Diversity and Infection Frequency of Symbiotic Bacteria in Different Populations of the Rice Brown Planthopper in China
Genetic sequences (16S rRNA) demonstrated that the symbiotic bacteria associated with different populations of the rice brown planthopper, Nilaparvata lugens Stål, were clustered into 18 operational taxonomic units. Molecular phylogenetic analyses revealed that the majority of these bacteria belong to Alphaproteobacteria (Wolbachia), Gammaproteobacteria (Arsenophonus, Acinetobacter, Serratia, Arthrobacter), and the Bacteroidetes (Chryseobacterium, Sphingobacterium). Bacteria of the genera Acinetobacter, Serratia, and Arthrobacter were present in all brown planthopper individuals analyzed. The infection frequency (IF) of Arsenophonus in brown planthopper reared on the rice variety ‘Taichun Native1′ was higher than that observed with the ‘Mudgo' and ‘ASD7′ varieties. The IF of Arsenophonus increased by 50% when the planthoppers were reared on Taichun Native1 for >15 generations, unrelated to the origin of that population of planthoppers. The IF of Wolbachia, however, declined when the brown planthoppers were maintained on a single rice variety under laboratory conditions for several generations. These results suggest that the symbiotic bacteria varied with brown planthopper populations and might have an effect on the adaptation and evolution of brown planthopper on different rice varieties.Abstract
The brown planthopper, Nilaparvata lugens Stål (Homoptera: Delphacidae), is a major pest of rice. Rice varieties resistant to the pest have been successfully developed and widely cultivated; however, virulent strains of N. lugens that are capable of feeding and/or reproducing on resistant rice varieties have appeared. For example, the ASD7 population of N. lugens is highly virulent to the gene bph2-carrying rice variety ‘ASD7’, while the Mudgo population is highly virulent to the Bph1-carrying rice variety ‘Mudgo' (Cruz et al. 2011, Pathak and Heinrichs 1982). The mechanisms underlying the adaptation of brown planthopper populations on different resistant rice varieties are not well known.
Symbiotic bacteria play important roles in the insect–plant interactions. A large number of symbiotic bacteria associated with herbivorous insects are commonly divided into two general categories: (a) primary symbionts, such as Buchnera aphidicola in aphids, Carsonella ruddii in psyllids, and Tremblaya princeps in mealybugs (Baumann 2005, Douglas 1998); and (b) secondary symbionts, such as Regiella insecticola, Serratia symbiotica, and Hamiltonella defensa in aphids (Chen et al. 2000, Clark et al. 2010, Darby et al. 2001, Fukatsu et al. 2000) and Cardinium, Wolbachia, Rickettsia, Arsenophonus, Enterobacter, Paracoccus, and Acinetobacter in whiteflies (Chiel et al. 2007, Singh et al. 2012).
In general, the role of primary symbionts in their hosts is thought to be nutritional enrichment. On the other hand, secondary endosymbionts have various roles such as acting as a mutualist or a reproductive manipulator (Moran et al. 2008) or by enhancing resistance to pathogens or parasitoids, thermal tolerance, and adaptation to new host plants (Feldhaar 2011). For example, H. defensa and S. symbiotica can increase resistance to parasitoids of the genus Aphidius (Ferrari et al. 2004, Oliver et al. 2003). Also, S. symbiotica is found to improve the thermal tolerance of aphids (Allen et al. 2007, Montllor et al. 2002, Russell and Moran 2006), synthesize essential amino acids in Acyrthosiphon pisum and Cinara cedri (Lamelas et al. 2011), and potentially contribute to cellulose digestion in termites (Thayer 1978). In the stable fly, Stomoxys calcitrans (L.), the survival rate of larvae was generally higher when the larvae were reared on egg yolk medium inoculated with Acinetobacter sp. and Flavobacterium odoratum compared with Escherichia coli and Empedobacte (Lysyk et al. 1999). Chryseobacterium sp. and Sphingobacterium sp. were associated with cellulose or xylan digestive systems, respectively, in the termite Reticulitermes speratus (Kolbe) (Cho et al. 2010) and the longhorn beetle Batocera horsfieldi (Hope) (Zhou et al. 2009). Regiella insecticola is strongly correlated with its host plant range (Tsuchida et al. 2002, 2004) and its resistance to entomopathogenic fungi (Scarborough et al. 2005). In the sweetpotato whitefly, Bemisia tabaci (Gennadius), the association between whitefly biotypes and secondary symbionts indicated a possible contribution of these bacteria to host characteristics, such as insecticide resistance, host range, and speciation (Chiel et al. 2007, Thierry et al. 2011). It is widely accepted that bacteria greatly facilitate both the evolution and the ecological diversification of major hemipteran lineages (Baumann 2005, Moran et al. 2008).
Brown planthopper individuals harbor eukaryotic yeast-like symbionts found in the fat body cells of the abdomen (Chien et al. 1981). These yeast-like symbionts can supply its host with proteins during embryonic and postembryonic development (Lee and Hou 1987) and play a key role in recycling uric acid (Hongoh et al. 2000). The density of these yeast-like symbionts decrease when brown planthoppers are transferred from a susceptible to a resistant rice variety. The yeast-like symbionts increase in abundance in the subsequent second and third generations feeding on the resistant plant variety (Lu et al. 2004). The occurrence of these symbionts, therefore, may be related to the variation in brown planthopper performance and adaptation to rice resistance. Recently, Wolbachia or Arsenophonus were detected in all 15 brown planthopper populations collected from China and southeastern Asian countries (Qu et al. 2013). Subsequent analysis of the symbiotic bacteria from the different brown planthopper populations using denatured gradient gel electrophoresis (Xu et al. 2014) and the 16S rRNA gene sequences (Tang et al. 2010) showed that these methods might be effective means to study the structure and diversity of bacterial communities in some brown planthopper populations. However, little is known about the infection frequency (IF) and the dynamics of these bacterial communities in different populations of brown planthopper. Moreover, the associations between specific bacterial microbes and brown planthopper populations and their virulence to rice are less understood. We present herein a study of the bacterial diversity associated with different brown planthopper populations and the bacterial IF in brown planthopper populations of different generations. The relation between bacteria and brown planthopper adaptation to resistant rice varieties will also be discussed.
Materials and Methods
Insect source and rearing.
Four populations of brown planthoppers were established by collecting adults from field-grown rice at the China National Rice Research Institute (CNRRI, Fuyang, Zhejiang, China). One thousand insects were transferred to each of four different varieties of rice grown in the laboratory and reared exclusively on the respective variety for at least 30 generations prior to use in this study. Each of these varieties possessed either the bph1 gene (Mudgo), the bhp2 gene (ASD7, IR42), or the bph3 gene (IR56) that impart host resistance for brown planthopper. The name designating each population corresponded to the name of the rice plant variety (Table 1). An additional four populations were established from field-collected insects at CNRRI in 1995 (population TN1-1995); at CNRRI in 2009 (population TN1-2009); Shunhua, Vietnam (population TN1-VN); and Los Baños, Philippines (population TN1-PP) (Table 1). These latter four populations were reared exclusively on the TN1 rice variety. The temperature in the insectary where these populations and plants were reared was maintained at 25°C. About 100 individuals were collected and frozen at −70°C from each generation.
DNA extraction.
Thirty planthoppers were randomly chosen from each of the populations TN1-1995, Mudgo, and ASD7, as well as individuals collected from field-grown rice at CNRRI. These were frozen at −70°C for 5 min, immersed in 70% ethanol for 1 min, and then rinsed with sterile distilled water five times to remove surface microorganisms. Total DNA from each planthopper population was extracted using a DNeasy blood and tissue kit (Qiagen, The Netherlands) and used for 16S rRNA amplification and quantitative real-time polymerase chain reaction (qRT-PCR). The protocol for DNA extraction from a single brown planthopper is as follows: An insect was crushed in 150 μl of extraction buffer (1.0% SDS, 0.1 M Tris-HCl, pH 9.0, 0.05 M EDTA, 0.1 M NaCl, and 6.486% sucrose) using a grinding rod in a 1.5-mL centrifuge tube and then lysed at 56°C for 1 h, after which 28 μL of 8.0 M KAC (pH 9.0) was added and the tube was placed on ice for 30 min. Total DNA was then extracted with equal volumes of chloroform–isoamyl alcohol (24:1) and precipitated with 2 volumes of cold ethanol. The DNA extracted from a single brown planthopper was used for IF analysis.
16S rDNA amplification and endonuclease digestion.
The 16S rRNA genes of bacteria associated with brown planthopper were amplified by polymerase chain reaction (PCR) with the 16S rDNA universal primers (Table 2). The PCR reaction mixture was 20 μL containing 100 ng of template DNA, 0.5 mmol/L primers, 1× PCR buffer, 0.2 mmol/L of dNTPs, and 0.125 U/μL Taq polymerase (Takara, Japan). The PCR program was as follows: 5 min at 95°C, 40 cycles of 45 s at 94°C, 1 min at 52°C, and 1 min at 72°C, followed by 10 min postamplification at 72°C. The template DNA was extracted, having been randomly selected from 30 brown planthoppers from the TN1-1995, Mudgo, ASD7, and CNRRI field-collected populations. Each DNA sample was repeated three times for PCR amplification to obtain sufficient PCR products for subsequent purification and cloning. Primers that are specific to the ß-actin gene (EU179846) of brown planthopper and 18S rDNA of the yeast-like symbiont were used to confirm DNA extraction. PCR products of almost full-length 16S rDNA were purified from agarose gel and ligated into TOPO TA cloning vector (Invitrogen, USA) and used to transform E. coli. Sixty E. coli–positive colonies for each brown planthopper population were subjected to an additional PCR with the standard vector primers M13, and bands of 1,500 bp were amplified. Enzymatic digestion was performed by incubating 10 μL of the amplified product with 10 U of the enzyme and 2 μL of the 10× buffer R in a total volume of 20 μL. The four endonucleases—MseI, EcoRI, HindIII, and MspI (MBI Fermentas, Germany)—were tested. The reaction mixture was incubated at 65°C for 3 h (MseI) and at 37°C for 1 h (MspI). Restriction fragments were separated on 3% agarose gels.
Sequencing and analyses of 16S rDNA.
The corresponding E. coli clones, which were identified to have different restriction patterns with MseI or MspI digestion, were then sequenced. Only sequences >1,200 bp were considered for this study. The CHECK_CHIMERA program (http://rdp.cme.msu.edu) was utilized to remove chimeric sequence. The remaining sequences were blasted at the National Center for Biotechnology Information (NCBI). Operational taxonomic units (OTUs) for the sequences were estimated by the computer program DOTUR (Schloss and Handelsman 2005). The homological sequences were selected and downloaded from NCBI. Multiple sequence alignment was performed using the software Clustal X 1.83 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/clustalx1.83.zip). Phylogenetic trees were constructed using the software mega4.1 (http://www.megasoftware.net) by the neighbor-joining method, and the robustness of the tree topology was tested by bootstrap analysis with 1,000 resamplings (Felsenstein 1985).
Analysis of bacterial density in different brown planthopper populations with qRT-PCR.
The density of total bacteria, Enterobacteria, Chryseobacterium, Arsenophonus, and yeast-like symbiont was assayed by qRT-PCR analysis of rDNA with specific primers listed in Table 2. qRT-PCR was conducted on ABI7500, and the accompanying software was used for data normalization and quantification. The PCR reaction mixture was 20 μL, which contains 100 ng of template DNA, 1× SYBR Green PCR mix (ABI), and 0.4 μM of primer. The amplification consisted of the following four steps: initial denaturation for 10 min at 95°C, 40 cycles of 20 s at 94°C, 1 min at 60°C (with single data acquisition), and followed by melting curve analysis starting from 60°C and slowly heating to 95°C with a transition rate of 0.1°C/s (with continuous data acquisition). Each population DNA has three biological replicates. Each DNA sample was extracted from 30 emergent adults (15 females, 15 males) of each population. A ß-actin gene (EU179846) in brown planthopper was used as internal control and the sample TN1 population for calibration. The data analysis was according to the 2-ΔΔCT method reported by Livak and Schmittgen (2001), where ΔCt = Ct (target) − Ct (ß-actin), ΔΔCt = ΔCt (Sample) − ΔCt (Calibrator), Relative quantity = 2-ΔΔCt. Thus, the calibrator becomes the 1× sample, and all other quantities are expressed as an n-fold difference relative to the calibrator.
Bacterial IFs in different brown planthopper populations.
The bacterial IF was tested by PCR amplification of rDNA using genus-specific PCR primers (Table 2). More than 95 adults (equal number of females and males) for each population were collected and ground individually for DNA extraction. The PCR reaction mixture was 20 μL containing 2.0 μL 10× PCR buffer, 0.2 mmol/L of each dNTP, 0.5 mmol/L of each primer, 0.5 U of Taq DNA polymerase (Takara, Japan), and 1.0 μL of template DNA. β-actin was used as the internal control. The PCR program was as follows: 5 min at 95°C, 40 cycles of 45 s at 94°C, 30 s at 58°C, and 45 s at 72°C, and followed by 5 min postamplification at 72°C. The PCR products were separated on 2% ethidium bromide–stained agarose gel by electrophoresis.
Results
Symbiotic bacterial diversity in brown planthopper.
PCR analysis of three laboratory populations (TN1-1995, ASD7, and Mudgo) and one field population from CNRRI yielded 200 positive E. coli clones verified by using the M13 primer and digested with four endonucleases (MseI, EcoRI, MspI, and HindIII). MseI and MspI digestion of these positive clones generated 13 and 22 restriction patterns, respectively, some of which are shown in Fig. 1. Digestion with HindIII indicated that no HindIII restriction site exists. Only two kinds of restriction patterns were obtained upon digestion with EcoRI.
Citation: Journal of Entomological Science 50, 1; 10.18474/0749-8004-50.1.47
Twenty-four sequences, ranging in length from 1,466 to 1,507 bp, were obtained and deposited in GenBank under accession numbers GU124492–GU124507 and JQ975877–JQ975885 (Table 3). The guanine-cytosine contents of those fragments were mostly from 50.2% to 56.6%, except one of 46.9%. These sequences were grouped into 18 OTUs based on 97% similarity cutoff values (Fig. 2). The symbiotic bacterial communities in the brown planthopper populations appeared to be dominated by Proteobacteria (164 clones, more specifically, gamma- and alpha-proteobacterial) and Bacteroidetes (31 clones) (Table 3, Fig. 2).
Citation: Journal of Entomological Science 50, 1; 10.18474/0749-8004-50.1.47
In the CNRRI field population, 41 positive E. coli clones were digested with 35 of those possessing the same restriction patterns. One of those, W269 shares >99% sequence similarity with AY754820 (Wolbachia endosymbiont of the phytoseiid mite Metaseiulus occidentalis (Nesbitt)), indicating that Wolbachia is the dominant community in the brown planthopper population collected from field-grown rice at CNRRI. The TN1-1995 population had 52 of 57 E. coli clones exhibiting the same restriction pattern. Among those, TN68 was found to share >99% similarity with EU043378 (Arsenophonus endosymbiont of the psyllid Glycaspis brimblecombei Moore), indicating that Arsenophonus is the dominant bacterial community in the TN1-1995 population. In the Mudgo population, two dominant communities (clones M157 and M142) were found to share 98% similarities with Bacteroidetes symbionts of Chryseobacterium and Sphingobacterium, respectively. The dominant bacterial communities were found belong to five genera—Arsenophonus (58 of 200), Wolbachia (35 of 200), Serratia (17 of 200), Acinetobacter (36 of 200), and Chryseobacterium (27 of 200).
Bacterial density in brown planthopper populations.
The qRT-PCR analysis showed that the densities of Enterobacteriace and Arsenophonus in TN1-1995 and ASD7 populations were significantly higher than those in Mudgo population (Table 4). There were no significant differences in density of Chryseobacterium and yeast-like symbiont among the four populations evaluated, although the average density of Chryseobacterium was slightly higher in the Mudgo and ASD7 populations than in the TN1-1995 population (Table 4). No Wolbachia was detected in the laboratory populations because it can only be cloned from the field population.
Bacterial IFs in brown planthopper populations.
Genus-specific primers for nine bacterial endosymbionts (five prevalent in brown planthoppers, four from other insects) were used in PCR analysis to assess infection frequency of the bacterial symbionts in brown planthopper. Based on these analyses, Arsenophonus occurred at a higher frequency (57.3%) in TN1 populations (TN1-1995, TN1-2009, TN1-VN, TN1-PP) than in the Mudgo (3.18%) and ASD7 (35.7%) populations. Furthermore, the IF of Arsenophonus increased by 50% when the planthoppers were reared on the TN1 rice variety for >15 generations, irrespective of where and when the original population was collected. The IF of Wolbachia, however, was reduced by continued experience and rearing on Mudgo (G161), TN1-1995 (G152), and ASD7 (G161) (Table 5). Using Student's t-test, no significant difference was observed in either Arsenophonus or Wolbachia IF in response to insect gender. All, or almost all, of the planthopper individuals (total 850) tested harbored Acinetobacter (848), Serratia (800), Chryseobacterium (847), and yeast-like symbiont (850). Hamiltonella defense, Rickettsia, Cardinium, and Flavobacterium, previously reported from whitefly or aphid, were not detected in brown planthopper.
The IF dynamics of Arsenophonus and Wolbachia were assessed in the TN-2009, IR42, and IR56 populations to explore whether harboring Arsenophonus and Wolbachia was related to virulence to planthopper-resistant varieties of rice. Approximately 100 insects were randomly selected and assayed from generation 0, 8, 18, 24, and 30 of those laboratory-established populations. The IF of Arsenophonus varied with respect to different planthopper populations. In TN1-2009, the IF of Arsenophonus increased from 36.7% in generation 9 to 63.9% in generation 30, and then remained at 50–60% thereafter (Table 3). Similar results were seen with the Arsenophonus IF dynamics in the IR56 population, which increased from 29.7% in generation 8 to 83.3% in generation 30, and then remained at 80% thereafter. However, the IF of Arsenophonus in IR42 did not change dramatically (Fig. 3A). The IF of Wolbachia was 35.8% in the CNRRI field population and 5.0% in the laboratory populations (IR56, IR42, TN1-2009) tested. A decline of Wolbachia IF was also observed when planthoppers were maintained under laboratory conditions (Fig. 3B). Occurrence of both Arsenophonus and Wolbachia in the same individual was extremely low, with detection of both in only 5 of 1,500 insects tested.
Citation: Journal of Entomological Science 50, 1; 10.18474/0749-8004-50.1.47
Discussion
Our results show that the symbiotic bacteria associated with the brown planthopper populations examined in this study were predominately proteobacteria, more specifically gamma- and alpha-proteobacteria and Bacteroidetes. Gene sequences were grouped into 18 OTUs (Fig. 2). The OTU1, OTU9, OTU12, and OTU15 identified were similar to OTU1, OTU9, OTU15, and OTU3 reported by Tang et al. (2010), which belong to genus Arsenophonus, Acinetobacter, Arthrobacter, and Wolbachia, respectively. We did not obtain OTU2, which corresponds to Asaia krungthepensis cloned from planthoppers on the rice variety Mudgo by Tang et al. (2010). The reason for this inconsistency is possibly due to an inadequate number of samples or contamination. In addition, different environmental conditions and nutritional supplies for brown planthopper may affect the diversity of bacteria they may harbor. The OTU4 and OTU5 were only cloned from the Mudgo population, but a diagnostic PCR-based survey confirmed that these bacteria were also present in TN1-1995 and ASD7 populations at frequencies of 98%, suggesting that the dominant bacterial communities have masked the detection of those in low abundance. Future use of high-throughput sequencing-based metagenomics approaches for individual insects are likely to provide more comprehensive information on the composition of the microbiota in brown planthopper.
The incidence of Wolbachia, Arsenophonus, and more recently Cardinium, has received much attention in studies of arthropod biology (Duron et al. 2008). The proportion of Wolbachia-infected species was estimated to be 66% (Hilgenboecker et al. 2008). The genera Arsenophonus and Cardinium are estimated to be harbored by approximately 5–6% of arthropods (Gherna et al. 1991, Zchori-Fein and Perlman 2004). These symbionts are manipulators of insect reproduction (Engelstädter and Gregory 2009), may be involved in physiological responses of hosts to external stress (e.g., host immune response to parasitoid and virus attacks) (Bian et al. 2010, Fytrou et al. 2006, Teixeira et al. 2008), and host cost related to insecticide resistance (Duron et al. 2006), phytopathogenicity (Zreik et al. 1998), or obligatory mutualism (Burke et al. 2010, Perotti et al. 2007). We did not detect Cardinium in this study, while Acinetobacter, Serratia, Chryseobacterium, Wolbachia, and Arsenophonus were the predominant symbionts detected. Serratia, Acinetobacter, and Chryseobacterium were harbored by almost all planthopper individuals tested in our study. In general, individuals harboring Arsenophonus were mostly from the IR56 or TN1 populations, while those harboring neither Arsenophonus nor Wolbachia were from the Mudgo population.
The Wolbachia and Arsenophonus IF factors differed among the brown planthopper populations examined in this study. The IF of Arsenophonus was higher in the TN1 and IR56 planthopper populations than in the IR42, Mudgo, or ASD7 populations (Fig. 3, Table 3). Furthermore, a decline in the IF of Wolbachia was observed when the planthoppers were maintained continuously under laboratory conditions. And, planthoppers from the TN1 population are virulent to nonresistant rice variety ‘Taichun Native1′, while the IR56 population insects are virulent to the IR56 variety which possesses the Bph3 resistant gene for brown planthopper. Therefore, this suggests that the maintenance of the high Arsenophonus IF in the TN1 and IR56 populations may not be related to brown planthopper virulence but rather to some unknown traits. Facultative symbionts with the capacity to supply host insects with essential nutrients might enable insect herbivores to feed on nutritionally poor plants. Conversely, symbionts that compete with insects for nutrients provided by the obligate symbiont might force insects to feed on plants of high nutritional quality (Clark et al. 2010). So, we proposed that the nutritional quality in rice variety TN1 and IR56 is similar, but different from rice variety Mudgo and IR42.
Our results reported herein demonstrate that not all brown planthoppers tested in this study harbored Arsenophonus or Wolbachia and, thus, we propose that these bacteria are not essential for survival of brown planthopper on rice varieties included in this study. We also provided a relatively complete profile of the bacterial communities associated with brown planthopper from these populations. While many questions remain unanswered, these bacteria may affect the host in a number of ways. These results will help to improve our understanding of the complicated symbiosis system in the brown planthopper. Further investigation is now required to document changes in bacterial profiles generated from brown planthopper populations to correlate these variations with virulence to rice varieties.
Restrictive patterns of 16S rDNAs of endosymbiotic bacteria associated with brown planthopper populations. Polymerase chain reaction (PCR) products digested with (A) Mse I or (B) Msp I, with the numbers 1 to 23 indicating positive clones.
Phylogenetic tree (without outgroups) of the endosymbiotic bacteria associated with brown planthopper based on 16S rRNA gene nucleotide sequences. Numbers above branches indicate parsimony percent bootstrap values after performing 1,000 bootstraps. Sequences in this study are indicated by a triangle. Other sequences were obtained from GenBank, with accession numbers in parentheses: Erwinia amylovora (GQ131594), Chryseobacterium sp. (HQ895718), Chryseobacterium jll (AY278484), Sphingobacterium sp1 (EU483665), Sphingobacterium sp2 (JN860316), Arthrobacter sp1 (EF379937), Arthrobacter sp2 (AY940423), Serratia marcescens 1 (EU233275), Serratia marcescens 2 (DQ417332), Enterobacter aerogenes (DQ857896), Pantoea ananatis (DQ517335), Arsenophonus (EU043378), Acidovorax sp. ( HQ857618), Acinetobacter sp1 (FR677019), Pseudomonas putida (HQ164547), Wolbachia (AY754820), Staphylococcus sciuri (AY940424), Massilia timonae (NR026014), uncultured bacterium (KC119316), Massilia sp. (FJ005057), Acinetobacter baumannii (FJ550344), Acinetobacter sp2 (EF031061).
Infection frequencies of (A) Arsenophonus and (B) Wolbachia in three brown planthopper laboratory populations (TN1, IR56, and IR42) reared on the respective rice varieties Taichung Native1, IR56, and IR42 for numbers of generations indicated.
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