*, were also analyzed with RNA interference technology through the design of suitable dsRNAs, whose silencing specificity was confirmed using qRT-PCR, and mortality was determined in nymphs treated with the dsRNAs combined with sulfoxaflor. The objectives of this study were to explore the risk of the development of resistance to sulfoxaflor, to conduct a preliminary investigation for the functional verification of P450 genes induced by sulfoxaflor, and to manage the development of resistance through the design of suitable dsRNAs for the silencing of significantly upregulated genes in the future.

#### **2. Results**

#### *2.1. Toxicity of Sulfoxaflor in S. furcifera*

The LC25 for the Sus-Lab strain was estimated at 2.102 μg/mL, and a 2.06-fold decrease in the susceptibility level was observed in the SF-Sel strain, which presented LC50 value of 7.284 μg/mL for sulfoxaflor, compared with the LC50 value of 3.544 μg/mL for Sus-Lab.

#### *2.2. Synergism Experiment*

According to the results of the synergism experiment, when the Sus-Lab strain was treated with the synergistic agents triphenyl phosphate (TPP) acting on CarEs, Diethyl maleate (DEM) on GSTs and piperonyl butoxide (PBO) on cytochrome P450 monooxygenases (P450s), all synergistic treatments showed strong synergistic effects compared with treatment without a synergistic agent, and their toxicities were clearly enhanced (not overlapping the 95% confidence interval, 95%CI), with synergism ratio (SR) values of 1.495, 1.205 and 1.211, respectively, while as the toxicity showed no difference between the synergistic treatments (overlapping 95% CI). The toxicity of the SF-Sel strain treated with the synergistic agent PBO was significantly enlarged compared with those in the other two treatments involving TPP and DEM (not overlapping 95% CIs for the other two synergistic agents). The SR value reached 5.328 and was significantly higher than those in the treatments involving TPP and DEM, for which SR values were 2.689 and 2.283, respectively (Table 1).


**Table 1.** Synergistic effect of three synergists and sulfoxaflor on *S. furcifera.*

\* SR (synergism ratio) = LC50 of a strain treated with sulfoxaflor alone divided by LC50 of the same strain treated with sulfoxaflor plus a synergist. The synergists of TPP, DEM and PBO stand for triphenyl phosphate, Diethyl maleateand piperonyl butoxide, respectively.
