Insecticide resistance governed by gut symbiosis in a rice pest, Cletus punctiger, under laboratory conditions

Abstract

Resistance to toxins in insects is generally thought of as their own genetic trait, but recent studies have revealed that gut microorganisms could mediate resistance by detoxifying phytotoxins and man-made insecticides. By laboratory experiments, we here discovered a striking example of gut symbiont-mediated insecticide resistance in a serious rice pest, Cletus punctiger. The rice bug horizontally acquired fenitrothion-degrading Burkholderia through oral infection and housed it in midgut crypts. Fenitrothion-degradation test revealed that the gut-colonizing Burkholderia retains a high degrading activity of the organophosphate compound in the insect gut. This gut symbiosis remarkably increased resistance against fenitrothion treatment in the host rice bug. Considering that many stinkbug pests are associated with soil-derived Burkholderia, our finding strongly supports that a number of stinkbug species could gain resistance against insecticide simply by acquiring insecticide-degrading gut bacteria.

1. Introduction

Chemical insecticides have been globally used to control agricultural and medically relevant pest insects. However, the abuse of insecticides has caused the emergence of insecticide resistance in pest insect populations [1,2]. The emergence of insecticide-resistant pests has caused numerous problems around the world, from the loss of crops, to public health problems due to insect disease vectors [3,4]. Insecticide resistance is generally caused by a mutation in genes involved in insecticide degradation and/or genes of target enzymes of insecticide [5–7]. In addition to the evolution of insecticide degradation directly by the insect host, symbiont-mediated detoxification of insecticides has recently been reported in diverse insects [8]. For instance, the bean bug Riptortus pedestris and the oriental chinch bug, Cavelerius saccharivorus, which are respectively notorious pests of leguminous crops and sugarcane, can acquire insecticide-degrading gut symbionts of the genus Burkholderia, conferring insecticide resistance on the host bugs [9–11]. Such insecticide resistance mediated by gut symbionts is also found in other insects such as Bactrocera dorsalis (oriental fruit fly), Spodoptera frugiperda (fall armyworm moth) and Plutella xylostella (diamondback moth) [12–14]. In addition, symbiont-mediated insecticide tolerance has been studied in disease vectors including mosquitoes [15,16].

Fenitrothion, or O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate (MEP), has been one of the most commonly used organophosphate insecticides worldwide since 1959 to control insect pests on diverse crops, including cotton, fruits, vegetables and rice [17–19]. Fenitrothion kills pest insects by inhibiting their acetylcholinesterase [20]. Some soil bacteria, including Burkholderia species, can hydrolyse fenitrothion into a less insecticidal molecule, 3-methyl-4-nitrophenol (3M4N), and use it as a carbon source [21–23] (figure 1a). In the bean bug R. pedestris, fenitrothion-degrading Burkholderia retains a high degrading ability in the midgut and confers fenitrothion resistance on the host bug [9,11], although infection rate of fenitrothion-degrading Burkholderia is low in field populations. Besides the bean bug and the chinch bug, most of the phytophagous species belonging to the stinkbug superfamilies Lygaeoidea and Coreoidea are also associated with the Burkholderia symbiont in the midgut [24]. However, Burkholderia-mediated insecticide resistance has still scarcely been investigated in other stinkbug species.

Figure 1.

Colonization in the midgut crypts of fenitrothion-degrading Burkholderia in Cletus punctiger. (a) Pathway of fenitrothion degradation in Burkholderia. (b) An adult male C. punctiger on sunflower seeds. (c) Dissected gut of C. punctiger: M1, midgut first section; M2, midgut second section; M3, midgut third section; M4B, the bulbous region prior to M4; M4, midgut fourth section. (d) Titre of gut-colonizing Burkholderia strains measured by quantitative PCR. The statistical significance was analysed by the Mann–Whitney U test with Bonferroni correction. n.s., non-significant. n = 8 insects were used in each group. (e) Fluorescence microscopy images of M4 infected with each Burkholderia strain. Green fluorescence is derived from GFP-expressing Burkholderia strains. Burkholderia insecticola RPE225 is a non-fenitrothion-degrading Burkholderia. SFA1, KM-A, KM-G, MDT-44 and MDT-50 are fenitrothion-degrading Burkholderia strains.

The symbiotic association between stinkbugs and Burkholderia has been well studied in the bean bug R. pedestris [25,26]. The bean bug acquires Burkholderia from environmental soil every generation and harbours the symbiont in midgut crypts. The symbiont acquisition mainly occurs at the second instar stage of the insect [27]. The Burkholderia symbiont is culturable by using bacterial standard media, and under laboratory conditions aposymbiotic nymphs become infected with the symbiont by drinking water containing cultured Burkholderia [28–31]. The genus Burkholderia consists of over 100 species and is widespread in the environment, primarily residing in the soil [32]. Burkholderia species are further phylogenetically classified into more than three groups: the pathogenic ‘B. cepacia complex and B. pseudomallei' (BCC&P), ‘plant-associated beneficial and environmental' (PBE) or Paraburkholderia, ‘stinkbug-associated beneficial and environment' (SBE) or Caballeronia, and other Burkholderia species [33,34]. Among the diverse Burkholderia species, the SBE species establish a symbiotic association with many stinkbugs of the superfamilies Lygaeoidea and Coreoidea [24,35]. Phylogenetic analyses strongly suggest that there is no or little host-symbiont specificity, and hence stinkbug species share the SBE Burkholderia symbionts [24].

Cletus punctiger (superfamily Coreoidea: family Coreidae) is a serious pest of rice in East Asia (figure 1b), which causes pecky rice injury and is thus known as a ‘pecky rice bug' [36,37]. Cletus punctiger also possesses gut symbionts of the genus Burkholderia in the crypt-bearing posterior midgut region [24] (figure 1c). Our previous study indicated that, as shown in the bean bug R. pedestris, C. punctiger horizontally acquires the SBE Burkholderia from ambient soil every generation [24]. In this study, we demonstrate that fenitrothion-degrading Burkholderia strains of the SBE group, isolated from crop field soils (Chinese cabbage and soya bean fields) and the midgut of field-collected bean bugs and oriental chinch bugs, colonize the midgut of C. punctiger well, conferring resistance against fenitrothion on the rice bug by degrading the insecticide in the midgut.

2. Material and methods

The rice bug C. punctiger was collected from Tsukuba, Ibaraki, Japan. Insects were reared in Petri dishes (90 mm in diameter and 20 mm high) at 25°C under a long-day regime (16 h light and 8 h dark) and supplied wheat seeds, sunflower seeds and distilled water containing 0.05% ascorbic acid (DWA). The strains of the gut symbiont Burkholderia used in this study are listed in electronic supplementary material, table S1 and their phylogenetic relationship is shown in electronic supplementary material, figure S1. The well-studied type species, Burkholderia insecticola strain RPE225, is a non-degrading symbiont, and SFA1, KM-A, KM-G, MDT-44 and MDT-50 are fenitrothion-degrading symbionts. To inoculate symbiont into the first instar nymph of the symbiont-free host insect, each Burkholderia strain was cultured at 27°C overnight in yeast–glucose (YG) medium (0.5% yeast extract, 0.4% glucose and 1% NaCl). A 10⁷ cells ml⁻¹ cultivated bacterial solution was mixed with DWA and orally administered to the first instar nymph of the insect as described in the previous study [38].

The GFP-expression vector pIN25 was transferred into the Burkholderia strains by electroporation [39]. To make electrocompetent cells, the overnight-cultured Burkholderia cells were transferred to fresh medium and further cultured until mid-log phase. The bacterial cells were washed three times with 10% glycerol and stored at −80°C until use. The pIN25 vector was transferred into the Burkholderia cells by electroporation using the Gene Pulser Xcell Electroporation System (Bio-Rad). GFP-expressing Burkholderia colonies were selected by spreading the electroporated cells on YG agar containing 15 µg ml⁻¹ of chloramphenicol. The GFP signal of the grown colonies was observed by LED-EXTRA with a green filter (OptoCode). The GFP-expressing Burkholderia strains were orally administered, and infected midgut crypts were observed under an epifluorescence microscope (DMI4000B, Leica).

To examine the population of gut-colonizing symbionts by quantitative PCR (qPCR), DNA was extracted from the midgut fourth section (M4) of six groups of fourth instar nymphs, each infected with a different Burkholderia symbiont listed in electronic supplementary material, table S1 (n = 8 per group were investigated) by using the QIAamp DNA mini kit (Qiagen) according to the manufacturer's instructions. The dnaA gene copies of Burkholderia symbiont were quantified using BSdnaA-F (5′-AGC GCG AGA TCA GAC GGT CGT CGA T-3′) and BSdnaA-R (5′-TCC GGC AAG TCG CGC ACG CA-3′) primer pairs by the KAPA SYBR FAST qPCR Master Mix (KAPA Biosystems) and the LightCycler 96 System (Roche). The number of Burkholderia symbionts was calculated based on a standard curve for the dnaA gene containing 10, 10², 10³, 10⁴, 10⁵, 10⁶ and 10⁷ copies per reaction of the target PCR product.

Six groups of fourth instar nymphs of C. punctiger, each infected by a different Burkholderia strain listed in electronic supplementary material, table S1 (n = 10 per group), were dissected in phosphate-buffered saline (PBS) using fine forceps, and the midgut fourth section (M4) was homogenized by using a pestle in a 1.5 ml microcentrifuge tube. Since the midgut is not shed at each moulting, the fourth instar nymph of C. punctiger still possesses a large number of the symbiont in the midgut crypts. Two microlitres of M4 lysate containing gut-colonizing Burkholderia symbionts was spotted on a minimal agar plate containing 0.08% fenitrothion emulsion and cultured at 27°C for 4 days. The fenitrothion-degrading activities were measured based on halo formation on the agar around a drop of M4 lysate. To quantify the size of haloes, the maximum diameter of the clear zone was measured. To test degrading ability in solution, fenitrothion emulsion was mixed with 2 µl of M4 lysate and incubated for 1 h. The degrading activities were measured by the colour change of the emulsion, which was quantified as absorbance at 405 nm (A₄₀₅) by a spectrophotometer (Nanodrop 1000, Thermo Fisher Scientific) [9].

Three wheat seeds and one sunflower seed were dipped in 0.2 mM fenitrothion for 5 s and dried at room temperature [9]. Six groups of insects (n = 40 or 50 per group) were each infected with a different Burkholderia strain listed in electronic supplementary material, table S1 and were reared in a clean plastic container with fenitrothion-applied seeds and DWA. The survival rate of the insects was measured by counting the number of dead insects every day.

All statistical analyses were performed using GraphPad Prism 8 and R v. 3.6.3. The statistical significance of gut-colonizing symbiont populations was analysed by the Mann–Whitney U test with Bonferroni correction. The statistical significance of the insects' survival rate was analysed by log-rank test with Bonferroni correction. Raw data of this study are available in the Dryad repository (https://doi.org/10.5061/dryad.t4b8gtj13).

3. Results

(a). Colonization of Cletus punctiger by fenitrothion-degrading Burkholderia

The midgut of C. punctiger consists of four separate regions: the midgut first section (M1), the midgut second section (M2), the midgut third section (M3), and the midgut fourth section (M4), which has numerous crypts that are densely populated by the Burkholderia gut symbionts (figure 1c). Since field-captured C. punctiger consistently harboured SBE clade Burkholderia in the midgut [24] (electronic supplementary material, figure S1), we first orally inoculated YG C. punctiger with a typical species from the SBE clade, B. insecticola. As a result, the GFP-expressing B. insecticola densely colonized the midgut crypts (figure 1d,e). We then tested five strains of fenitrothion-degrading Burkholderia, with the result that all of the fenitrothion-degrading Burkholderia strains colonized the M4 crypts of C. punctiger well (figure 1d,e). qPCR analyses confirmed that all of the Burkholderia strains colonized the M4 region well (greater than 10⁷ cells), demonstrating that C. punctiger accepts a broad range of Burkholderia in the SBE clade as gut symbionts (figure 1d).

(b). Fenitrothion resistance in Cletus punctiger infected with degrading symbionts

To clarify if fenitrothion-degrading Burkholderia symbionts increase the survival rate of the host C. punctiger against fenitrothion treatment via their degradation ability, fenitrothion was orally administered to fourth instar nymphs of C. punctiger possessing one of each of the different Burkholderia strains. Cletus punctiger infected with the non-fenitrothion-degrading B. insecticola showed high mortality (75% dead), whereas the insects infected with fenitrothion-degrading Burkholderia strains showed resistance against fenitrothion (figure 2a). These results clearly demonstrated that the fenitrothion-degrading Burkholderia contribute to insecticide resistance in the rice bug C. punctiger.

Figure 2.

Fenitrothion-degrading ability of gut-colonizing Burkholderia. (a) Survival rate of C. punctiger after feeding on fenitrothion-treated seeds. n.s., non-significant. Asterisk indicates statistically significant differences from the RPE225-infected insect group (p < 0.05). Data were analysed using the log-rank test with Bonferroni correction. Fenitrothion-degrading activity of gut-colonizing Burkholderia (b) in fenitrothion emulsion-containing, and (c) on minimal agar. Colour change from white to yellow (b) and formation of haloes (c) indicate the degradation of fenitrothion. n = 10 insects were used in each group.

(c). Fenitrothion degradation by gut-colonizing Burkholderia

Next, we investigated the fenitrothion-degrading abilities of gut-colonizing Burkholderia symbionts. Fenitrothion is a colourless insecticide, but it becomes yellowish when it is degraded into the byproduct 3M4N [9]. Thus, we measured the optical density of yellowish 3M4N by spectrophotometry to determine the fenitrothion-degrading ability of gut-colonizing Burkholderia. Co-incubation of fenitrothion emulsion with the M4 lysates of insects containing the fenitrothion-degrading Burkholderia strains produced a colour change to yellow, but M4 lysate of insects infected with the non-degrading B. insecticola did not (average A₄₀₅ values ± s.d. are 0.005 ± 0.004, 0.517 ± 0.029, 0.510 ± 0.029, 0.516 ± 0.042, 0.513 ± 0.030 and 0.522 ± 0.028 for RPE225, SFA1, KM-A, KM-G, MDT-44 and MDT-50, respectively) (figure 2b and electronic supplementary material, figure S2a). Furthermore, while the M4 lysate of insects infected with the non-fenitrothion-degrading B. insecticola did not form any halo on the minimal agar containing emulsified fenitrothion (figure 2c and electronic supplementary material, figure S2b), the M4 lysate of insects infected with the fenitrothion-degrading Burkholderia strains showed haloes (average diameters ± s.d. are 7.820 ± 0.512, 7.720 ± 0.598, 7.560 ± 1.053, 7.000 ± 0.581, and 7.380 ± 0.592 for SFA1, KM-A, KM-G, MDT-44, and MDT-50, respectively) (figure 2c and electronic supplementary material, figure S2b). These results demonstrated that the fenitrothion-degrading Burkholderia strains retain their degrading ability in vivo and actively degrade the chemical insecticide in the midgut of C. punctiger.

4. Discussion

In this study, we revealed that (i) C. punctiger horizontally acquires fenitrothion-degrading SBE clade Burkholderia through oral infection and houses large numbers of them in M4 crypts (figure 1d,e), (ii) fenitrothion-degrading Burkholderia symbionts confer fenitrothion resistance on C. punctiger (figure 2a), and (iii) all of the tested fenitrothion-degrading Burkholderia symbionts retain their degrading ability in the midgut (figure 2b,c and electronic supplementary material, figure S2). Our previous study reported Burkholderia-mediated fenitrothion resistance in the bean bug R. pedestris and the oriental chinch bug, C. saccharivorus [9,11], which are notorious pests of leguminous crops and sugarcane, respectively. Considering that many stinkbug species of the superfamilies Coreoidea and Lygaeoidea are associated with a broad range of SBE clade Burkholderia symbionts [24], here our findings strongly support that a number of stinkbug species could gain resistance against insecticide simply by acquiring insecticide-degrading gut bacteria.

Since fenitrothion-degrading bacteria use the chemical compound as a carbon source, in laboratory conditions, fenitrothion spraying efficiently enriches fenitrothion-degrading bacteria in the soil [9–11]. When the bean bug R. pedestris was reared on such fenitrothion-sprayed soil, insects frequently acquired fenitrothion-degrading Burkholderia symbionts from the soil [11]. Moreover, in field surveys, fenitrothion-degrading Burkholderia has been found in agricultural soils where fenitrothion was frequently used [9,22,40], and the oriental chinch bug, C. saccharivorus, collected in fenitrothion-sprayed sugarcane fields, harboured fenitrothion-degrading Burkholderia [9]. However, it should be noted that only a low percentage of insects were infected with fenitrothion-degrading Burkholderia strains in natural fields [9]. Since fenitrothion has been used in paddy fields in Asia [17,18], C. punctiger living in those regions also could acquire fenitrothion-degrading Burkholderia from the field soils, although there has been no report of the occurrence of fenitrothion resistance in C. punctiger. A broad field survey of C. punctiger is necessary to understand how symbiosis affects its resistance against insecticide in Asian paddy fields.

In addition to the genus Burkholderia, diverse soil bacteria, including members of the genera Achromobacter, Arthrobacter, Corynebacterium, Cupriavidus, Dyella, Pandoraea, Pseudomonas, Ralstonia, Sphingobium and Sphingomonas, have also been detected as fenitrothion-degraders [10,11,23,41,42]. Interestingly, in some bacteria, insecticide-degrading genes are encoded on plasmids and horizontally transmitted between phylogenetically divergent bacterial species via interspecific conjugation [8,23]. If gut symbionts acquire such an insecticide-degrading plasmid, these symbionts could confer insecticide resistance on their host insects. Considering that most insects are associated with diverse gut microbiota [43], further research on microbe-mediated insecticide resistance is required in diverse pest insects that harm crops or human health.

Besides fenitrothion, other organophosphorus insecticides, such as acephate and trichlorphon, are also extensively used in the field to control pest insects. Long-term use of these pesticides has eventually caused the appearance of resistant pest insects such as flies, moths, aphids and beetles [12,14,44–46]. In addition to the genetic evolution of insects' own traits, as in the case of stinkbugs, recent studies have revealed that gut microbes considerably contribute to the degradation of insecticides in these insect species. For example, the gut symbiont Citrobacter sp. of the oriental fruit fly, B. dorsalis, confers trichlorphon resistance on the host by degrading insecticide using phosphatase hydrolases [12]. Also, diverse gut symbionts isolated from diamondback moth, P. xylostella, including Bacillus, Enterobacter and Pantoea, possessed acephate-degrading activities [14]. Although it remains unclear how these pest insects acquire insecticide-degrading bacteria, recent studies have revealed that some insect species' gut bacteria are established from the environmental soil [47,48]. To control pest insects more efficiently, this study on C. punctiger as well as the above recent studies highlight how important it is to better understand the soil microbiota in crop fields.

Data accessibility

Raw data of this study have been deposited to Dryad: https://dx.doi.org/10.5061/dryad.t4b8gtj13 [49].

Authors' contributions

K.I., S.J. and Y.K. designed and developed the study. K.I. and S.J. performed fenitrothion-degrading assay and microscope observation. H.I. performed phylogenetic analysis. K.I. performed other experiments and statistical analysis. K.I., S.J., H.I. and Y.K. wrote the manuscript. All authors approved the final version of the manuscript and agree to be held accountable for the content therein.

Competing interests

We declare we have no competing interests.

Funding

This study was supported by JSPS Research Fellowships for Young Scientist to S.J. (201911493) and by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) KAKENHI to Y.K. (20H03303).