Skip to main content

The gut symbiont Sphingomonas mediates imidacloprid resistance in the important agricultural insect pest Aphis gossypii Glover

Abstract

Background

Neonicotinoid insecticides are applied worldwide for the control of agricultural insect pests. The evolution of neonicotinoid resistance has led to the failure of pest control in the field. The enhanced detoxifying enzyme activity and target mutations play important roles in the resistance of insects to neonicotinoid resistance. Emerging evidence indicates a central role of the gut symbiont in insect pest resistance to pesticides. Existing reports suggest that symbiotic microorganisms could mediate pesticide resistance by degrading pesticides in insect pests.

Results

The 16S rDNA sequencing results showed that the richness and diversity of the gut community between the imidacloprid-resistant (IMI-R) and imidacloprid-susceptible (IMI-S) strains of the cotton aphid Aphis gossypii showed no significant difference, while the abundance of the gut symbiont Sphingomonas was significantly higher in the IMI-R strain. Antibiotic treatment deprived Sphingomonas of the gut, followed by an increase in susceptibility to imidacloprid in the IMI-R strain. The susceptibility of the IMI-S strain to imidacloprid was significantly decreased as expected after supplementation with Sphingomonas. In addition, the imidacloprid susceptibility in nine field populations, which were all infected with Sphingomonas, increased to different degrees after treatment with antibiotics. Then, we demonstrated that Sphingomonas isolated from the gut of the IMI-R strain could subsist only with imidacloprid as a carbon source. The metabolic efficiency of imidacloprid by Sphingomonas reached 56% by HPLC detection. This further proved that Sphingomonas could mediate A. gossypii resistance to imidacloprid by hydroxylation and nitroreduction.

Conclusions

Our findings suggest that the gut symbiont Sphingomonas, with detoxification properties, could offer an opportunity for insect pests to metabolize imidacloprid. These findings enriched our knowledge of mechanisms of insecticide resistance and provided new symbiont-based strategies for control of insecticide-resistant insect pests with high Sphingomonas abundance.

Background

Neonicotinoid insecticides have been applied worldwide, and by 2015, they accounted for approximately 25% of total global insecticide sales [1]. As selective agonists of insect nicotinic acetylcholine receptors (nAChRs), neonicotinoids are very effective in controlling sucking pests and several coleopteran, dipteran, and lepidopteran pest species [2]. Imidacloprid, which was launched in 1991 by Bayer Crop Science, was the first neonicotinoid insecticide. Due to its low mammalian toxicity, imidacloprid (IMI) has replaced many carbamates and organophosphates in crop protection [3]. It was reported that imidacloprid failed to control the cotton whitefly, Bemisia tabaci, in Spain, the first field failure of neonicotinoids worldwide [4]. With the widespread use of imidacloprid, issues of imidacloprid resistance are often reported. It has been detected that the resistance ratio of field populations of Nilaparvata lugens to imidacloprid has been up to 248-fold in Thailand [5]. In China, imidacloprid was applied to control Aphis gossypii in most regions, and high selection pressure has resulted in high imidacloprid resistance in this pest [6, 7].

The cotton aphid, A. gossypii (Hemiptera: Aphididae), is a sap-feeding insect species that can cause considerable economic damage to cotton and numerous other crops worldwide [8]. With the widespread cultivation of transgenic Bt cotton, major devastating lepidopteran pests, such as Helicoverpa armigera, have been successfully controlled [9]. The occurrence of secondary pests such as A. gossypii increased and gradually increased to become the main pests in the cotton fields of China [10]. Some research has shown that the resistance mechanisms of insect pests to neonicotinoids are related to an increase in detoxification enzyme activity and/or mutations in the nicotinic acetylcholine receptor (nAChR) molecular targets [11, 12]. Cytochrome P450-mediated detoxification might play a primary role in insect resistance to neonicotinoids [13]. CYP6CY22 and CYP6CY13 of cytochrome P450s in A. gossypii could metabolize imidacloprid, which explained its high resistance level to imidacloprid [14]. The overexpression of uridine 5-diphosphate glucuronosyltransferases (UGTs) was also found to be associated with imidacloprid resistance in field populations of A. gossypii [15]. Another important mechanism of insect resistance to neonicotinoids is mutations in the β1 subunit of the nAChR. The mutation R81T was reported by Koo et al. [16], and then the mutations L80S [17], V62I, and K264E [7] were reported successively. Some researchers have shown that gut symbiotic bacteria in insect pests can degrade pesticides, which improves the resistance level of hosts to such compounds [18, 19].

It has been reported that Pseudomonas species could degrade imidacloprid, such as Pseudomonas putida [20], Pseudoxanthomonas indica CGMCC 6648 [21], Pseudomonas sp. 1G, and Pseudomonas sp. RPT 52 [22]. In addition, other genera of bacteria have also been found to metabolize imidacloprid, such as Stenotrophomonas maltophilia CGMCC 1.1788 [23], Leifsonia sp. PC-21[24], Klebsiella pneumonia BCH1 [25], Bacillus alkalinitrilicus [26], Mycobacterium sp. MK6 [27], Ochrobactrum thiophenivorans, and Sphingomonas melonis [28]. Since the metabolites of imidacloprid showed a relatively low affinity for nAChR, their insecticidal activity was significantly decreased in insects [29]. Notably, many insects harbor symbionts within their gut lumen, body cavity, or cells [30]. A number of insects have developed symbiotic microorganisms that can degrade toxic compounds, and these symbiotic microorganisms could contribute to host resistance against phytotoxins and pesticides [31]. Thus, the degradation of pesticides by insect symbionts could be an important resistance mechanism of insects to pesticides.

Insects can maintain endosymbiotic bacteria in their body [32]. Detoxifying symbioses can enhance the resistance of the host to pesticides [33]. Susceptible Riptortus pedestris developed resistance to fenitrothion via the acquisition of gut symbionts of the genus Burkholderia, which could degrade fenitrothion [18]. The symbionts of Bacillus cereus isolated from the gut of the diamondback moth Plutella xylostella were identified to degrade indoxacarb with high efficiency, which resulted in the resistance of P. xylostella to indoxacarb [34]. The gut symbiont Citrobacter sp. of the oriental fruit fly Bactrocera dorsalis played a key role in the degradation of trichlorfon, which contributed to the decreased susceptibility of the host to trichlorfon [19]. Moreover, the gut bacteria isolated from the resistant strains of Spodoptera frugiperda possessed pesticide-degrading capacity, such as cyhalothrin, deltamethrin, chlorpyrifos-ethyl, spinosad, and lufenuron, which could be degraded efficiently [35].

In our study, we hypothesized that gut symbionts of A. gossypii could increase its resistance to imidacloprid by degrading such compounds. 16S rDNA sequencing was performed to evaluate the differential abundance of the gut symbionts at the genus level between the imidacloprid-resistant (IMI-R) and imidacloprid-susceptible (IMI-S) strains of A. gossypii. We then isolated and cultivated the gut symbiont Sphingomonas to examine its ability to degrade imidacloprid. Both deprivation or supplementation of Sphingomonas was conducted in the IMI-R and IMI-S strains, and then the susceptibility of A. gossypii to imidacloprid was analyzed by the leaf-dipping method. The prevalence of infection with Sphingomonas in nine field populations was detected by quantitative real-time PCR (qPCR). The changes in the susceptibility of field populations to imidacloprid were further investigated after antibiotic treatment. Finally, the metabolic capacity and metabolic pathway of imidacloprid by Sphingomonas were determined by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC–MS).

Results

The higher relative abundance of Sphingomonas in the imidacloprid-resistant strain

The toxicity of imidacloprid to IMI-S and IMI-R strains of A. gossypii was determined (Table 1). Compared to the IMI-S strain with an LC50 value of only 3.14 mg/L, the IMI-R strain developed high resistance to imidacloprid with an LC50 value greater than 5000 mg/L, and the resistance ratio was more than 1592.

Table 1 Toxicity of imidacloprid to IMI-S and IMI-R strains of Aphis gossypii after treatment with ampicillin or Sphingomonas for seven days

To identify which gut symbiotic bacteria in A. gossypii might contribute to imidacloprid metabolism, we analyzed the bacterial composition of gut communities in both IMI-S and IMI-R strains by sequencing the V4 variable region of 16S rDNA with high-throughput amplicon sequencing (GenBank accession No. PRJNA929464). As shown in Additional file 1: Table S1, the reads generated from the samples of IMI-R and IMI-S strains were all more than 80,000. The value of Good’s coverage from all the samples was closer to 1, indicating that the sequencing depth basically covered all species in the samples (Table S1). Rarefaction curves were generated to avoid biases in the downstream analyses, which directly reflected that the amount of sequencing data of all the samples was rational (Additional file 2: Fig. S1).

At the genus level, the relative abundance was evaluated for all observed genera. The gut microbiota of both IMI-S and IMI-R strains was mainly composed of Buchnera, Faecalibacterium, Arsenophonus, and Sphingomonas (Fig. 1A). The relative abundance of Sphingomonas was significantly higher in the IMI-R strain than in the IMI-S strain (Fig. 1B). Principal coordinate analysis (PCoA) showed that the IMI-S and IMI-R strains were clustered together, indicating that the structure and composition of gut symbionts in the IMI-R strain were not significantly different from those of the IMI-S strain (Fig. 1C). Shannon index analysis demonstrated that there was no significant difference in the diversity of the gut symbionts between the two strains (Fig. 1D). The Chao1 index (reflecting species richness of bacteria) in the IMI-R strain was also similar to that in the IMI-S strains (Fig. 1E).

Fig. 1
figure 1

Different symbiotic bacterial communities in the Aphis gossypii guts of IMI-S and IMI-R strains. A Relative abundances of the top 10 genera in the guts of the IMI-S and IMI-R strains. B The relative abundance of Sphingomonas in the guts of the IMI-S and IMI-R strains. Asterisks indicate significant differences between strains followed by t test, n = 5; “*”, P < 0.05. C The PCoA plot determined by the structure and composition of gut symbionts in the IMI-S and IMI-R strains based on weighted UniFrac analysis. Alpha diversity indices of species for the gut symbiont in the IMI-S and IMI-R strains, Shannon index (D) and Chao 1 index (E) based on the Wilcoxon test, n = 5

Sphingomonas isolation and identification

Guts of A. gossypii were dissected on sterilized ice-cold slides and plated on 2xYT agar plates to isolate Sphingomonas. According to the 16S rDNA sequencing results, bacteria were identified as Sphingomonas (Fig. 2A). Based on a BLAST search against GenBank, the 16S rDNA sequence exhibited 99% identity with that of Sphingomonas (Fig. 2B). The 16S rDNA fragment of the bacteria was 1314 bp, and the sequence was deposited in GenBank (accession number: OQ359156).

Fig. 2
figure 2

Isolation, identification, and location of Sphingomonas. A The colony characteristics of Sphingomonas on 2xYT agar plates. B Phylogenetic relationships of the symbiotic Sphingomonas strain. The red star indicates the Sphingomonas strain isolated from the gut of the IMI-R strain. C The colony characteristics of Sphingomonas on mineral media. D, E The gut organization of A. gossypii and localization of Sphingomonas in the gut of the IMI-R strain. Red signals indicate Sphingomonas symbionts, whereas blue signals show host insect nuclei

Imidacloprid-degrading bacteria of Sphingomonas in the gut of Aphis gossypi i

Carbon and nitrogen sources are indispensable to bacterial subsistence. Sphingomonas could subsist on the mineral media, which used imidacloprid as a sole carbon and nitrogen source (Fig. 2C). According to Fig.S2, the OD600 value of Sphingomonas was significantly increased after being cultivated in the liquid MM with IMI as the only carbon source and nitrogen source for 2 days. but no significant change of OD600 value was observed for Sphingomonas cultivated in liquid MM without IMI as the only carbon source and nitrogen source during the 3-day experiment. The above results indicated that carbon and nitrogen from imidacloprid were utilized by Sphingomonas to subsist.

Localization of Sphingomonas symbionts in guts

Fluorescence in situ hybridization (FISH) targeting 16S rDNA of Sphingomonas symbionts was performed to identify its location in the gut of A. gossypii. The fluorescent signal of Sphingomonas was consistently localized in the foregut of A. gossypii (Fig. 2D, E).

Deprivation of Sphingomonas increased A. gossypii susceptibility to imidacloprid

We examined whether the deprivation or supplementation of Sphingomonas affects the susceptibility of IMI-R and IMI-S strains to imidacloprid. Thus, 16S rDNA gene sequencing and toxicity bioassays were performed on both strains after treatment with antibiotics or Sphingomonas for seven days (Fig. 3A). The susceptibility of Sphingomonas to antibiotics was tested by comparison of the inhibiting zone. The results demonstrated that Sphingomonas is highly sensitive to ampicillin (Additional file 3: Fig. S2). Therefore, ampicillin was used to remove Sphingomonas from the guts of the IMI-R and IMI-S strains. According to the sequencing result of 16S rDNA (accession number PRJNA929464), the reads generated from the samples of IMI-R and IMI-S strains after being treated with ampicillin or Sphingomonas for 7 days were all more than 60,000 (Table S1). The Good’s coverage values suggested that the sequencing was sufficiently deep to cover all species in the samples (Table S1). Rarefaction curves were generated, indicating that the amount of sequencing data was rational in the samples (Additional file 2: Fig. S1).

Fig. 3
figure 3

Deprivation and supplementation of Sphingomonas in the gut of the IMI-S and IMI-R strains and detection of Sphingomonas in nine field populations of Aphis gossypii collected in 2019. A Experimental design: apterous adult aphids of the IMI-S and IMI-R strains were treated with ampicillin for 7 days to deprive Sphingomonas or treated with Sphingomonas for 7 days for Sphingomonas supplementation. Afterward, the guts of apterous aphids from the IMI-S strain or IMI-R strain were dissected for DNA extraction and then for 16S rDNA sequencing. In addition, after treatment with ampicillin or Sphingomonas for 7 days, the changes in the susceptibility of the IMI-S and IMI-R strains to imidacloprid were determined by the leaf-dipping method. B Histogram showing the relative abundance of the 10 most dominant bacterial genera of the gut symbiont in the IMI-R and IMI-S strains. The abundance of Sphingomonas in the gut of the IMI-R and IMI-S strains (C, D). The different lowercase letters (a, b, c) on the bars indicate significant differences according to one-way ANOVA (n = 5, P < 0.05, Tukey’s test). The analysis of alpha diversity indices of species for the gut symbiont in the IMI-S and IMI-R strains, Chao 1 index (E, F) and Shannon index (G, H) based on the Wilcoxon test, n = 5

The top 10 bacterial genera in Fig. 3B revealed that the shifted structure and composition of gut symbionts were observed in the IMI-R strain or IMI-S strain after being treated with ampicillin or Sphingomonas for 7 days. It is true that the abundance of Sphingomonas from the gut of the IMI-R strain was significantly reduced after treatment with ampicillin for 7 days compared with the control of the IMI-R strain, which was treated with sterile water only (Fig. 3C). After the supplementation of Sphingomonas for 7 days in the IMI-R and IMI-S strains, the abundance of Sphingomonas from the gut of the IMI-S strain was significantly increased compared with the control treated with sterile water only (Fig. 3D). Measurement of within-sample diversity (α-diversity) by Chao 1 and Shannon index revealed no significant difference in the IMI-R strain or IMI-S strain after being treated with ampicillin or Sphingomonas for 7 days (Fig. 3E–H).

Although the LC50 value of the IMI-S strain decreased from 3.14 to 1.85 mg/L after treatment with ampicillin for 7 days, the overlapping 95% confidence limits indicated that no significant difference existed between the two LC50 values (Table 1). The abundance of Sphingomonas in the gut of the IMI-S strain was lower, and it could not be further reduced by antibiotic exposure. Thus, no significant increase in susceptibility was observed in the IMI-S strain. The LC50 value of the IMI-R strain decreased from > 5000 to 1656.71 mg/L after ampicillin treatment for 7 days, and the resistance ratio decreased from > 1592 to 528, indicating that the significantly reduced abundance of Sphingomonas in the gut could increase the susceptibility of the IMI-R strain to imidacloprid (Table 1).

Supplementation with Sphingomonas decreased A. gossypii susceptibility to imidacloprid

The LC50 value of the IMI-S strain increased significantly from 3.14 to 19.99 mg/L after supplementation with Sphingomonas for 7 days (Table 1). Moreover, the supplementation of Sphingomonas increased the resistance of the IMI-S strain to 6.37-fold, indicating that the gut possessed a higher abundance of Sphingomonas, dramatically decreasing the susceptibility of A. gossypii to imidacloprid. There was no obviously increased abundance of Sphingomonas observed in the gut of the IMI-R strain after supplementation. As expected, the LC50 value and the resistance ratio for the IMI-R strain showed little change. (Table 1).

Detection and deprivation of Sphingomonas symbionts in the field populations

The results showed that the nine field populations from various provinces in China were all infected with Sphingomonas (Additional file 4: Fig. S3). After these field populations were treated with ampicillin for 7 days for the deprivation of Sphingomonas, their susceptibilities to imidacloprid were all decreased to different degrees (Table 2). Among these nine field populations, the SXYC population possessed the highest LC50 value of 19,830 mg/L with a resistance ratio of 6315 but dramatically decreased to 844 mg/L (RR = 269) after treatment with ampicillin for 7 days. The resistance ratio of the SXYC population showed the greatest decrease compared to other field populations treated with antibiotics. In addition to the SXYC population, the resistance ratios of the XJWS and SDBZ populations also showed a greater decrease after exposure to ampicillin for 7 days, and their resistance ratios decreased from 55 to 18 and 134 to 37, respectively. However, no obvious decrease in the resistance ratio was observed in the remaining six populations after ampicillin treatment. 16S rDNA sequencing was also performed to determine the abundance of Sphingomonas in the gut of SXYC, SDBZ, XJSW, and HBHS field populations (accession No. PRJNA929464). The abundance of Sphingomonas in the gut of these four populations was also compared with that of the IMI-S and IMI-R strains. As shown in Additional file 5: Fig. S4, the highest abundance of Sphingomonas was observed in the IMI-R strain and SXYC field population, and the lowest was found in the gut of the IMI-S strain, XJSW, and HBHS field populations. Furthermore, the abundance of Sphingomonas in the gut of SXYC and SDBZ populations was significantly higher than that in the IMI-S strain.

Table 2 Toxicity of imidacloprid to field populations of Aphis gossypii after treatment with ampicillin for 7 days

Metabolism of imidacloprid by Sphingomonas

LC/MS was performed to analyze the metabolic pathway of imidacloprid by Sphingomonas. Figure 4A–E shows the molecular ion peaks of imidacloprid (IMI), 5-OH IMI, nitroso IMI, guanidine IMI, and urea IMI. The mass charge ratio (MCR) of ion peak A was 256 and was determined to be IMI. The MCR of ion peak B was 272 with one more oxygen atom than A, indicating that B was 5-OH IMI. The MCR of ion peak C was 240 and was one mass of oxygen atom less than that of ion peak A, indicating that it was nitroso IMI. The MCR of ion peak D was 211, which was two oxygen atoms and one nitrogen atom less than that of ion peak A and one hydrogen atom more than that of ion peak A, indicating that ion peak D was guanidine IMI. The MCR of ion peak E was 212, which was one mass of nitrogen atom and one mass hydrogen atom less than that of ion peak D but one more oxygen atom than that of ion peak D, indicating that ion peak E was urea IMI. Thus, the metabolic pathway of imidacloprid by Sphingomonas was determined and is shown in Fig. 4H.

Fig. 4
figure 4

Degradation of imidacloprid by Sphingomonas. AE HPLC–MS chromatograms and probable structures of metabolites of imidacloprid after incubation with Sphingomonas for 8 days. F, G Changes in imidacloprid and its metabolite concentrations during the 8-day incubation with Sphingomonas. The initial concentrations of imidacloprid were 25 ppm (F) and 50 ppm (G). H Proposed pathways of the biodegradation of imidacloprid by Sphingomonas

The standard curves of IMI, 5-OH IMI, and urea IMI are shown in Additional file 6: Fig. S5. The metabolic ability of Sphingomonas to imidacloprid was examined by HPLC, and samples were collected on days 2, 4, 6, and 8 to analyze the concentration of imidacloprid and its metabolites. The concentration of IMI decreased to a minimum, and the concentrations of 5-OH IMI and urea IMI increased to a maximum on day 8. Specifically, the concentration of IMI was decreased to 11 mg/L and 28 mg/L on day 8, achieving maximum metabolic efficiencies of 44% and 56%, respectively, when the initial IMI concentration was 25 mg/L and 50 mg/L (Fig. 4F, G). In the control group, however, no significant degradation of IMI was observed, regardless of whether the initial concentration of IMI was 25 mg/L or 50 mg/L (Fig. 4G).

Discussion

Symbiont-mediated detoxification of pesticides has attracted increasing attention. Generally, toxin-degrading symbiotic bacteria defend hosts against pesticides in the environment [33]. In this study, the genus Sphingomonas isolated from the gut of the IMI-R strain can utilize imidacloprid as a sole carbon source and nitrogen source. During degradation, the Sphingomonas symbiont was identified to metabolize imidacloprid via hydroxylation and nitro-reduction pathways with high efficiency. This further provided evidence that Sphingomonas could enhance A. gossypii resistance to imidacloprid.

Notably, obligate symbionts usually provide key nutrients to compensate for their host’s unbalanced diets [32]. For example, Buchnera could provide aphids with the essential amino acids and vitamins missing in plant phloem sap [36], and Wigglesworthia could synthesize vitamin B to contribute to tsetse host fitness [37]. From Fig. 1A, the highest abundance of gut symbionts was Buchnera, and it was the obligate symbiont in A. gossypii. The main role of facultative mutualists is to confer fitness benefits upon hosts, which extend the lifespan and stimulate the fecundity of their carriers [38]. In addition, facultative symbionts can protect their host against parasitoids [39], pathogens [40], and pesticides [41]. Here, we demonstrated that the gut symbiont Sphingomonas plays a key role in the degradation of imidacloprid. Thus, it was supposed to act as a facultative symbiont in A. gossypii.

Sphingomonas was identified as a new genus in 1990 due to its outer membrane component of sphingosine [42]. In 2001, Sphingomonas was divided into four new genera, Sphingomonas, Sphingobium, Novosphingobium, and Sphingopyxis [43]. Sphingomonas is characterized by its degradation of various organic pollutants, such as biphenyl, naphthalene, phenanthrene, dioxin compounds, carbazole, chlorophenol, and various pesticides [44]. It has been reported that Sphingomonas sp. DC-6, isolated from soil, can hydrolyze the organophosphorus pesticide dimethoate into dimethoate carboxylic acid and methylamine [45]. Sphingomonas sp. CDS-1 could degrade carbamate insecticide carbofuran into carbofuran phenol in soil [46]. Symbiont Sphingomonas sp. HJY from chives, in which chlorpyrifos was applied for a long time, was reported to effectively degrade chlorpyrifos [47]. Thus, Sphingomonas in imidacloprid-resistant A. gossypii may have the potential to degrade imidacloprid.

The results of 16S rDNA sequencing demonstrated that the richness and diversity of the gut community showed no significant difference between the IMI-R and IMI-S strains of Aphis gossypii. However, the abundance of Sphingomonas in the IMI-R strain was significantly higher than that in the IMI-S strain. Indeed, the titer of individual symbiont species could be regulated by insects to respond to pesticide pressures [48]. Furthermore, we found that imidacloprid could be used as a carbon and nitrogen source by Sphingomonas (Fig. 2C, Additional file 7: Fig. S6), indicating the potential metabolic capacity of Sphingomonas to imidacloprid. Then, the ability of Sphingomonas to metabolize imidacloprid was analyzed by HPLC, and high metabolic efficiency (56%) of imidacloprid was observed. Both decreased imidacloprid concentrations and increased concentrations of its two metabolites (5-OH IMI and urea IMI) were also recorded, which provided solid evidence for the involvement of Sphingomonas in imidacloprid resistance. It has been reported that the resistant strain of R. pedestris possessed a higher abundance of the gut symbiont Burkholderia, which could degrade fenitrothion with high efficiency, and susceptible R. pedestris developed resistance to fenitrothion via the acquisition of Burkholderia [18]. Supplementation of Citrobacter from the gut of B. dorsalis, which was proven to degrade trichlorfon with high efficiency, could result in decreased susceptibility of B. dorsalis to trichlorfon [19]. In our study, after supplementation of Sphingomonas, the abundance of Sphingomonas in the gut of IMI-S strain significantly increased, and the susceptibility to imidacloprid was significantly decreased as expected. These results suggested that the acquisition of insecticides degrading symbionts could increase insecticide resistance in insects.

However, supplementation with Sphingomonas had no effect on the abundance of Sphingomonas in the gut of the IMI-R strain and its susceptibility to imidacloprid. The possible reason is that the link between detoxifying symbionts infection and insecticide resistance could be a product of physiological tradeoffs [49]. That is, the increase of Sphingomonas abundance may play a role in insecticide resistance to some extent, but the abundance will not increase unlimitedly, which may impair the physiological function of the gut. Our results demonstrated that though SXYC population showed almost four times higher resistance to IMI than the IMI-R strain, the Sphingomonas abundance is similar in the guts of both SXYC and IMI-R, which also supported our hypothesis that the abundance of Sphingomonas in the gut of A. gossypii will not continuous increase because of the physiological tradeoffs.

The resistance level of pests to insecticides could be inhibited by antibiotic treatment [50, 51]. Moreover, pests become more susceptibility to toxins after deprivation of detoxifying microorganisms from the gut [19, 52]. Here, we demonstrated that the susceptibility of the IMI-R strain to imidacloprid was significantly increased after suppressing the abundance of Sphingomonas in the gut by antibiotic treatment. Besides, Sphingomonas infection was prevalent in the field populations of A. gossypii, of which the SXYC, XJWS, and SDBZ populations showed a decreased susceptibility to imidacloprid after antibiotic treatment. However, treatment with ampicillin did not increase the susceptibility to imidacloprid in the remaining six populations. The possible reason is that these populations possess a lower abundance of Sphingomonas, and its contribution to imidacloprid resistance is negligible. The Sphingomonas abundance data shown in Additional file 5: Fig. S4 partly supported our speculation, that is, among all four tested field populations, the SXYC and SDBZ populations had higher Sphingomonas abundance and ampicillin treatment could increase their susceptibility, which is similar to that in the IMI-R strain, while the abundance of Sphingomonas in the XJSW and HBHS populations was as low as that in the IMI-S strain, and ampicillin treatment did not affect their susceptibility to imidacloprid. In addition, the origin of Sphingomonas in the A. gossypii gut may be acquired from the soil because both Burkholderia and Sphingomonas could be detected in the soil of cotton fields [53], and the gut symbiont Burkholderia in a stinkbug has been proven to be acquired by nymphs from the environment every generation [54].

The metabolites of imidacloprid by Sphingomonas were further determined by HPLC–MS. The main transformation of IMI occurred via the hydroxylation and nitro-reduction pathways. In the hydroxylation pathway, IMI is metabolized to 5-hydroxy IMI by hydroxylase, and 5-hydroxy IMI is further dehydrated to olefin IMI by dehydratase [55]. Olefin IMI is prone to degradation and eventually decomposes into CO2 due to its unsaturated double bonds [56]. In the nitro-reduction pathway, the metabolites of IMI were nitroso IMI, guanidine IMI, and urea IMI under the action of aldehyde oxidase (AOX) [57]. Cytochrome P450s (P450s) and AOX are known to be involved in IMI hydroxylase and nitroreductase, respectively [58]. The metabolites of IMI by Sphingomonas were 5-OH IMI, nitroso IMI, guanidine IMI, and urea IMI. We confirmed that IMI was metabolized by Sphingomonas via nitroreduction and/or hydroxylation pathways. Metabolite toxicity studies have revealed that the guanidine IMI and urea IMI products formed via the nitro-reduction pathway did not possess any insecticidal activity [59]. It can be concluded that IMI metabolism by Sphingomonas via the nitro-reduction pathway was a detoxification process. The results further revealed the mechanisms by which Sphingomonas enhances the resistance of A. gossypii to imidacloprid. A previous study showed that IMI was metabolized mainly through the hydroxylation pathway in mammals, in which IMI was metabolized to 5-hydroxy IMI and 4,5-dihydroxy IMI by P450s and then converted to olefin IMI after dehydration [60]. Another common metabolic pathway of IMI in mammals is the oxidative cleavage of the methylene group between the pyridine ring and the imidazole ring to generate 6-chloropicolinol, which is further oxidized to 6-chloronicotinic acid [61]. In soil, IMI is metabolized by microbes through the hydroxylation pathway and nitro reduction pathway [62]. Olefin and urea metabolites of IMI were detected in soil samples collected from West Bengal, India [63], and olefin IMI and guanidine IMI were detected in soil collected from Nanjing, China [64]. Our findings showed that the metabolic pathway of IMI by Sphingomonas was the same as that of IMI by soil microbes.

Conclusion

It has been reported that the enhanced detoxifying enzyme activity and target mutations play important roles in imidacloprid resistance in insect pests. Here, we further demonstrated that as a ubiquitous genus in the gut of A. gossypii, Sphingomonas could enhance the resistance of A. gossypii by metabolizing IMI directly. The high metabolic efficiency of Sphingomonas to IMI indicated that it may play an important role in defending A. gossypii against imidacloprid. These findings enriched our knowledge of mechanisms of insecticide resistance and provided new symbiont-based strategies for control of insecticide-resistant insect pests with high Sphingomonas abundance.

Methods

Aphis gossypii strains

The cotton aphid Aphis gossypii Glover was used in this study. The aphid population collected in 2008 from cotton fields in Changchun City of Jilin Province, China, was susceptible to imidacloprid, which was named the imidacloprid-susceptible (IMI-S) strain and maintained in our laboratory without exposure to any insecticides since collection [7]. The imidacloprid-resistant (IMI-R) strain was selected from a field population originally collected from Yuncheng of Shanxi Province by 1000 mg/L imidacloprid for ten generations using the leaf-dipping method. For the leaf-dipping method, approximately 5000 apterous adults were carefully transferred onto cotton leaves and then dipped in 1000 mg/L imidacloprid. In addition, nine field populations of A. gossypii collected in August 2020 were also used in this study, including Shanxi Yuncheng (SXYC), Xinjiang Aksu (XJAKS), Shandong Dongying (SDDY), Hebei Hengshui (HBHS), Xinjiang Shawan (XJSW), Xinjiang Wusu (XJWS), Hubei Jingzhou (HBJZ), Shandong Jinan (SDJN), and Shandong Binzhou (SDBZ) populations. Their collection information is listed in Additional file 1: Table S1. All A. gossypii strains and populations were reared on cotton seedlings, Gossypium hirsutum (L.), under controlled conditions of 20–23 °C, 60% relative humidity, and a photoperiod of 16:8 h (light:dark).

Toxicity bioassays

The toxicity of imidacloprid to A. gossypii was determined using a leaf-dipping method [65] with slight modifications [66]. Stocks of imidacloprid (95.3%) were obtained from DuPont (USA), prepared in acetone and adjusted to final concentrations by serial dilution with distilled water containing 0.05% (v/v) Triton X-100 for the bioassays. The 20-mm diameter cotton leaf discs were dipped in the desired concentration of insecticide or in 0.05% (v/v) Triton X-100 water for 15 s as a control. The treated leaf discs were allowed to air dry and then placed upside down onto the agar beds in 12-well cell culture plates. Apterous adult aphids were carefully transferred onto the discs and covered with Chinese art paper to prevent escape. Bioassays were maintained in the laboratory at 20–23 °C with a photoperiod of 16:8 h (light: dark). The treatment for each concentration was performed with three replicates, and at least 30 aphids were used for each replicate. Mortality was assessed at 48 h after treatment. The LC50 values were calculated by probit analysis using POLO Plus 2.0 statistical software (LeOra Software Inc., Berkeley, CA).

Extraction of DNA from guts of the IMI-S and IMI-R strains and field populations

The DNA of the gut was extracted from IMI-S and IMI-R strains and field populations following previous research [19]. Apterous adults were selected and soaked in 75% ethanol for 90 s to remove surface bacteria. The entire guts were dissected from soaked aphids under a stereomicroscope and were directly transferred into centrifuge tubes containing DNA extraction buffer. For each sample, 90 aphids were dissected, and five independent biological replicates were conducted. Gut DNA was extracted using a DNA extraction kit (Qingke, Beijing, China) following the manufacturer’s instructions, and the DNA was used for gut symbiont high-throughput sequencing analysis.

V4 amplicon sequencing of the 16S rDNA genes and data analysis

The bacteria were profiled by sequencing the V4 region of the 16S rDNA gene. Primers 505F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACN NGGGTATCTAA-3′) were employed for the PCR. All PCRs were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs), and 400–450 bp were chosen for further experiments. Sequencing libraries were generated using TruSeq DNA PCR-Free Sample Preparation Kits (Illumina, USA) following the manufacturer’s recommendations. Index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina HiSeq 2500 platform, and 250-bp paired-end reads were generated.

Paired-end reads were merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/), and the splicing sequences were called raw tags. After quality filtering of the raw tags, the effective tags were obtained. For species annotation, sequences with 97% similarity were assigned to the same OTUs, and the representative sequence for each OTU was screened for further annotation. For each representative sequence, the GreenGene Database (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi) was used based on the RDP classifier (Version 2.2, http://sourceforge.net/projects/rdp-classifier/) algorithm to annotate taxonomic information.

Sphingomonas isolation and identification

Apterous adult aphids were collected and soaked in 75% ethanol for 90 s, followed by three rinses with sterile water to remove surface bacteria. The guts of A. gossypii were dissected and homogenized with 200 μL of sterile water. After five repetitions of tenfold gradient dilutions, all six samples were plated on 2xYT agar plates (0.5% peptone, 0.3% yeast extract, 0.5% NaCl, 1.5% agar, pH 6.8) and cultivated at 27 °C for 48 h. Colonies with distinct morphology were selected for purification on 2xYT medium at least three times and then used for subculturing.

Isolated colonies were transferred to centrifuge tubes containing 5 ml of 2xYT liquid medium, shaken, and incubated at 27 °C for 48 h for DNA extraction. Members of the Sphingomonas genus are gram-negative [67]. The bacterial DNA was extracted with Ezup Column Bacteria Genomic DNA Purification Kits (Shenggong, Shanghai, China) following the protocol for gram-negative bacteria. The DNA was used for the amplification of the complete 16S rDNA genes, and primers 27F (5′-GTTTG ATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were employed. PCR amplification was performed using 2xTaq PCR MasterMix (Aidlab, Beijing China), with an initial denaturation at 95 °C for 4 min followed by 30 cycles of 30 s at 95 °C, 30 s at 57 °C, 2 min at 72 °C, and a final extension at 72 °C for 10 min. The sequences of 16S rDNA were subjected to a BLAST search against the NCBI database for sequence homology analysis and bacterial species identification. Liquid cultures of Sphingomonas were stored in 25% glycerol solution at − 80 °C.

Fluorescence in situ hybridization

The location of Sphingomonas in the gut of A. gossypii was determined by fluorescence in situ hybridization (FISH). Sphingomonas symbionts were visualized with a probe SPH120 targeting a specific region of the 16S rDNA (5′-GGGCAGATTCCCACGCGT-3′) [68]. Oligonucleotides were labeled with the fluorophore Texas red by the manufacturer (EXON Biological Technology, Guangzhou, China). Ten gut samples were collected directly into Carnoy’s solution (ethanol-chloroform–acetic acid, 6:3:1) for fixation. After fixation, the samples were bleached in a 6% H2O2 ethanol solution and then hybridized overnight at 42 °C in hybridization buffer (20 mM Tris–HCl pH 8.0, 0.9 M NaCl, 0.01% SDS, and 35% (wt/vol) formamide) containing 40 nM oligonucleotide probe; the samples were placed in a humid and dark chamber. To remove nonspecific probe binding, the samples were washed in washing buffer (20 mM Tris/HCl pH 7.5, 70 mM NaCl, 0.01% SDS, 5 mM EDTA) for 30 min at room temperature. In addition, 4′,6-diamidino-2-phenylindole (DAPI) was supplied to counter-stain eukaryotic and bacterial DNA for 10 min, which contained an anti-fluorescence quencher. Subsequently, the samples were observed under an inverted fluorescence microscope (Nikon, USA).

Identification of the imidacloprid-degrading function of Sphingomonas

To preliminarily identify the ability of Sphingomonas to degrade imidacloprid, isolated colonies were lined on mineral media (MM) (9.5 mM KH2PO4, 4.8 mM MgSO4, 0.1 mM CaCl2, 0.8 mM Na2HPO4, and 20 g/L bacto agar), and 5 mM imidacloprid was added and used as the sole carbon and nitrogen source. The growth of bacteria was monitored daily to confirm whether Sphingomonas could subsist on the mineral media. In addition, the changes in OD value of Sphingomonas cultivated in the liquid mineral media (MM) (9.5 mM KH2PO4, 4.8 mM MgSO4, 0.1 mM CaCl2, 0.8 mM Na2HPO4) with IMI as the only carbon source and nitrogen source was observed, and the liquid MM without carbon and nitrogen source was used as the control. The inoculum of Sphingomonas was centrifuged at 8000 rpm and then suspended into 50-mL liquid MM with 25 ppm IMI as the only carbon source and adjusted the initial OD600 0.8. The inoculated media were incubated at 28 °C, 180 rpm for 3 days. Take out 1-mL cultures every 24 h and use ultraviolet spectrophotometer (Spectra Max; Molecular Devices) to measure the OD value of Sphingomonas at 600 nm. The MM (50 mL) without carbon source was used as the control. Three replicates were prepared for each treatment.

Antibiotic sensitivity testing of Sphingomonas

The sensitivity of Sphingomonas to antibiotics was investigated using the method of inhibiting zone. Ten microliters of the inoculated Sphingomonas with an OD600 of 0.8 was mixed with 20 mL 2xYT agar plates, and 60 μl of antibiotics with concentrations of 0.5 mg/L, 1 mg/L, 2 mg/L, and 4 mg/L were added to a hole of 0.3-cm diameter in the center of the agar plate. After cultivation at 27 °C for 48 h, the diameter of the inhibiting zones was measured. The types of antibiotics are shown in Additional file 5: Fig. S4.

Antibiotic treatment and Sphingomonas inoculation

According to the results of antibiotic sensitivity testing, a 10 mg/ml ampicillin solution was selected to remove Sphingomonas. Here, 100 apterous adult aphids were carefully transferred onto 80-mm diameter cotton leaf discs and then treated with 100 μl ampicillin by nanospraying for 7 days, and A. gossypii in the control group were treated with ampicillin-free water. Each antibiotic or ampicillin-free water treatment was performed with ten replicates. Then, the guts of apterous adult aphids from both the IMI-S and IMI-R strains were dissected for 16S rDNA sequencing. The changes in toxicity of imidacloprid to IMI-S and IMI-R strains after being treated with antibiotics were examined using the leaf-dipping method, and the biometric data were analyzed to determine the changes in imidacloprid susceptibility compared to the control.

For the Sphingomonas inoculation experiments, isolated Sphingomonas were propagated in 2xYT liquid medium at 27 °C and 200 rpm until an OD600 of 0.8 was reached. One hundred apterous adult aphids were carefully transferred onto 80-mm diameter cotton leaf discs and then treated with 100 μl Sphingomonas inoculum by nanospraying for 7 days, and A. gossypii in the control group were treated with sterilized water. Each Sphingomonas or sterilized water treatment was performed with ten replicates. The guts of apterous adult aphids were dissected for 16S rDNA sequencing. After being treated with Sphingomonas for 7 days, the susceptibility of the IMI-S and IMI-R strains to imidacloprid was determined by the leaf-dipping method.

Antibiotic treatment in field populations

Nine field populations from different provinces of China were collected in 2019, and the collection information is listed in Additional file 8: Table S2. The infection of Sphingomonas was detected by the cycle threshold (Ct) value for each population according to the result of amplification of the 16S rDNA gene by quantitative real-time PCR (qPCR). For qPCR detection, we isolated genomic DNA from the nine populations of apterous adults using a DNA extraction kit (Qingke, Beijing, China), and qPCR was performed on an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara Biotechnology, Dalian, China). The qPCRs were prepared as follows: 1 μg of DNA as template, 10 μL of SYBR Green mix, 0.4 μL of ROX Reference Dye II, 0.4 μL of each primer and DEPC-treated water to a final volume of 20 μl. The specific primers of the 16S rDNA gene to amplify Sphingomonas were SP-190 (5′-CGGACCAAAGATTTATCG-3′) and SP-853 (5′-CCAATCACCAAGTGACCCGGA-3′) [67]. The qPCR conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. After amplification, one dissociation step cycle of 95 °C for 15 s, 60 °C for 1 min and 95 °C for 30 s, and 60 °C for 15 s was performed to ensure the specificity of the amplified product. The experiment was conducted with three technical replications and three independent biological replicates.

Nine field populations from different provinces of China in 2019 were treated with ampicillin to examine the changes in susceptibility to imidacloprid. A 10 mg/ml ampicillin solution was sprayed onto the infected populations evenly for 7 days. Afterward, the leaf-dipping method was performed to determine the changes in susceptibility to imidacloprid in these nine field populations of A. gossypii.

Degradation of imidacloprid by Sphingomonas

Degradation tests were performed using imidacloprid as a sole source of carbon in Erlenmeyer flasks containing 50 mL liquid MM supplemented with 25 mg/L or 50 mg/L imidacloprid. To measure the degradation ability of Sphingomonas, 10-mL inoculum with an OD600 of 0.8 was centrifuged at 8000 rpm, and then the collected bacteria were added to MM. The MM without Sphingomonas was used as the control. Three replicates were prepared for each treatment. The flasks were incubated at 28 °C and 180 rpm for 8 days. Every 2 days, 1 mL of the cultures was filtered through a 45-μm bacterial membrane filter to detect the concentration of imidacloprid and its metabolites. The concentration of imidacloprid and its metabolites was determined at 269 nm by HPLC (1260 series; Agilent) on a Zorbax SB-Aq C18 column (5 μm, 4.6 mm 250 mm; Agilent). The mobile phase consisted of a mixture of 75% ultrapure water: 25% acetonitrile at a flow rate of 1.0 mL/min; the injection volume was 10 μL. To determine the degradation ability of Sphingomonas, the concentrations of imidacloprid and its metabolites were calculated by standard curves of imidacloprid, urea-imidacloprid, and 5-OH imidacloprid. Imidacloprid (DuPont, USA), urea imidacloprid (Standards, Shanghai, China), and 5-OH imidacloprid (Xiyuan, Shanghai, China) were prepared in acetonitrile and adjusted to final concentrations by serial dilution with acetonitrile. The standard curve was obtained according to the peak area corresponding to the concentration of compounds.

The metabolic pathways of imidacloprid by Sphingomonas were determined by an Agilent 1260/6520 liquid chromatography-mass spectrometer. MS analysis was performed in electrospray ionization (ESI) mode with an Agilent 1290 LC–MSD to analyze and identify imidacloprid and its metabolites. LC conditions: the chromatographic column and mobile phase were the same as those of HPLC; otherwise, the flow rate was 0.6 mL/min. A diode array detector (DAD) was utilized. In the MS analysis, the metabolites were separated and ionized by electrospray ionization to obtain a positive polarity. Characteristic fragment ions were identified by second-order MS and compared to those generated with authentic or structural analog standards.

Availability of data and materials

16 s amplicon sequencing data are available from NCBI with accession No. PRJNA929464 [69]. The 16S rDNA fragment of Sphingomonas was deposited in GenBank (accession No. OQ359156)[70].

References

  1. Bass C, Denholm I, Williamson MS, Nauen R. The global status of insect resistance to neonicotinoid insecticides. Pestic Biochem Physiol. 2015;121:78–87.

    Article  CAS  PubMed  Google Scholar 

  2. Elbert A, Haas M, Springer B, Thielert W, Nauen R. Applied aspects of neonicotinoid uses in crop protection. Pest Manage Sci. 2008;64(11):1099–105.

    Article  CAS  Google Scholar 

  3. Jeschke P, Nauen R, Schindler M, Elbert A. Overview of the status and global strategy for neonicotinoids. J Agric Food Chem. 2011;59(7):2897–908.

    Article  CAS  PubMed  Google Scholar 

  4. Cahill M, Gorman K, Day S, Denholm I, Elbert A, Nauen R. Baseline determination and detection of resistance to imidacloprid in Bemisia tabaci (Homoptera: Aleyrodidae). Bull Entomol Res. 1996;86(4):343–9.

    Article  CAS  Google Scholar 

  5. Wang KY, Guo QL, Xia XM, Wang HY, Liu TX. Resistance of Aphis gossypii (Homoptera: Aphididae) to selected insecticides on cotton from five cotton production regions in Shandong, China. J Pestic Sci. 2007;32(4):372–8.

  6. Bass C, Carvalho R, Oliphant L, Puinean A, Field L, Nauen R, Williamson M, Moores G. and Gorman K. Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens. Insect Mol Biol. 2021;20:763–73.

  7. Chen XW, Li F, Chen A, Ma KS, Liang PZ, Liu Y, Song DL, Gao XW. Both point mutations and low expression levels of the nicotinic acetylcholine receptor β1 subunit are associated with imidacloprid resistance in an Aphis gossypii (Glover) population from a Bt cotton field in China. Pestic Biochem Physiol. 2017;141:1–8.

    Article  CAS  PubMed  Google Scholar 

  8. Blackman R, Eastop VF. Aphids on the world’s crops. An identification guide. John Wiley and Sons; NY, 1984.

  9. Lu Y, Wu K, Jiang Y, Xia B, Li P, Feng H, Wyckhuys KA, Guo Y. Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science. 2010;328(5982):1151–4.

    Article  CAS  PubMed  Google Scholar 

  10. Lu Y, Wu K, Jiang Y, Guo Y, Desneux N. Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services. Nature. 2012;487(7407):362–5.

    Article  CAS  PubMed  Google Scholar 

  11. Riaz MA, Poupardin R, Reynaud S, Strode C, Ranson H, David J-P. Impact of glyphosate and benzo [a] pyrene on the tolerance of mosquito larvae to chemical insecticides. Role of detoxification genes in response to xenobiotics. Aquatic Toxicology. 2009;93(1):61–9.

    Article  CAS  PubMed  Google Scholar 

  12. Ilias A, Lagnel J, Kapantaidaki DE, Roditakis E, Tsigenopoulos CS, Vontas J, Tsagkarakou A. Transcription analysis of neonicotinoid resistance in Mediterranean (MED) populations of B. tabaci reveal novel cytochrome P450s, but no nAChR mutations associated with the phenotype. BMC Gen. 2015;16(1):1–23.

    Article  Google Scholar 

  13. Rauch N, Nauen R. Identification of biochemical markers linked to neonicotinoid cross resistance in Bemisia tabaci (Hemiptera: Aleyrodidae). Arch Insect Biochem Physiol. 2003;54(4):165–76.

    Article  CAS  PubMed  Google Scholar 

  14. Hirata K, Jouraku A, Kuwazaki S, Shimomura H, Iwasa T. Studies on Aphis gossypii cytochrome P450s CYP6CY22 and CYP6CY13 using an in vitro system. J Pestic Sci. 2017;42(3):1–7.

    Article  Google Scholar 

  15. Chen XW, Tang C, Ma KS, Xia J, Song DL, Gao XW. Overexpression of UDP-glycosyltransferase potentially involved in insecticide resistance in Aphis gossypii Glover collected from Bt cotton fields in China. Pest Manag Sci. 2020;76(4):1371–7.

    Article  CAS  PubMed  Google Scholar 

  16. Koo HN, An JJ, Park SE, Kim JI, Kim GH. Regional susceptibilities to 12 insecticides of melon and cotton aphid, Aphis gossypii (Hemiptera: Aphididae) and a point mutation associated with imidacloprid resistance. Crop Prot. 2014;55:91–7.

    Article  CAS  Google Scholar 

  17. Kim JI, Kwon M, Kim G-H, Kim SY, Lee SH. Two mutations in nAChR beta subunit is associated with imidacloprid resistance in the Aphis gossypii. J Asia-Pacific Entomol. 2015;18(2):291–6.

    Article  CAS  Google Scholar 

  18. Kikuchi Y, Hayatsu M, Hosokawa T, Nagayama A, Tago K, Fukatsu T. Symbiont-mediated insecticide resistance. Proc Natl Acad Sci USA. 2012;109(22):8618–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cheng D, Guo Z, Riegler M, Xi Z, Liang G, Xu Y. Gut symbiont enhances insecticide resistance in a significant pest, the oriental fruit fly Bactrocera dorsalis (Hendel). Microbiome. 2017;5(1):1–12.

    Article  Google Scholar 

  20. Lu TQ, Mao SY, Sun SL, Yang WL, Ge F, Dai YJ. Regulation of hydroxylation and nitroreduction pathways during metabolism of the neonicotinoid insecticide imidacloprid by Pseudomonas putida. J Agric Food Chem. 2016;64(24):4866–75.

    Article  CAS  PubMed  Google Scholar 

  21. Ma Y, Zhai S, Mao SY, Sun SL, Wang Y, Liu ZH, Dai YJ, Yuan S. Co-metabolic transformation of the neonicotinoid insecticide imidacloprid by the new soil isolate Pseudoxanthomonas indica CGMCC 6648. J Environ Sci Health B. 2014;49(9):661–70.

    Article  CAS  PubMed  Google Scholar 

  22. Gupta M, Mathur S, Sharma TK, Rana M, Gairola A, Navani NK, Pathania R. A study on metabolic prowess of Pseudomonas sp. RPT 52 to degrade imidacloprid, endosulfan and coragen. J Hazard Mater. 2016;301:250–8.

    Article  CAS  PubMed  Google Scholar 

  23. Yj D. Yuan S, Ge F, Chen T, Xu SC, Ni JP: Microbial hydroxylation of imidacloprid for the synthesis of highly insecticidal olefin imidacloprid. Appl Microbiol Biotechnol. 2006;71(6):927–34.

    Article  Google Scholar 

  24. Anhalt JC, Moorman TB, Koskinen WC. Biodegradation of imidacloprid by an isolated soil microorganism. J Environ Sci Health B. 2007;42(5):509–14.

    Article  CAS  PubMed  Google Scholar 

  25. Phugare SS, Kalyani DC, Gaikwad YB, Jadhav JP. Microbial degradation of imidacloprid and toxicological analysis of its biodegradation metabolites in silkworm (Bombyx mori). Chem Eng J. 2013;230:27–35.

    Article  CAS  Google Scholar 

  26. Sharma S, Singh B, Gupta V. Assessment of imidacloprid degradation by soil-isolated Bacillus alkalinitrilicus. Environ Monit Assess. 2014;186(11):7183–93.

    Article  CAS  PubMed  Google Scholar 

  27. Kandil MM, Trigo C, Koskinen WC, Sadowsky MJ. Isolation and characterization of a novel imidacloprid-degrading Mycobacterium sp. strain MK6 from an Egyptian soil. J Agri Food Chem. 2015;63(19):4721–7.

    Article  CAS  Google Scholar 

  28. Onder Erguven G, Demirci U. Statistical evaluation of the bioremediation performance of Ochrobactrum thiophenivorans and Sphingomonas melonis bacteria on Imidacloprid insecticide in artificial agricultural field. J Environ Health Sci Eng. 2020;18(2):395–402.

    Article  CAS  PubMed  Google Scholar 

  29. Nishiwaki H, Sato K, Nakagawa Y, Miyashita M, Miyagawa H. Metabolism of imidacloprid in houseflies. J Pesticide Sci. 2004;29(2):110–6.

    Article  CAS  Google Scholar 

  30. Lee JB, Byeon JH, Am Jang H, Kim JK, Yoo JW, Kikuchi Y, Lee BL. Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut. FEBS Lett. 2015;589(19):2784–90.

    Article  CAS  PubMed  Google Scholar 

  31. Itoh H, Tago K, Hayatsu M, Kikuchi Y. Detoxifying symbiosis: microbe-mediated detoxification of phytotoxins and pesticides in insects. Nat Prod Rep. 2018;35(5):434–54.

    Article  CAS  PubMed  Google Scholar 

  32. López-Madrigal S, Gil R. Et tu, brute? not even intracellular mutualistic symbionts escape horizontal gene transfer. Genes. 2017;8(10):247–55.

    Article  PubMed  PubMed Central  Google Scholar 

  33. van den Bosch TJ, Welte CU. Detoxifying symbionts in agriculturally important pest insects. Microb Biotechnol. 2017;10(3):531–40.

    Article  PubMed  Google Scholar 

  34. Ramya SL, Venkatesan T, Srinivasa Murthy K, Jalali SK, Verghese A. Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation. Braz J Microbiol. 2016;47(2):327–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Almeida LGd, Moraes LABd, Trigo JR, Omoto C, Consoli FL. The gut microbiota of insecticide-resistant insects houses insecticide-degrading bacteria: A potential source for biotechnological exploitation. Plos One. 2017;12(3):e0174754.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Shigenobu S, Wilson AC. Genomic revelations of a mutualism: the pea aphid and its obligate bacterial symbiont. Cell Mol Life Sci. 2011;68(8):1297–309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Snyder AK, Rio RV. “Wigglesworthia morsitans” folate (vitamin B9) biosynthesis contributes to tsetse host fitness. Appl Environ Microbiol. 2015;81(16):5375–86.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Moran NA, McCutcheon JP, Nakabachi A. Genomics and evolution of heritable bacterial symbionts. Annu Rev Genet. 2008;42:165–90.

    Article  CAS  PubMed  Google Scholar 

  39. Oliver KM, Russell JA, Moran NA, Hunter MS. Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proc Natl Acad Sci USA. 2003;100(4):1803–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Goettler W, Kaltenpoth M, Herzner G, Strohm E. Morphology and ultrastructure of a bacteria cultivation organ: the antennal glands of female European beewolves, Philanthus triangulum (Hymenoptera, Crabronidae). Arthropod Struct Dev. 2007;36(1):1–9.

    Article  PubMed  Google Scholar 

  41. Zhang Y, Cai T, Ren Z, Liu Y, Yuan M, Cai Y, Yu C, Shu R, He S, Li J. Decline in symbiont-dependent host detoxification metabolism contributes to increased insecticide susceptibility of insects under high temperature. ISME J. 2021;15(12):3693–703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Yabuuchi E, Yano I, Oyaizu H, Hashimoto Y, Ezaki T, Yamamoto H. Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb, nov., and two genospecies of the genus Sphingomonas. Microbiol Immunol. 1990;34(2):99–119.

    Article  CAS  PubMed  Google Scholar 

  43. Takeuchi M, Hamana K, Hiraishi A. Proposal of the genus Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol. 2001;51(4):1405–17.

    Article  CAS  PubMed  Google Scholar 

  44. Pinyakong O, Habe H, Kouzuma A, Nojiri H, Yamane H, Omori T. Isolation and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading Sphingomonas sp. strain A4. FEMS Microbiol Lett. 2004;238(2):297–305.

    CAS  PubMed  Google Scholar 

  45. Chen Q, Chen K, Ni H, Zhuang W, Wang H, Zhu J, He Q, He J. A novel amidohydrolase (DmhA) from Sphingomonas sp. that can hydrolyze the organophosphorus pesticide dimethoate to dimethoate carboxylic acid and methylamine. Biotechnol Lett. 2016;38(4):703–10.

    Article  CAS  PubMed  Google Scholar 

  46. Yan X, Jin W, Wu G, Jiang W, Yang Z, Ji J, Qiu J, He J, Jiang J, Hong Q. Hydrolase CehA and monooxygenase CfdC are responsible for carbofuran degradation in Sphingomonas sp. strain CDS-1. Appl Environ Microbiol. 2018;84(16):e00805-00818.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Feng F, Ge J, Li Y, Cheng J, Zhong J, Yu X. Isolation, colonization, and chlorpyrifos degradation mediation of the endophytic bacterium Sphingomonas strain HJY in Chinese Chives (Allium tuberosum). J Agric Food Chem. 2017;65:1131–8.

    Article  CAS  PubMed  Google Scholar 

  48. Berticat C, Rousset F, Raymond M, Berthomieu A, Weill M. High Wolbachia density in insecticide–resistant mosquitoes. Proc R Soc Lond B. 2002;269:1413–6.

    Article  Google Scholar 

  49. Pietri JE, Liang D. The links between insect symbionts and insecticide resistance: causal relationships and physiological tradeoffs. Ann Entomol Soc Am. 2018;111:92–7.

    Article  CAS  Google Scholar 

  50. Wu YQ, Zheng YF, Chen Y, Wang S, Chen YP, Hu FL, Zheng HQ. Honey bee (Apis mellifera) gut microbiota promotes host endogenous detoxification capability via regulation of P450 gene expression in the digestive tract. Microb Biotechnol. 2020;13:1201–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Sangare AK, Rolain JM, Gaudart J, Weber P, Raoult D. Synergistic activity of antibiotics combined with ivermectin to kill body lice. Int J Antimicrob Agents. 2016;47:217–23.

    Article  CAS  PubMed  Google Scholar 

  52. Ceja-Navarro JA, Vega FE, Karaoz U, Hao Z, Jenkins S, Lim HC, Kosina P, Infante F, Northen TR, Brodie EL. Gut microbiota mediate caffeine detoxification in the primary insect pest of coffee. Nat Commun. 2015;6:7618.

    Article  CAS  PubMed  Google Scholar 

  53. Lv NN, Liu Y, Guo TF, Liang PZ, Li R, Liang P, Gao XW. The influence of Bt cotton cultivation on the structure and functions of the soil bacterial community by soil metagenomics. Ecotoxicol Environ Saf. 2022;236: 113452.

    Article  CAS  PubMed  Google Scholar 

  54. Kikuchi Y, Hosokawa T, Fukatsu T. Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation. Appl Environ Microbiol. 2007;73(13):4308–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Dai YJ, Chen T, Ge F, Huan Y, Yuan S, Zhu FF. Enhanced hydroxylation of imidacloprid by Stenotrophomonas maltophilia upon addition of sucrose. Appl Microbiol Biotechnol. 2007;74(5):995–1000.

    Article  CAS  PubMed  Google Scholar 

  56. Pá D. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans. Curr Drug Metab. 2002;3(6):561–97.

    Article  Google Scholar 

  57. Suchail S, De Sousa G, Rahmani R, Belzunces LP. In vivo distribution and metabolisation of 14C-imidacloprid in different compartments of Apis mellifera L. Pest Management Science: formerly Pesticide Science. 2004;60(11):1056–62.

    Article  CAS  Google Scholar 

  58. Casida JE. Neonicotinoid metabolism: compounds, substituents, pathways, enzymes, organisms, and relevance. J Agric Food Chem. 2011;59(7):2923–31.

    Article  CAS  PubMed  Google Scholar 

  59. Tomizawa M, Zhang N, Durkin KA, Olmstead MM, Casida JE. The neonicotinoid electronegative pharmacophore plays the crucial role in the high affinity and selectivity for the Drosophila nicotinic receptor: An anomaly for the nicotinoid cation− π interaction model. Biochemistry. 2003;42(25):7819–27.

    Article  CAS  PubMed  Google Scholar 

  60. Schulz-Jander DA, Casida JE. Imidacloprid insecticide metabolism: human cytochrome P450 isozymes differ in selectivity for imidazolidine oxidation versus nitroimine reduction. Toxicol Lett. 2002;132(1):65–70.

    Article  CAS  PubMed  Google Scholar 

  61. Suchail S, Guez D, Belzunces LP. Discrepancy between acute and chronic toxicity induced by imidacloprid and its metabolites in Apis mellifera. Environ Toxicol Chem. 2001;20(11):2482–6.

    Article  CAS  PubMed  Google Scholar 

  62. Cheng X, Chen KX, Jiang ND, Wang L, Jiang HY, Zhao YX, Dai ZL, Dai YJ. Nitroreduction of imidacloprid by the actinomycete Gordonia alkanivorans and the stability and acute toxicity of the nitroso metabolite. Chemosphere. 2022;291: 132885.

    Article  CAS  PubMed  Google Scholar 

  63. Sarkar MA, Roy S, Kole RK, Chowdhury A. Persistence and metabolism of imidacloprid in different soils of West Bengal. Pest Manage Sci. 2001;57(7):598–602.

    Article  CAS  Google Scholar 

  64. Liu Z, Dai Y, Huang G, Gu Y, Ni J, Wei H, Yuan S. Soil microbial degradation of neonicotinoid insecticides imidacloprid, acetamiprid, thiacloprid and imidaclothiz and its effect on the persistence of bioefficacy against horsebean aphid Aphis craccivora Koch after soil application. Pest Manag Sci. 2011;67(10):1245–52.

    Article  CAS  PubMed  Google Scholar 

  65. Moores GD, Gao X, Denholm I, Devonshire AL. Characterisation of insensitive acetylcholinesterase in insecticide-resistant cotton aphids, Aphis gossypiiglover (homoptera: Aphididae). Pestic Biochem Physiol. 1996;56(2):102–10.

    Article  CAS  Google Scholar 

  66. Ma KS, Tang QL, Xia J, Lv NN, Gao XW. Fitness costs of sulfoxaflor resistance in the cotton aphid, Aphis gossypii Glover. Pestic Biochem Physiol. 2019;158:40–6.

    Article  CAS  PubMed  Google Scholar 

  67. Leung K, Chang Y, Gan Y, Peacock A, Macnaughton S, Stephen J, Burkhalter R, Flemming C, White D. Detection of Sphingomonas spp in soil by PCR and sphingolipid biomarker analysis. J Ind Microbiol Biotechnol. 1999;23(4–5):252–60.

    Article  CAS  PubMed  Google Scholar 

  68. Neef A, Witzenberger R, Kämpfer P. Detection of sphingomonads and in situ identification in activated sludge using 16S rDNA-targeted oligonucleotide probes. J Ind Microbiol Biotechnol. 1999;23(4–5):261–7.

    Article  CAS  PubMed  Google Scholar 

  69. Gut symbiont 16S rDNA sequences on Aphis gossypii Glover. NCBI BioProject accession: PRJNA929464. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA929464 (2023)

  70. Sphingomonas sp. strain SPY1 16S ribosomal RNA gene, partial sequence. GenBank accession: OQ359156.1. https://www.ncbi.nlm.nih.gov/nuccore/OQ359156 (2023)

Download references

Acknowledgements

We want to thank Key Laboratory of Surveillance and Management for Plant Quarantine Pests, Ministry of Agriculture and Rural Affairs, P. R. China, for providing the experimental apparatus. Furthermore, we would like to thank other former and present members from the Insect Toxicology Laboratory of China Agricultural University for helping with Aphis gossypii collections.

Funding

This work is supported by the National Key Research and Development Program of China (2022YFD1400901).

Key Technologies Research and Development Program,2022YFD1400901,Pei Liang

Author information

Authors and Affiliations

Authors

Contributions

The authors listed in the paper all contribute to the paper. NL and XG designed the study. NL, RL, SC, LZ, PL, and XG performed the research. NL and RL collected the field populations and identified the gut symbionts species. NL and SC analyzed the data. NL, PL, and XG wrote and edited the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Pei Liang.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1.

Total reads and good coverage in different samples. 

Additional file 2:

Fig. S1. Rarefaction curves obtained from all samples.

Additional file 3: Fig. S2.

The susceptibility of Sphingomonas to different antibiotics. (A) The inhibition zone of different antibiotics against Sphingomonas (a-f): sodium sulfate, gentamicin, chloramphenicol, tetracycline, amoxicillin, and ampicillin. (B) The line chart of different antibiotics to Sphingomonas.

Additional file 4: Fig. S3.

Detection of Sphingomonas in nine field populations in 2019. Bars represent the mean ± SE (P < 0.05, Tukey’s test).

Additional file 5: Fig. S4.

The abundance of Sphingomonas in the gut of the IMI-S and IMI-R strains and SXYC, SDBZ, XJSW and HBHS field populations. The bars with lowercase letters (a, b, c) are significantly different according to one-way ANOVA, followed by Tukey's multiple comparison test (P< 0.05).

Additional file 6: Fig. S5.

The stand curve of IMI (A), urea IMI (B) and 5-OH IMI (C).

Additional file 7: Fig. S6.

The changes in OD600 value of Sphingomonas in the control group (A) and IMI group (B) during the 3-day cultivation. The bars with different lowercase letters (a, b, c) are significantly different (one-way ANOVA followed by Tukey's multiple comparison, P< 0.05).

Additional file 8: Table S2.

Collecting information of Aphis gossypii field populations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lv, N., Li, R., Cheng, S. et al. The gut symbiont Sphingomonas mediates imidacloprid resistance in the important agricultural insect pest Aphis gossypii Glover. BMC Biol 21, 86 (2023). https://doi.org/10.1186/s12915-023-01586-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12915-023-01586-2

Keyword