Key message

  • Sul-R strain showed cross-resistance to neonicotinoid, pyrethroid and carbamate insecticides.

  • Enhanced activities of P450, CarE and GST enzymes were detected in the Sul-R strain.

  • Twenty-five P450 genes were overexpressed in the Sul-R strain.

  • CYP6CY13-2 played a key role in sulfoxaflor resistance.

  • This Sul-R strain made more intercellular apoplastic stylet pathway events than Sus strain.

  • This Sul-R strain showed an increased relative fitness of 1.19.

Introduction

The cotton aphid, Aphis gossypii (Glover) (Homoptera: Aphididae), is a worldwide insect pest on cotton and numerous other crops worldwide (Kim 2007). It causes serious damage through direct feeding and virus disease transmission (Koo et al. 2014). Nowadays, cotton aphids have developed high resistance to numerous common insecticides, such as neonicotinoids, carbamates, organophosphates, and pyrethroids (Wang et al. 2007; Gao and Zheng 1990; Zheng et al. 1989, 1988). Thus, novel insecticides are urgently needed to control this sap-feeding insect pest.

Sulfoxaflor, a sulfoximine insecticide, acts on nicotinic acetylcholine receptor (nAChR) in insect nervous to control sap-feeding insect pests (Christopher et al. 2013; Sparks et al. 2013). Like other insecticides (neonicotinoids, spinosyns, nicotine, nereistoxin analogs), sulfoxaflor acts on insect nAChRs, but in a manner that is distinct from other chemistries (Watson et al. 2011; Babcock et al. 2011). Due to its unique properties, it has been demonstrated to have excellent control efficacy against sucking pests, including those which are difficult to control owing to resistance to currently registered insecticides (Zhen et al. 2018; Babcock et al. 2011). Thus, sulfoxaflor could be a new tool for the management of sap-feeding pests which are resistant to established insecticide groups all over the world. However, long-term use of sulfoxaflor may cause the likelihood of resistance developing and increasing. For example, previous studies reported that A. gossypii has developed different levels of resistance to sulfoxaflor in many countries, including China (Chen et al. 2017a), Korea (Koo et al. 2014) and Japan (Hirata et al. 2017). Therefore, it is necessary to study the resistance mechanisms and related biological changes in sulfoxaflor-resistant A. gossypii.

Insect pests can develop resistance to insecticides by various ways, including metabolic resistance, target-site resistance, penetration resistance and behavioral resistance (Dawkar et al. 2013). So far, the elevated detoxification by cytochrome P450s has been reported most frequently to underpin neonicotinoid resistance in insect pests. For example, the overexpression of one or multiple P450 genes (such as CYP6CY14, CYP6A2, CYP6DA2 and CYP380C6) has been found to be involved in A. gossypii resistance to thiamethoxam, spirotetramat or gossypol (Wu et al. 2018; Pan et al. 2018; Peng et al. 2016a, b). Similar results can also be found in other pests, such as Plutella xylostella (Li et al. 2018) and Nilaparvata lugens (Zhang et al. 2016; Ding et al. 2013; Bao et al. 2016; Liao et al. 2019). Moreover, the overexpression of P450 genes CYP6CY13 and CYP6CY19 have been identified to confer sulfoxaflor resistance in A. gossypii (Ma et al. 2019).

With the development of insect resistance, the resistant insects may have certain differences in morphological characteristics, physiological responses and biological characteristics. Thus, the feeding behavior and life history changes associated with sulfoxaflor resistance in A. gossypii were also evaluated through electrical penetration graph (EPG) technique and life-table analyses. The EPG is a useful technique that was first developed by McLean and Kinsey (1964) and later modified to a direct current (DC) system by Tjallingii (1978). This technique has been widely used to investigate the characteristics of host plant resistance (Alvarez et al. 2012), the mechanisms of plant pathogen transmission and the mode of action of insecticides (Moreno et al. 2012; Garzo et al. 2016). Life-table analysis is widely accepted as a powerful tool for the ecological studies, including those on the timing of pest control procedures (Yu et al. 2013), life history (Chi and Su. 2006), host preference and the fitness of insects (Alami et al. 2014; Naseri et al. 2014).

In the present study, the sulfoxaflor-selected A. gossypii was used to elucidate the potential resistance mechanisms and the changes in biological characteristics. For example, the cross-resistance, the synergistic effects of PBO, TTP and DMF against sulfoxaflor, the activities of detoxification enzymes (GST, CarE and P450), the gene expression of the P450s, the feeding behavior and life history in Sul-R A. gossypii were studied. These results were useful for understanding sulfoxaflor resistance mechanisms in A. gossypii and might be helpful for the management of sulfoxaflor-resistant cotton aphid.

Materials and methods

Insecticides and chemicals

The technical grade of sulfoxaflor (99.99%) was obtained from Dow AgroSciences Inc. (Indianapolis, IN, USA). Dimethylsulfoxide (DMSO), dimethylformamide (DMF), piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP) and Tween 80 were purchased from Beijing Chemical Reagent Co., Ltd. Bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), Coomassie brilliant blue G-250, glutathione (GSH), α-naphthyl acetate (α-NA), 2,4-dinitrochlorobenzene (CDNB) and dithionitrobenzoic acid (DTNB) were purchased from Sigma Chemical Corporation (St. Louis, MO, USA).

Insects

A laboratory-reared relatively susceptible strain (Sus Strain) of A. gossypii was kindly provided by Nanjing Agricultural University, and it was maintained in the laboratory under insecticide-free conditions. The Sul-R strain was established from the relatively Sus strain via continual laboratory selection by increasing sulfoxaflor concentrations based on LC50 values of their parental generations for total 20 generations. About 7000 apterous adult aphids were selected in each generation by leaf-dipping method, and the mortality was controlled at 60–80%. All insects were reared on cotton seedlings (Zhongzhi 8). Cultures were maintained under controlled environment laboratory conditions (25 ± 1 °C with 70 ± 10% relative humidity) with a photoperiod of 14:10 h (L/D).

Bioassays

The leaf-dipping method (Cui et al. 2018) was used to evaluate the toxicity of sulfoxaflor against A. gossypii under laboratory conditions. The stock solution of sulfoxaflor (10,000 mg L−1 in DMSO) was diluted using an aqueous solution of 0.1% (w/v) Tween 80 to five concentrations. Each concentration replicated thrice. Individual cotton leaves infested with about fifty A. gossypii were dipped in sulfoxaflor solutions for 3 s and dried on tissue paper. Then individual leaves were transferred to 90-mm petri dishes which contain a water-moistened filter paper. To provide ventilation, each petri dish was covered by a perforated lid with fine mesh, and then all petri dishes were stored in an incubator at 25 ± 1 °C, 70 ± 10% RH and a 14:10 h light: dark photoperiod for 24 h until mortality was assessed. Control aphids treated with 0.1% tween 80 aqueous containing DMSO (20 mg L−1) showed mortality < 10% in all bioassays.

Synergism bioassays

The synergism of PBO (P450 inhibitor), DEM (GST inhibitor) and TPP (esterase inhibitor) in combination with sulfoxaflor was evaluated using the leaf-dipping bioassay method described above. The concentrations of PBO, DEM and TPP that led to < 10% mortality in the susceptible strain were adopted as the maximum sublethal concentrations. And the final adopted concentrations of PBO, TPP and DEM were 80 mg L−1, 40 mg L−1 and 40 mg L−1, respectively. Apterous adult aphids infested in cotton leaves were treated with PBO (80 mg L−1), DEM (40 mg L−1) or TPP (40 mg L−1) and the sulfoxaflor mixtures, and at least five concentrations of sulfoxaflor were conducted. The mortality was recorded at 24 h after treatment. The synergistic ratio was calculated by dividing the LC50 values without the synergist by the LC50 values with the synergist.

Enzyme assays

Protein assay

A. gossypii samples (7.5 mg) from Sus or Sul-R strains were collected at three-day adult stage. The samples were homogenized with 2 mL of 0.1 M pH 7.6 phosphate buffer on ice, containing 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 1 mM N-phenylthiourea (PTU) and 1 mM phenylmethylsulfonyl fluoride (PMSF). After homogenization, the homogenates were centrifuged at 12, 000 g for 10 min at 4 °C, and the supernatant was collected as the enzyme source. The content of total protein was detected using Bradford's method (Bradford 1976).

CarE assay

CarE activity was measured using α-naphthyl acetate (α-NA) as substrate according to the method described by Van (1962), with slight modification. Five milliliters of a substrate solution containing 3 × 10–4 M α-NA and 1 × 10–4 M physostigmine, an inhibitor of acetylcholinesterase, was prepared. Then, 0–0.5 mL of enzyme source (diluted 20-fold) and 1–0.5 mL of phosphate-buffered saline (PBS: 0.04 M, pH 7.0) were added into the substrate. The mixture was incubated with shaking for 30 min at 30 °C. The reaction was stopped by addition of 1 mL distilled water containing 2.9 mg of fast blue B salt and 35.7 mg of sodium dodecyl sulfate (SDS). The absorbance at 600 nm was read after 30 min using a Synergy HT multi-mode microplate reader (BioTek, Winooski, VT, USA). The results were expressed as ΔmOD600 min−1 mg protein−1. At least three replicates of enzyme sources were tested with five individuals for each replicate.

GST assay

GST activity was calculated using 2,4-dinitrochlorobenzene (CDNB) as the substrate (Habig et al. 1974). The enzyme solution (0.2 mL) was hatched with CDNB (0.1 mL, 30 mM), GSH (0.3 mL, 50 mM) and PBS (2.4 mL, 66 mM, pH 7.0). Enzyme activity was measured using a Synergy HT multi-mode microplate reader at 340 nm at 27 °C using the kinetic model for 5 min. The results are expressed as ΔmOD340 min−1 mg protein−1. Three replicates of enzyme sources were detected with five individuals for each replicate.

P450 assays

The P450 levels in the samples were measured via an insect function oxidase ELISA kit following the manufacturer’s protocols. Sample ODs were measured at 450 nm using a microplate reader, and the concentrations were calculated by comparing the sample ODs to the standard curve (y = 0.0055x + 0.1343). Three replicates of enzyme sources were tested and the results were expressed as ΔmOD450 min−1 mg protein−1.

The genes expression levels

Approximately 100 adults of Sus or Sul-R A. gossypii were put on cotton leaves and only neonate nymphs were kept after 12 h. When the majority of neonate nymphs had developed into the three-day adult stage, they were collected gently with a soft brush into a 1.5-mL centrifuge tube. Total RNA was extracted from apterous adult using RNeasy® Mini Kit (Qiagen, Germany) and first-strand cDNA was synthesized from 1 µg of total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China) following the manufacturer’s protocols. TB GreenTM Premix Ex TaqTM II (Tli RNaseH Plus) (Takara, Shiga, Japan) was used to conduct the RT-qPCR reactions. The mRNA expression levels of 35 P450 genes were detected in Sul-R and Sus strains. All the primers are presented in Table S1.

RT-qPCR reactions were carried out using the ABI 7500 qPCR System (Applied Biosystems 7500) with a 20 μL reaction system containing: 10 μL TB Green Premix Ex Taq II (Tli RNaseH Plus), 2 μL of diluted cDNA, 0.8 μL (10 μM) of each primer, 0.4 μL ROX Reference Dye II and 6 μL ddH2O. The thermal cycling conditions were as follows: 95 ℃ for 10 min, 40 cycles of 95 ℃ for 15 s, 60 ℃ for 30 s and 72 ℃ for 30 s. After amplification, one dissociation step cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 30 s, 60 °C for 15 s was performed to ensure the specificity of the amplified product. A standard curve was established for each primer pair to determine the amplification efficiencies. The house keeping genes, elongation factor1-alpha (EF1α), was used as internal reference genes for A. gossypii (Ma et al. 2019). RT-qPCR reactions were independently performed three times for each strain. Relative gene expression was calculated using the 2−ΔΔCT method (Pfaffl 2001).

RNAi and bioassays

Based on the full-length open reading frame (ORF) sequences of CYP6CY13-2 (Supplementary data 1) and the predicted possible interference sites obtained from online prediction software (http://www.dkfz.de/signaling/e-rnai3/), we designed the specific primers using Premier 5.0 software. The gene fragments were amplified from cDNA and cloned into pClone 007 Versatile Simple Vector (Tsingke Biotech Co., Ltd. Beijing, China), then sequenced by Tsingke Biotech Co., Ltd. (Beijing, China). The purified plasmids served as templates for RNA synthesis using the T7 RiboMAX™ Express RNAi System (Promega, USA). GFP dsRNA was synthesized under the same conditions, and it was used as the control (Table S1). The artificial diet and the rearing method applied for our study were reported previously (Peng et al. 2016b). To ensure the absence of RNase activity, the diet was prepared in DEPC-treated water. For the dsRNA feeding experiments, dsRNA at the concentration of 150 ng μL−1 was added to the artificial diet. An artificial diet containing dsRNA-GFP was applied for control. Eighty apterous adults from the Sul-R strain were transferred onto the artificial diet for rearing. To analyze the knockdown efficiency of this dsRNA, the aphids were collected after feeding for 48 h, and the samples were used for RT-qPCR. To assess the sensitivity of A. gossypii to sulfoxaflor after P450 RNAi, eighty apterous adults were transferred to the artificial diet containing sulfoxaflor (1.0 mg L−1), dsRNA-CYP6CY13-2 (150 ng μL−1). Aphids fed with the artificial diet containing sulfoxaflor (1.0 mg L−1) and dsRNA-GFP (150 ng μL−1) were used as the control. Aphids mortality was recorded after exposure to insecticide for 48 h. Each treatment included three replicates.

Feeding behavior

The aim of this experiment was to find the difference in probing and feeding behavior between Sus and Sul-R A. gossypii. The feeding behavior experiments of Sus and Sul-R A. gossypii were conducted on cotton plants. All treatments were monitored using one Giga-8 DC-EPG device with 1 GΩ (EPG-Systems Wageningen, The Netherlands), and all EPG signals were recorded onto a computer hard disk and converted through an analog/digital (A/D) converter card (Di 710, EPG-systems, Wageningen, Netherlands). The monitor and record method in this study were reported previously (Garzo et al. 2016; Wang et al. 2020). Each individual was monitored continuously for 6 h. A minimum of 20 recordings were made for each treatment, and new aphids and plants were used for each replicate.

Life table

Life tables were constructed separately for the Sus and Sul-R strains. The rearing method of adults used in this study was described previously (Wang et al. 2020). After 12 h, all adults were removed and only one neonate nymph was left on the cotton leaf. Totally, least 100 neonate nymphs for both strains were observed individually. During the reproductive period, the newly nymphs were counted and then discarded every 24 h until the adult was dead. During this study, the following observations were made: the developmental time of every stage, the emergence of adults, survival, the oviposition period, longevity and fecundity. These data were then used to establish the age-stage, two-sex life table.

Statistical analysis

Adjusted mortality was calculated using the Schneider-Orelli formula. The LC50 values and their 95% confidence intervals (CIs) were calculated using software DPS (v 7.05) (Refine Information Tech. Co. Ltd, Hangzhou, China). The NWEI (number of waveform events per insect) was calculated as the sum of the number of events of a particular waveform divided by the total number of insects under each treatment. The WDI (waveform duration per insect) was calculated using the sum of durations of each event of a particular waveform made by each individual insect divided by the total number of insects under each treatment (Garzo et al. 2016). Student's t-test in SAS 9.2 software (SAS Inc., Cary, NC, USA) was used to compare significant differences between Sus and Sul-R strains. The raw life history data, such as survival, fecundity, longevity, instinsic rate of increase (rm), finite rate of increase (λ), net reproductive rate (R0), mean generation time (T), age-stage survival rate (sxj) (where x is the age and j is the stage), age-specific fecundity (mx), age-specific survival rate (lx), age-specific maternity (lxmx), age-stage specific life expectancy (exj), reproductive value (vxj), of A. gossypii individuals were analyzed by the TWOSEX-MS Chart program, which is based on the age-stage, two-sex life-table theory and methods described by Chi and Liu (1985) and Chi (1988). The developmental time, adult longevity, oviposition days, fecundity, adult pre-reproductive period (APOP), total pre-reproductive period (TPOP) and the strain parameters (rm, λ, R0, and T) were compared using bootstrapping methods in TWOSEX-MSChart (10 000 replications were used in the bootstrapping procedures) (Akköprü et al. 2015).

Results

Sulfoxaflor toxicity and cross-resistance to different classes of insecticides in the Sul-R strain

The toxicities of sulfoxaflor to both susceptible and resistant strains are shown in Table 1. The laboratory-selected Sul-R strain developed medium resistance to sulfoxaflor with 40.19-fold of resistance ratio and its LC50 value was 25.72 mg L−1 in contrast to the relatively Sus strain with a LC50 value of 0.64 mg L−1. In addition, after releasing the selection pressure for six generations, the LC50 value of sulfoxaflor was 26.29 mg L−1 for the Sul-R strain, and the level of resistance was not decreased in the absence of selection pressure. Compared with the Sus strain, the Sul-R strain developed cross-resistance to thiamethoxam (52.69-fold), imidacloprid (35.90-fold), nitenpyram (32.81-fold), acetamiprid (29.41-fold), cycloxaprid (10.16-fold), alpha-cypermethrin (11.91-fold) and carbosulfan (5.62-fold), while no cross-resistance was detected between sulfoxaflor and chlorpyrifos.

Table 1 Cross-resistance of Sul-R strain to other insecticides
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Synergism bioassays and Metabolic enzyme activity

The synergism results of A. gossypii by leaf-dipping method are summarized in Table 2. Three synergists, PBO, DEM and TPP, exhibited no significant synergism to sulfoxaflor in the Sus strain, while they obviously increased the toxicity of sulfoxaflor by 7.37-, 2.60- and 1.76-fold in the Sul-R strain, respectively. (Table 2). These results indicated that P450s, GST and CarE might be related with the medium resistance to sulfoxaflor in cotton aphids.

Table 2 Toxicity of sulfoxaflor against A, gossypii of Sul-R and Sus strains after synergists
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The activities of detoxification enzymes, such as P450, CarE and GST in two strains of A. gossypii are presented in Fig. 1. Sulfoxaflor selection significantly increased the activities of P450 (1.50-fold) (P = 0.0034), CarE (4.76-fold) (P = 0.0001) and GST (1.13-fold) (P = 0.0310) of A. gossypii. These results also demonstrated that three detoxification enzymes, P450, CarE and GST, were elevated in the sulfoxaflor resistance A. gossypii strain.

Fig. 1
figure 1

The activity of P450, GST and CarE of A. gossypii in Sus and Sul-R strain. The P450 results are expressed as ΔmOD450 min−1 mg protein−1. The GST results are expressed as ΔmOD340 min−1 mg protein−1. The CarE results are expressed as ΔmOD600 min−1 mg protein−1. Error bars indicate 95% confidence intervals (n = 3). *Significant difference as determined by Student's t test (SAS 9.2 software) (P < 0.05)

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Dynamic changes in P450 gene expression in the Sul-R and Sus strains

To determine which P450 genes may be associated with Sulfoxaflor resistance, the mRNA transcriptional levels of thirty-five P450 genes in Sus and Sul-R strains were analyzed by quantitative real-time PCR. Compared with the Sus strain, the gene (in CYP3 clade) expression level of CYP6CZ1, CYP6DC1, CYP6DD1, CYP6CY13-2, CYP6CY9, CYP6CY18, CYP6CY13-1, CYP6CY7, CYP6DA1, CYP6DA2, CYP6CY4, CYP6Y16 were up-regulated 2.06-, 1.95-, 2.74-, 4.89-, 2.37-, 1.24-, 2.32-, 1.71-, 2.04-, 1.97-, 1.21- and 2.05-fold, respectively, in the Sul-R strain. Expression of CYP353B1, CYP301A1, CYP49A1 (in CYP2 clade), CYP4G51, CYP4CK1, CYP6CJ3, CYP6CJ1 (in CYP4 clade) and CYP305E1 (in the Mito clade) were up-regulated 1.68-, 1.51-, 3.63-, 3.00-, 3.49-, 1.15-, 1.42-, 1.90- and 2.49-fold, respectively, in the Sul-R strain (Fig. 2). The transcripts of ecdysone synthesis-related P450 genes, including CYP315A1, CYP302A1 (in CYP2 clade) and CYP306A1, CYP307A1, CYP18A1 (in the Mito clade) were increased to 4.23-, 3.86-, 1.16-, 3.71- and 1.91-fold, respectively, in the Sul-R strain (Fig. 2). In contrast, four genes in CYP3 Clade (CYP6CY14, CYP6UN1, CYP6CY12, CYP6DB1), one gene in CYP2 Clade (CYP314A1) and two genes in CYP4 Clade (CYP380C6, CYP6CJ2) were down-regulated in the Sul-R strain.

Fig. 2
figure 2

Transcription levels of P450s in the Sus and Sul-R strains determined via real-time PCR. EF1a was employed as internal reference genes. Error bars indicate 95% confidence intervals (n = 3). *Significant difference as determined by Student's t-test (SAS 9.2 software) (P < 0.05)

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Knockdown of P450 gene increases sulfoxaflor toxicity

To evaluate the functional roles of CYP6CY13-2 gene in sulfoxaflor resistance, the toxicity of sulfoxaflor to the Sul-R strain was bioassayed after suppressing the expression levels of CYP6CY13-2 via RNAi. Under the RNAi treatments, the expression level of CYP6CY13-2 gene was reduced to 0.36-fold in the dsRNA-CYP6CY13-2-treated aphids compared to the control (dsRNA-GFP). Mortality significantly increased from 34.04% in the control aphids to 74.39% in the dsRNA-CYP6CY13-2-treated aphids under 1.0 mg L−1 sulfoxaflor treatment (Fig. 3).

Fig. 3
figure 3

dsRNA-mediated suppression of CYP6CY13-2 transcripts and its effect on sulfoxaflor toxicity in A. gossypii. a dsRNA-mediated suppression of CYP6CY13-2 transcripts in resistant aphids fed an artificial diet with dsRNA (150 ng μL−1). b Mean mortality ± SE (n = 3) of resistant cotton aphids after they were fed sulfoxaflor (1.0 mg L−1) and a dsRNA mixture (150 ng μL−1 of dsRNA) for 48 h. Each treatment included three replicates, and eighty adults of resistant aphids were used in each replication. Error bars indicate 95% confidence intervals (n = 3). *Significant difference as determined by Student's t-test (SAS 9.2 software). (P < 0.05)

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Probing and feeding behavior of two aphid strains

The probing and feeding behavior of Sus and Sul-R A. gossypii on cotton plants is shown in Table 3. During the 6 h of recording, no significant differences were found in the number and duration of non-probing (Np) waveform between Sus and Sul-R strains. The number and duration of intercellular apoplastic stylet probes (waveform C) of per insect was significantly increased in the Sul-R aphids (NWEI: 141.10 ± 12.78, WDI: 156.20 ± 10.21 min) when compared with Sus strain (NWEI: 90.95 ± 6.04, WDI: 125.60±9.15 min). The number of brief intracellular stylet punctures (Pd) waveforms of Sul-R aphids (NWEI: 126.60 ± 11.38) was significantly higher than that of the Sus strain (NWEI: 78.00 ± 5.39), although the duration of the Pd phase was not significantly increased in this Sul-R strain. The number of short probes (C < 3 min) was significantly lower in the Sul-R strain compared with the Sus strain. In addition, the Sul-R strain spent less time in both phloem ingestion phases (E2) and phloem salivation phases (E1) (WDI: E2: 117.90 ± 16.09 min, E1: 7.09 ± 2.06 min) than the Sus strain (WDI: E2: 133.10 ± 18.76 min, E1: 24.41 ± 8.60 min), although no significant differences were observed between two strains. However, the Sul-R A. gossypii ingested from the phloem (time from the beginning C to the first E2) (WDI: 96.26 ± 13.66 min) more rapidly than the Sus strain (WDI: 144.70 ± 17.47 min). Moreover, the percentage of E1 in the phloem phase did not differ significantly between Sul-R strain (7.59 ± 2.18%) and Sus strain (9.40 ± 3.03%).

Table 3 Probing and feeding behavior of Sus and Sul-R A. gossypii on cotton plants
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Life history parameters of two A. gossypii strains

The developmental time, longevity and fecundity of Sus and Sul-R A. gossypii are shown in Table 4. The fecundity of adults was obviously higher in the Sul-R strain (41.02 ± 1.55 offspring) than in the Sus strain (34.36 ± 1.39 offspring). Furthermore, the developmental time of the third-instar (1.42 ± 0.08 d) and fourth-instar (1.52 ± 0.07 d), total preoviposition period (TPOP) (6.16 ± 0.10) were significantly prolonged in the Sul-R strain. Although the Sul-R strain exhibited an increase in the developmental time of adults, the mean longevity of aphids and adult preoviposition period (APOP), no significant differences were observed between the two strains.

The development of strain dynamics was estimated by bootstrap methods based on the life table. The net reproductive rate (R0), mean generation time (T), finite rate of increase (λ), intrinsic rate of increase (rm) and gross reproduction rate (GRR) were calculated and analyzed (Table 5). The R0 (41.02 ± 1.55 d−1), T (11.98 ± 0.17 d−1) and GRR (44.59 ± 2.29 offspring/individual) were significantly higher in Sul-R A. gossypii. However, no statistically significant differences were found in the rm and λ between the two strains.

Age-stage-specific survival rate (sxj) curves represent the probability that a newborn nymph will survive to age x and stage j (Fig. 4). Because of the variable developmental rates of individuals, obvious overlaps among stages were found in Sus and Sul-R A. gossypii. The relative numbers of second-instar (N2) and third-instar (N3) nymphs of the Sus strain were larger than those of the Sul-R strain. However, the relative numbers of fourth-instar (N4) of the Sul-R strain were larger than those of the Sus strain. Furthermore, the probability that a newborn nymph survived to an adult in the Sul-R strain (0.96) was slightly lower than the Sus strain (0.97).

Table 4 Developmental time and fecundity of Sus and Sul-R A. gossypii on cotton leaves
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Table 5 Population parameters of Sus and Sul-R A. gossypii on cotton leaves
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Fig. 4
figure 4

Age-stage survival rate (Sxj) of Sus and Sul-R A. gossypii. Sxj curve reveals the probability that a newborn nymph will survive to age x and stage j

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The age-specific survival rate (lx) curve indicates the probability that a newborn nymph will survive to age x, and lx provides a simplified summary of the survival history. Figure 5 shows that the lx for the Sul-R strain was higher than that for the Sus strain from 11 to 30 d and after 34 d. The age-specific fecundity curve (mx) reveals the fecundity of individuals at age x. Based on the mx curve, both the highest age-specific fecundity peak of Sul-R strain (2.98 offspring/24 h) and Sus strain (2.61 offspring/24 h) all occurred at the age of 9 d. Based on lx and mx, the maximum lxmx values were 2.98 and 2.6 offspring for the Sul-R and Sus strains, respectively (Fig. 5). The age-specific reproductive value (vx) of the Sul-R strain was considerably higher than that of the Sus strain in the all adult stage. Moreover, the maximum vx value of the Sul-R strain (9.48 at the age of 8 d) was higher than that of the Sus strain (8.08 at the age of 9 d) (Fig. 5).

Fig. 5
figure 5

Age-specific survival rate (lx), age-specific fecundity (mx), lxmx and age-specific reproductive value (vx) of Sus and Sul-R A. gossypii. lx curve represents the probability that a newborn nymph will survive to age x; mx curve represents the fecundity of individuals at age x

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Age-stage life expectancy (exj) is the time that an individual of age x and stage y is expected to live, and life expectancy is reduced as age increases (Fig. 6). The life expectancy of first-instar (N1), second-instar (N2), third-instar (N3) fourth-instar (N4) nymphs and adult were 35.20, 34.20, 33.20, 32.20 and 31.20 d, respectively, for the Sul-R strain, while these values were only 33.23, 32.23, 31.23, 30.23 and 29.23 d, respectively, for the Sus strain. The age-specific life expectancy (ex) curve represents the expected life span of an individual of age x. The ex curve showed that Sul-R A. gossypii had a longer life expectancy than the Sus strain. (Fig. 5).

Fig. 6
figure 6

Age-stage life expectancy (exj) and age-specific life expectancy (ex) of Sus and Sul-R A. gossypii. exj curve is the time that an individual of age x and stage y is expected to live. ex curve reveals the expected life span of an individual of age x

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Discussion

Sulfoxaflor is the initial commercial product from the new sulfoximine class of insect control agents (Sparks et al. 2012) and exhibits high activity against a broad range of insect species in field, including those resistant to neonicotinoids and other currently available insecticides. (Zhen et al. 2018; Babcock et al. 2011). Thus, sulfoxaflor has been used as an alternative insecticide to control a range of sucking insects, including A. gossypii. Unfortunately, medium to high-level resistance (RR values ranged from 42.9-fold to 464.3-fold) to sulfoxaflor has been observed in four field strains of A. gossypii from Korea (Koo et al. 2014). Similarly, Chen et al. (2017a) have reported a case of the high cross-resistance to sulfoxaflor (260-fold) in imidacloprid-resistant A. gossypii (1215-fold). Moreover, the field strains of N. lugens had already developed a low level of resistance to sulfoxaflor (resistance ratio, RR = 0.8–6.8-fold), and the resistance level showed an increasing trend from 2013 (Liao et al. 2017). In the present study, a colony of A. gossypii with 40.19-fold resistance to sulfoxaflor was established by continuous selection using sulfoxaflor. These results suggested that A. gossypii possess high resistance risk to sulfoxaflor.

Understanding cross-resistance between sulfoxaflor and other pesticides can provide useful information for pest resistance management. In our study, the resistant strain has developed different cross-resistance to neonicotinoid, pyrethroid and carbamate insecticides (RR values ranged from 5.62-fold to 35.90-fold). Similarly, Ma et al. (2019) reported that a high level of sulfoxaflor-resistant A. gossypii was established by continuous selection with sulfoxaflor for 15 generations from a relatively susceptible strain, and this sulfoxaflor-resistant strain showed medium to high level of cross-resistance to imidacloprid, acetamiprid, thiamethoxam and flupyradifurone (RR values ranged from 10.0-fold to 107.5-fold). Moreover, Liao et al. reported that the sulfoxaflor-resistant N. lugens (147.67-fold) shows high-level cross-resistance to dinotefuran, nitenpyram and thiamethoxam (Liao et al. 2019). However, the cross-resistance mechanisms between sulfoxaflor and neonicotinoids are complex. For example, Mezei et al. (2020) reported that no direct and consistent link was found between target-site resistance to neonicotinoids and reduced efficacy toward sulfoxaflor, although sulfoxaflor and neonicotinoids, such as imidacloprid, were unquestionably nAChR agonists. Moreover, Watson et al. (2017) reported that although the neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid) displayed relatively high affinity for the [3H] sulfoxaflor binding site, no significant correlation was found between neonicotinoid affinity and Myzus persicae toxicity. Therefore, the resistance observed to neonicotinoids in our Sul-R strain may be due to increased detoxification. A possible candidate is cytochrome P450 monooxygenases which are known to confer resistance to neonicotinoids in many insects (Liao et al. 2019; Wu et al. 2018; Ma et al. 2019). Our results suggested that there is a risk of cross-resistance between sulfoxaflor and neonicotinoids in cotton aphid and other insect pests. Therefore, the cross-resistance between sulfoxaflor and other insecticides, especially neonicotinoids, should be of concern. And in order to delay or prevent the development of resistance, sulfoxaflor cannot be mixed or alternated with neonicotinoid insecticides in the control of sucking insect pests (Mezei et al. 2020).

Insecticide resistance in insect pests is mainly attributed to enhanced detoxification by metabolic enzymes and target insensitivity caused by key amino acid mutation or target-site (Casida and Durkin 2013; Ding et al. 2013; Bass et al. 2011; Chen et al. 2017b; Ma et al. 2019; Pan et al. 2020). For example, enhanced detoxification by P450 contributing to insecticides resistance in A. gossypii has been commonly reported (Pan et al. 2018; Peng et al. 2016a, b; Shang et al. 2012). In our study, the activity of P450 in Sul-R strain was significantly higher (1.5-fold) than that in Sus strain. Additionally, a significant synergistic effect of PBO to sulfoxaflor in the Sul-R strain was observed (synergism ratio: 7.37). Both results suggested that the elevated detoxification of P450 enzymes play an important role in A. gossypii resistance to sulfoxaflor. Meanwhile, we also found that DEM and TPP have significant synergism to sulfoxaflor in the Sul-R strain. Moreover, the activities of GST and CarE were significantly higher in the Sul-R strain, suggesting that GST and CarE enzymes may also be involved in A. gossypii resistance to sulfoxaflor. Interestingly, in our study, PBO showed the biggest synergistic effects (7.37-fold) on sulfoxaflor toxicity in resistant strain, while the activity of CarE had the biggest change (4.76-fold) in Sul-R strain when compared with Sus strain. Similarly, Ma et al. also reported that the sulfoxaflor-resistant strain possessed significantly higher CarE activity (1.53-fold) compared to the susceptible strain although sulfoxaflor lacks the carboxylic ester group and cannot be hydrolyzed by CarE directly. It was speculated that carboxylesterases cause sulfoxaflor resistance in A. gossypii primarily via sequestration (Ma et al. 2019). Meanwhile, DEM increased the toxicity of sulfoxaflor by 2.60-fold in our Sul-R strain with medium resistance. However, Ma et al. (2019) reported that DEM showed no synergistic effect to sulfoxaflor in the highly resistant strain with 245-fold of resistance ratio. This phenomenon may be related to different resistance levels or mechanisms in aphids. And GST-mediated resistance mechanism might be not absolutely correlated with resistance levels. The highly resistant strain of Ma et al. was obtained by sulfoxaflor selection for only 15 generations, while P450 and CarE were much more important than GST for the resistance. Our study demonstrated that P450 plays the most important role in sulfoxaflor medium-resistance; meanwhile, CarE and GST can take part in the sulfoxaflor resistance in A. gossypii.

Generally, the overexpression of the cytochrome P450 monooxygenase genes has been considered to be a general molecular mechanism for the increasing levels of enzymes activities in resistant pests. (Karunker et al. 2008; Bao et al. 2016; Huang et al. 2019; Pan et al. 2018). To determine a comprehensive view of the expression changes in P450 genes in the development of sulfoxaflor resistance, thirty-five P450 genes were compared in both strains. Our results showed that twenty-five genes were significantly up-regulated in the Sul-R strain A. gossypii (the values ranged from 1.15-fold to 4.89-fold), among which CYP6CY13-2 and CYP315A1 were up-regulated more than 4.0-fold. Similarly, the overexpression of nine of twenty-eight P450 genes (CYP6CY13, CYP6CY19, CYP6CY4, CYP6CY18, CYP4CJ1, CYP6A2, CYP6J1, CYP380C6, CYP3323A1) was observed in a high level of sulfoxaflor-resistant A. gossypii (Ma et al. 2019). Furthermore, the RNAi of CYP6CY13-2, in the Sul-R strain of A. gossypii, not only suppressed the genes expression to 0.36-fold, also significantly increased the mortality of the Sul-R A gossypii under sulfoxaflor treatment. These results indicated the overexpression of CYP6CY13-2 contributed to sulfoxaflor resistance in A. gossypii. However, the metabolic capacity to sulfoxaflor of this P450 gene CYP6CY13-2 through eukaryotic expression in vitro also should be studied in future.

The development of resistance to sulfoxaflor has led to certain changes in physiological responses and biological characteristics. Our work showed that there were significant differences between Sus and Sul-R aphids in the probing and feeding behavior. However, no deterrent effects or delays in probing were found in the Sul-R aphids, as the time elapsed from the beginning of the EPG recording until the first probe was the same as the susceptible aphids. Moreover, there were no significant differences in the number and duration of non-probing activities. Similar results were found in studies on M. persicae, which started feeding normally when M. persicae (Mp1989) were exposed to sulfoxaflor-, imidacloprid-treated or water-treat plants (Garzo et al. 2016). Before the insect stylet arrives at the phloem, it often requires multiple explorations to find a suitable site for feeding (Lei et al. 1997). In the present study, the number and duration of waveform C were significantly increased in the Sul-R strain. Moreover, the number of Pd waveform was significantly higher in the Sul-R strain, although the duration of the Pd phase was not significantly increased in this population. These results were consistent with our previous study, the imidacloprid-resistant aphids made more waveform C than the susceptible population and the number of Pd waveforms significantly increased in the resistance strain (Wang et al. 2020). These results suggested that Sul-R strain became more active to find an appropriate position for feeding (Tjallingii 1985). Furthermore, the aphids ingested from the phloem (time from the beginning C to the first E2) more rapidly in the Sul-R strain (WDI: 96.26 ± 13.66 min) than the Sus strain (WDI: 144.70 ± 17.47 min), and values for the number of short probes (C < 3 min) were significantly decreased in the Sul-R strain compared with the Sus strain. These results suggested that the Sul-R strain aphids had higher ability to get to the phloem than the Sus strain. And the increased ability to get to the phloem might be beneficial for Sul-R aphids to obtain nutrients effectively. Because phloem sap provides amino acids, saccharides and other nutrients to insects, and these nutrients are key energy source to the growth and development of insects (Nardone et al. 2013). However, no significant differences were observed in the number and duration of phloem salivation and sustained phloem ingestion in two strains. In addition, the weight of Sul-R and Sus aphids was further studied. And no significant differences were observed in two aphid strains (not shown). Therefore, we can conclude that aphid resistance to sulfoxaflor does not result in large differences in nutrient uptake.

Chemicals may alter mate location, courtship and oviposition of insect (Haynes. 1988). In the present study, we found that sulfoxaflor has a positive impact on the oviposition and embryogenesis process of A. gossypii. The demographic growth parameters of A. gossypii, e.g., the developmental time of 3rd- and 4th-instar, fecundity, total pre-reproductive period (TPOP), gross reproduction rate (GRR), the net reproductive rate (R0) and the mean generation time (T), were significantly increased in the Sul-R strain. Moreover, the relative fitness increased to 1.19 in the Sul-R strain, although some previous reports demonstrated that resistant strains were less competitive and associated with high fitness costs (Shah et al. 2017; Ejaz and Shad 2017; Zhang et al. 2018; Castellanos et al. 2019). However, Wang et al. (2020) found that the fecundity of imidacloprid-resistant A. gossypii was dramatically higher than that of susceptible strain and the resistant strain showed an increased relative fitness of 1.36. In addition, the fenpropathrin-resistant strain of Tetranychus cinnabarinus showed an increased fecundity and the expression and content of vitellogenin (vg) and vitellogenin receptor (vgr) were significantly higher in the resistant strain (Liu et al. 2016). Our results suggested that nutrients might not be the important reason for the increased relative fitness in the Sul-R aphids. In insects, vg and vgr play extremely important roles in reproduction (Zhao et al. 2018). Therefore, vg and vgr might be the key factors for the increased fitness in the Sul-R aphids and further studies about vg and vgr are required to clarify the mechanism of the increased fitness in Sul-R A. gossypii. Overall, our results suggested that the Sul-R strain is more competitive than the Sus strain. More individuals will survive when exposure to sulfoxaflor, and subsequent strain resurgence may occur in the field. Although the Sul-R strain became more capable in reaching to the phloem, making more pds and had better demographic parameters than the Sus strain, only one resistant genotype was studied in the present study. Therefore, the correlation between resistance and behavioral/demographic patterns in more resistant and susceptible genotypes should be studied in the future.

In conclusion, our results suggested that Sul-R A. gossypii possess the high cross-resistance risk to other insecticides and higher P450 enzyme activity and multiple overexpression of P450 genes, especially the CYP6CY13-2 gene, likely contribute to sulfoxaflor resistance in A. gossypii. Moreover, this Sul-R A. gossypii made more intercellular apoplastic stylet pathway events and had an increased relative fitness. Therefore, resistance to sulfoxaflor could result in stimulated fecundity and subsequent strain outbreaks, thereby making the control of Sul-R A. gossypii more challenging. The results will provide a foundation for developing better strategies to control A. gossypii.

Author contributions

LW, LC and CHR conceived and designed the experiments. LW and LC wrote the manuscript. LW, YPC, QQW and WLH analyzed the data. LC, HZY and CHR supervised this research.