Symbiont Diversity in Imidacloprid-Resistant and Imidacloprid-Susceptible Populations of Nilaparvata lugens (Hemiptera: Delphacidae)¹

The brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), is an economically important pest that damages Asian rice, Oryza sativa L., crops by direct injury and by transmitting rice virus disease. The brown planthopper is distributed over approximately half of the rice-growing area of China and causes annual losses of approximately 1–1.5 million tons of rice (Lou and Cheng 2011).

Effective control of the brown planthopper has been seriously compromised in recent years by the widespread appearance of resistance to imidacloprid, a neonicotinoid insecticide that has been used as the primary control of brown planthopper infestations in eastern and southeastern Asia since the mid-1990s. Imidacloprid resistance was first reported in Thailand but has since been reported in other countries, including China (Lou and Cheng 2011, Matsumura et al. 2008, Wang et al. 2008). The detoxification mechanisms of insects in response to neonicotinoid insecticides mainly include the up-regulation of P450 detoxification enzyme activity and the enhancement of transport metabolism ability.

Microbial symbionts benefit their insect hosts (Gibson and Hunter 2010) by impacting nutrition (Douglas 2009, Hosokawa et al. 2010), reproduction (Wilkinson et al. 2001), virulence regulation (Lu et al. 2004), and detoxification (Dowd and Shen 1990, Xu et al. 2009), with detoxification indicating possible links between insect gut symbionts and resistance to chemical toxins. Cheng et al. (2017) found that the gut symbiont Citrobacter sp. plays a key role in the degradation of trichlorfon. DNA sequencing revealed that members of the Lactobacillales or other scarcer taxa play a role in conferring insecticide resistance to Plutella xylostella (L.) (Lepidoptera: Plutellidae) (Xia et al. 2013). The susceptibility of Myzus persicae Sulzer (Hemiptera: Aphididae) to nine insecticides was determined after treatment with ampicillin; the susceptibility of M. persicae to imidacloprid, cyantraniliprole, and clothianidin decreased significantly (3.4 times, 2.2 times, and 2.0 times, respectively) (Yang et al. 2020). Kikuchi et al. (2012) found that fenitrothion-degrading Burkholderia strains established a symbiosis with the hemipteran Riptortus pedestris (F.), conferring resistance to fenitrothion.

Bacterial and yeast-like symbiotes are important symbionts of the brown planthopper that provide their hosts with cholesterol, vitamins, and essential amino acids that the host cannot produce (Sasaki et al. 1996). Exposure of neonatal planthoppers to high temperature, antibiotics, lysozyme via injections, and insecticides reduced yeast-like symbiont abundance and thus influenced host growth and development (Raguraman et al. 1988, Shankar and Baskaran 1988). These symbionts also play crucial roles in changes in the virulence of brown planthopper populations for resistant rice varieties (Lu et al. 2004) because the rate of change in endosymbiotic genes is much more rapid than that in host genes (Campbell 1990, Ishikawa et al. 1986). The symbiont-related mechanisms underlying high resistance to imidacloprid in the brown planthopper have received relatively little attention. Analysis of strains revealed a single point mutation (Y151S) in two nAChR subunits that were associated with a dramatic reduction in binding to imidacloprid (Liu et al. 2005). Enhanced detoxification of imidacloprid by carboxyesterase, glutathione-S-transferase, and cytochrome P450 monooxygenase appears to be the predominant mechanism of resistance in field-selected populations (Puinean et al. 2010, Wen et al. 2009).

Zhang et al. (2013) used polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) to study the mid-gut bacterial communities of the larvae of the striped rice stem borer (Chilo suppressalis (Walker)). Hou et al. (2013) analyzed yeast-like symbiont diversity in N. lugens with PCR-DGGE and found several previously detected, undetected, and uncultured fungi. Xu et al. (2014) used DGGE to elucidate the structures of bacterial communities in N. lugens from different geographic and resistant virulent populations. In this study, we used PCR-DGGE to assess bacterial and yeast-like symbiont communities in imidacloprid-resistant and imidacloprid-susceptible brown planthopper populations, with the goal of revealing the possible functions of these endosymbionts in the development of imidacloprid resistance.

Rice varieties, including TN1 (brown planthopper susceptible), were planted in clay pots (15 cm in diameter) in the greenhouse of the Zhejiang Academy of Agricultural Sciences, Hangzhou, China. The experiments described below were conducted with 45-d-old rice plants.

The susceptible N. lugens population, provided by the Zhejiang Research Institute of Chemical Industry, had not been exposed to any insecticide for at least 10 yr before the study. The resistant N. lugens population was selected by spraying imidacloprid (at the LC₅₀) for more than 50 generations at the Zhejiang Academy of Agricultural Science. The resistance ratio of the resistant N. lugens population was nearly 400 times greater than that of the susceptible population. Female adult N. lugens from the imidacloprid-susceptible and imidacloprid-resistant populations were collected and used for tests.

Fifty adult female brown planthoppers from each population were used to collect total DNA. Each planthopper was surface sterilized with 75% ethanol for 1 min. Genomic DNA was extracted with a bacterial DNA kit (Omega Bio-Tek Company Ltd., Guangzhou, China) or a yeast DNA kit (Omega Bio-Tek). DNA purity and concentration were measured with a protein nucleic acid spectrophotometer (DU800, Beckman Coulter Life Sciences, Indianapolis, IN).

All primers used in this study are shown in Table 1 and were synthesized by Shanghai Shenggong Bioengineering Company, Ltd. (Shanghai, China). For analysis of specific bacterial diversity, PCR amplification of the 16S rRNA gene was conducted using bacteria-specific primers 49f and 1525r (Henckel et al. 1999, Muyzer et al. 1993). For general bacterial diversity analysis, PCR amplification of the 16S rRNA gene was performed using the 341f-GC and 534r primers (Nakagawa and Fukui 2003). For analysis of yeast diversity, PCR amplification of the 26S rRNA gene was performed as described previously (Prakitchaiwattana et al. 2004) with initial amplification of the D1/D2 region with eukaryotic universal primers NL-1 and NL-4 (Taylor et al. 2002) followed by nested PCR using primers GCNL-1 and LS2 (Cocolin et al. 2002). DNA from each sample was subjected to DGGE following PCR amplification with each primer set (Table 1). All PCR amplification was conducted in a final volume of 25 µl containing 0.5 µl (50 ng/µl) of template, 1 μl of template DNA, 0.5 μl of primer NL-1 and primer NL-4 (10 μM), 21.5 μl of Platinum PCR Supermix High Fidelity, and 1.5 μl of sterile double-distilled water. Reactions were performed in a PTC-220 DNA Engine Dyad thermocycler (MJ Research, Saint-Bruno-de-Montarville, Quebec, Canada).

DGGE was performed using the D Code Universal Mutation System (Bio-Rad Laboratories, Hercules, CA) for separation of PCR products. PCR products were applied directly onto 8% (w/v) polyacrylamide gels in a running buffer containing 1× TAE buffer (20 mM Tris, 10 mM acetate, 0.5 mM EDTA, pH 8.3) and a denaturing gradient of 30–60% urea and formamide (for bacteria and yeast) or 35–55% urea and formamide (for Archaea, where 100% denaturant contains 7 M urea and 40% formamide). Electrophoresis was performed at 80 V for 14 h at a constant temperature of 60°C. After electrophoresis, the gels were stained with SYBR Green I nucleic acid stain (Thermo Fisher Scientific, Waltham, MA) and photographed under ultraviolet transillumination. Sterile blades were used to excise bands from the gels, and each band was mixed with 20 µl of 0.1× TE buffer solution, incubated overnight at 4°C, and used for PCR amplification with the appropriate primer set.

The significance of differences between the two insect populations was analyzed with a one-way analysis of variance with a significance threshold of P < 0.05.

The Shannon-Weaver diversity index values for both bacterial and yeast-like symbionts of the resistant population (Table 2) were close to that of those values for the susceptible population, thus suggesting that the richness and diversity of symbiotic microorganisms of the two populations were similar. The evenness index values for both bacterial and yeast-like symbionts of the two populations were all close to 1, meaning the relative abundance of microbial species in imidacloprid-resistant and imidacloprid-susceptible N. lugens populations were even (e.g., no difference in the evenness of resistant and susceptible microbial community). The similarity coefficients comparing the imidacloprid-resistant and imidacloprid-susceptible populations were 0.53 and 0.56 for the bacterial and yeast-like symbiont communities, respectively (Table 2), indicating differences in the main types of microorganisms in the two populations. Fifteen bacterial bands and 12 yeast-like bands from gels were selected for partial sequencing (Fig. 1). Sequence comparison analysis was conducted based on sequences in the National Center for Biotechnology Information GenBank database. All selected clones were closely related (≥97% sequence identity) to the species reported in GenBank. The number of bacterial species in the imidacloprid-susceptible and -resistant populations were not significantly different. The bacteria in the susceptible population were members of the Enterobacteriaceae and Moraxellaceae families. The bacteria in the resistant population belonged to more families: Oxalobacteriaceae, Rhodobacteriaceae, Sphingomonadaceae, and Enterobacteriaceae (Table 3). Yeast-like fungi Cryptococcus luteolus, Pseudozyma aphidis, Pseudozyma antarctica, Capnodiales species, and Cladosporium perangustum were identified for the first time in N. lugens. The imidacloprid-susceptible population contained more yeast-like symbiont species than did the imidacloprid-resistant population. Cryptococcus luteolus, P. aphidis, and P. antarctica were detected in only the susceptible population, C. perangustum was detected in only the resistant population, and Capnodiales species and some unknown species existed in both populations (Table 4).

Insects harbor symbionts that enhance fitness by contributing to digestion, nutrition, reproduction, and resistance to xenobiotics (Douglas 2011). However, few studies on the contributions of symbionts to insecticide resistance have been performed because of the difficulty of separating symbionts from host insects. The evolution of insecticide resistance is accompanied by a series of physiological changes in the host (Klepzig et al. 2009), which alter the structure and function of the microorganism community (Gimonneau et al. 2014). In this study, the Shannon-Weaver diversity and evenness index values of the imidacloprid-susceptible and -resistant populations were not significantly different. These findings could indicate that the development of insecticide resistance by N. lugens is not closely related to the richness and evenness of the symbiotic microorganism population; instead, insecticide resistance seems to be related to the types of symbiotic microorganisms.

Sequencing analysis revealed that common bacteria (Arsenophonus nasoniae and Enterobacter asburiae) existed in both the imidacloprid-resistant and -susceptible populations, whereas Herbaspirillum sp., Sphingomonas sp., and Amaricoccus sp. were detected in only the resistant population. We speculated that aromatic compound degradation (Bacosa et al. 2010, Baraniecki et al. 2002, Lafortune et al. 2009, Singleton et al. 2008) resulted in these species transitioning from secondary bacterial species to dominant species under imidacloprid exposure, perhaps enhancing imidacloprid resistance. Herbaspirillum sp. and Amaricoccus sp. also function in nitrogen fixation (Elbeltagy et al. 2001, Valverde et al. 2003) and intracellular storage of synthesized polymers (Falvo et al. 2001, Lemos et al. 2008) and could contribute to raw material storage by synthesizing amino acids and proteins.

In this study, the yeasts C. luteolus, P. aphidis, P. antarctica, Capnodiales species, and C. perangustum were found in N. lugens for the first time. The yeast-like symbiont population of N. lugens was a mixture of many types of yeasts, indicating that the microbial species in imidacloprid-susceptible and imidacloprid-resistant populations is variable. Capnodiales species was detected in both N. lugens populations and might have been carried into the body when these insects fed on infected rice plants. Cladosporium perangustum was detected in the resistant population, whereas the other three yeasts (C. luteolus, P. aphidis, and P. antarctica) were found in only the susceptible population.

Cladosporium perangustum is abundant in the air, from which this organism can be absorbed by insects (Hsu et al. 2012). The basidiomycetous yeast C. luteolus produces polysaccharides (Vorotynskaya et al. 1992), and P. antarctica is an excellent source of edible single-cell protein and facilitates utilization of waste glycerol (Morita et al. 2007). Pseudozyma aphidis has biocontrol activity and provides a natural barrier against some plant pathogens (Avis and Bélanger 2001, Urquhart and Punja 2002). The manner in which differences in the microorganism distributions in the populations are related to insecticide resistance merits further study.

The results reported here highlight the significant differences in the microbial symbiont communities of imidacloprid-susceptible and imidacloprid-resistant brown planthopper populations. More evidence is required to assess whether changes in microbial community structure are related to insecticide resistance.

We thank Xin Wang from the Institute of Plant Protection and Microbiology (Zhejiang Academy of Agricultural Sciences) for his help in analyzing the data, and we are grateful to Guangjie Liu from the AgriLife Research and Extension Center (Texas A&M University, College Station) for suggestions that improved this manuscript. This research was funded by the Zhejiang Provincial Programs for Science and Technology Development (2023C02030) and the Zhejiang Provincial Natural Science Foundation of China (LY23C140002). We confirm that all authors have approved the manuscript and agree with submission to the Journal of Entomological Science. J.F.Z. carried out the experiments and drafted the manuscript. F.L. and H.Y.Z. helped with experimental procedures and manuscript preparation. J.M.C. designed the study and critically revised the manuscript. The authors declare there were no conflicts of interest.