The salivary glands of insects are paired structures and immersed in haemolymph inside the head and thorax^{[1, 2]}. Researches on insect salivary glands have been involved in many aspects,including its structure,functions,saliva composition and properties,etc^{[3, 4, 5, 6]}. Saliva of insects contains a mixture of amino acid,protein,and many digestive enzymes,which plays an important role in both food ingestion and interactions between herbivores and their host plants^{[7, 8]}. Salivary glands of the Hemiptera are the most complex glands among insect groups,which are closely associated with the peculiar pierce-sucking feeding mode^{[9, 10]}. Regarding phytophagous hemipterans subsisting exclusively on plant xylem or phloem sap,the saliva plays an important physiochemical role during the mechanical penetration of plant tissues by the piercing-sucking mouthparts,and the saliva may vary in its chemical composition and physical consistency from one moment to the next to accomplish penetration^{[11, 12, 13]}.

Moreover,the salivary glands of some hemipteran insects are also one of the sites harboring microorganisms^[14]. For example,the endosymbionts Candidatus Cardinium hertigii and Candidatus Phytoplasma vitis ofthe ‘Bacteroidetes’ were found in the salivary glands of the leafhopper Scaphoideus titanus^[15]. Acetic acid bacteria (AAB),especially members of the genera Acetobacter and Gluconacetobacter,and the versatile acetic acid bacterial symbiont Asaia sp.,were reported in the salivary glands of S. titanus as well^[16]. Coccoid or rod-shaped bacteria were consistently found in the salivary glands of planthopper Pentastiridius leporinus (L.)^[17]. The endosymbionts Candidatus Liberibacter asiaticus has been densely aggregated within the cells of salivary glands of psyllid Diaphorina citri Kuwayama,which is the insect vector of citrus huanglongbing disease^[18]. Facultative secondary symbionts Rickettsia were found in the salivary glands of whitefly Bemisia tabaci (Gennadius)^[19]. Two groups of betaproteobacterial endosymbionts were reported in the salivary glands of aphid Adelges tsugae Annand^[20]. Athough the microbial community in salivary glands of hemipteran insects may play potential importance for their hosts,few data on the complexity of this biota are available.

Like other sap-feeding hemipterans,cicadas habitat obligate vertically transmitted bacterial symbionts Candidatus Sulcia muelleri in their bacteriomes to supply the host with nutrients that are limiting in their diet^{[21, 22]},but it is unknown if the endosymbionts and/or other bacteria were distributed in their salivary glands. Zhong et al^[23] and Zhong et al^[24] studied the ultrastructure of salivary glands in cicadas K. caelatata Distant and Platypleura kaempferi Fabricius,respectively,and rod-shaped microorganisms were unexpectedly observed in K. caelatata,but no microorganisms were observed in that of P. kaempferi. However,the identity of the microorganisms residing in the salivary glands of K. caelatata remains unknown^[23].

Considering information on the bacteria associated with cicada salivary glands was very limited,we investigated the bacterial communities in the salivary glands of a common cicada,Hyalessa maculaticollis (Motschulsky),in China using 16S rRNA-RFLP analysis,for the specific purpose of clarifying the occurrence of bacteria and/or special endosymbionts and their community in this special microenvironment. The results may be formative for further understanding the microbes in the salivary glands of cicadas and their potential roles in cicadas’ nutrition and feeding behavior.

Three male and three female adults of the cicada H. maculaticollis were collected from the host plants Salix babylonica in Mts. Taibai (Meixian County,Shaanxi,China) in August of 2012. The cicadas were put into a voile-cage and transferred alive to the lab for dissection as soon as possible.

Before dissection,the cicadas were first narcotized by putting in 4 °C for a few minutes,and then the body were externally sterilized with 75% ethanol and rinsed with sterile water for several times. Cicadas were fixed on a wax-plate with sterilized 4# insect needles,and the cuticula were cut off along the back middle line from the anus to the head with sterilized 10 cm scissors under a Stereoscopic Zoom Microscope (Motic SMZ168,Xiamen,China). The salivary glands were carefully separated from other organs with sterilized fine 14 cm forceps,washed three times with sterilized water to reduce the contamination,and then put into a 1.5 ml microcentrifuge tube for DNA extraction. All works were done in the laminar flow cabinet (Taisite,Tianjin,China).

Genomic DNA was extracted from the pooled salivary glands tissue by using TIANamp Genomic DNA kit (TIANGEN Inc.) according to the manufacturer’s directions. 16S rRNA gene was amplified from genomic DNA extracted from salivary glands of H.maculaticollis with paired primers 27F (5¢-AGAGTTTGATCMTGGCTCAG-3¢) and 1492R (5¢-TAGGGYTACCTT GTTACGACTT-3¢). PCR was performed using a Peltier Thermal Cycler (Eppendorf,Germany). The amplification was performed in 25 ml PCR reactions system,containing: 1 ml Temple DNA,2.5 ml of 10×PCR buffer,1.5 ml of MgCl₂ (25 mmol/L),2 ml of dNTP Mix (10 mmol/L),1 ml of each primer (10 mmol/L),0.25 ml of Taq DNA polymerase (5 U/mL,Takara Bio. Inc.),and adding sterile ddH₂O to 25 ml. The PCR reaction condition was as follows: initial denaturation (2 min at 94 °C) followed by 25 cycles of denaturation (0.5 min at 94 °C),primer annealing (0.4 min at 55 °C),and primer extension (1 min at 72 °C) and a final extension step (7 min at 72 °C).

PCR products were purified using TaKaRa Agarose Gel DNA Purification Kit (Takara Bio. Inc.) following its instructions. In order to construct 16S rRNA gene clone library,PCR product was ligated into Enterobactercoli DH5α competent cell (TIANGEN Inc.) and then inserted into pMD19-T vector using heat-shock method according to the specification of the manufacturer (Takara Bio. Inc.). A dilution series of transformed Enterobactercoli cells ligated 16S rRNA inserts were plated on Luria-Bertani agar plates with 100 g/ml Ampicillin and 40 µl of 40 m/l X-gal and IPTG (Takara Bio. Inc.). The plates were incubated at 37 °C for 24 h,and then performed blue-white screening. Two hundred sixty of white clones were selected randomly and amplified using M13 vector primers to check if they were recombined plasmids. The PCR products amplified by M13 vector primers were digested independently with restriction endonucleases Afa I and Hha I for 4 h in 37 °C according to the specifications of manufacturer (TaKaRa Bio. Inc.). The restriction PCR fragments were separated by 2% agarose gel electrophoresis in 1×TAE buffer. The gel were stained with ethidium bromide and visualized under UV light. The restriction patterns of clones were compared and visually grouped. One to three representative clones for each unique RFLP pattern was selected for sequencing.

All sequencing was done by Sangon Biotech Company (Shanghai,China). Sequences were manually corrected and assembled by SeqMan program (DNAStar,Inc.). Sequences were blasted both in GenBank and Ribosomal Database Project (http://rdp.cme.msu.edu/) to find their closest relatives. All sequences we obtained and their best matched sequences were aligned using Clustal 2.1^[25] and then they were added to construct Maximum Likelihood tree in MEGA 5.0^[26]. Sequences with above 97% identity were defined as same operational taxonomic unit (OTU),and their taxonomic status was determined based on the position of each sequence in the phylogenetic tree. Rarefaction curve was conducted using Analytic Rarefaction 1.3 (http://strata.uga.edu/software/) to evaluate the efficiency of clones sampled. The diversity indices were calculated with the following equations^[27].

Shannon-Wiener index:

Where N is the number of total clones in the clone library; n_i is the clone number of ith bacteria; p_i is the proportion of clones in the ith bacteria; S is the number of bacteria species.

Simpson index:

Where N is the number of total clones in the clone library; n_i is the clone number of ith bacteria; p_i is the proportion of clones in the ith bacteria; S is the number of bacteria species.

The 16S rRNA gene sequences of the bacteria isolated from salivary glands of H. maculaticollis are available under accession numbers KJ569762− KJ569768.

We analyzed bacteria in the salivary glands of the cicada H.maculaticollis using PCR-RFLP analysis. Thirty-eight (38) different RFLP patterns were identified from 235 positive clones after digesting by two restriction endonucleases Afa I and Hha I (Figure1). The representative clones of the 38 RFLP patterns were sequenced,and their sequence blasting results indicated that they were belonging to Proteobacteria and Firmicutes,representing seven bacterial species: Pseudomonas aeruginosa,Thermomonas brevis,Enterobacter sp.,Sphingomonas sp.,Methyloversatilis sp.,Anaerococcus sp.,and Bacillus sp. (Table1).

图1  斑透翅蝉唾液腺细菌克隆文库的PCR-RFLP图谱 Figure 1  PCR-RFLP profiles of the 16S rRNA gene of the bacteria from the salivary glands of H.maculaticollis Note: The restriction profiles were obtained after sample digestion with the restriction enzymes Afa I and Hha I,and Afa I restriction map is on the upper layer; Hha I restriction map is on the under layer; lanes 1 to 230 show representative clones of different restriction profiles. 注：该图谱由两种限制性内切酶Afa I和Hha I消化而成；Afa I酶切图谱位于上层，Hha I酶切图谱位于下层；条带标号代表了不同的限制性片段条带类型.

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Among the detected bacterial species,P. aeruginosa andEnterobacter sp. were the most frequently species obtained in the clone library of the salivary glands of H. maculaticollis (each of them accounting for 48.7% of the clone library). Whereas other five bacterial species were only discovered at very low levels with the same relative abundance which accounting totally for 2.05% of all clones. The bacterial diversity of the library was low (Shannon index: H′=0.82,1-Simpson index: 1-D=0.52,which indicated that two individuals picked at random from this community would be different species in 52% of all cases). The endosymbiont Candidatus Sulcia muelleri were not detected in the salivary glands of H. maculaticollis which has been found in other cicadas^[22].

Although the rarefaction curve has a rising trend at 3% difference between sequences (95% confidence intervals) (Figure2),nearly 97.4% clones were found to belong to the two dominant bacterial species Enterobacter sp. and Pseudomonas aeruginosa. Hence,the number of clones sampled was sufficient to provide an accurate estimation of bacterial diversity in salivary glands of H.maculaticollis.

图2  斑透翅蝉唾液腺细菌16S rRNA基因克隆文库稀有度曲线 Figure 2  Rarefaction curve for 16S rRNA gene libraries constructed from salivary glands of H.maculaticouis Note: The rarefaction curve describe the equilibration of discovering bacteria species in the clone library of cicada H. maculaticollis; the error bars represent 95% confidence intervals which were automatically generated by the software Analytic Rarefaction for richness values. 注：稀有度曲线描述了斑透翅蝉唾液腺细菌克隆文库中所发现细菌种类的饱和度；误差线代表了稀有度分析软件自动生成的用于评价丰富度的95%置信区间.

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The Maximum Likelihood (ML) phylogenetic tree was constructed using the sequences of the seven representative clones isolated from the salivary glands of H.maculaticollis and their best matched sequences downloaded from GenBank (Figure3). The results of phylogenetic analysis indicate that all these bacteria were belonging to four distinct groups,i.e.,Gammaproteobacteria,Alphaproteobacteria,Betaproteobacteria and Firmicutes.

图3  斑透翅蝉唾液腺细菌16S rRNA基因序列以及数据库中比对序列的最大似然发育树 Figure 3  The Maximum likelihood tree based on bacteria 16S rRNA gene sequences of the salivary glands of H. maculaticollis,including selected database sequences Note: The tree was constructed through MEGA 5.0 with Tamura-Nei parameter model and complete deletion treatment for gaps and missing data; GenBank accession Numbers are provided in the parentheses; Numbers at each branch point indicate the percentage supported by bootstrap values based on 2 000 replications; The representative clones identified in this study are listed in boldface type followed by GenBank accession numbers. 注：发育树由MEGA 5.0软件根据Tamura-Nei参数模型自展2 000次构建，比对时对序列进行校正；括号内为该序列的序列号；分支节点处的数字为置信度；代表克隆子序号以及Genbank序列号用粗体表示.

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Clone-219 (KJ569768) was identified as bacterium Thermomonas brevis (NR_025578.1) with 97% support. Clone-54 (KJ569763) was identified as bacterium Pseudomonas aeruginosa (JF513147.1) with 99% support. Clone-202 (KJ569767) was clustered with bacteria Enterobacter cloacae (JQ993364.1),Enterobacter sp. (EU545406.1) and Enterobacter cancerogenus (JN644607.1) with 99% support,but its identity was unclear. All the above-mentioned clones formed a unique,well-supported branch,belonging to the class Gammaproteobacteria.

Clone-152 (KJ569765) was grouped with Methyloversatilis sp. (KC860260.1) with 99% support; while clone-116 (KJ569764) was grouped with Sphingomonas sp. (JQ396558.1) and Sphingomonas hankookensis (JQ660164.1) with 99% support. These two isolated bacteria were identified in the class Betaproteobacteria and Alphaproteobacteria,respectively.

Clone-164 (KJ569766) was identified as Anaerococcus sp. (HM587322.1) with 96% support. Clone-6 (KJ569762) was clustered with bacteria of Bacillus,Planococcus and Chryseomicrobium with 99% support,but its identity remains unknown. Clones-164 and clone-6 are members of the class Firmicutes,but belong to different families,viz. Clostridiales and Planococcaceae,respectively.

The microbiota inhabiting in the salivary glands of the cicada H. maculaticollis were investigated and characterized using culture-independent PCR-RPLP techniques for the first time. Seven bacterial species were identified in the clone library,and bacteria in the genera Pseudomonas and Enterobacter were dominant. The salivary glands of insects are known as one of the most important barriers to the transmission of pathogens^{[18, 28]}. Many antimicrobial peptide and proteins have been found in the salivary glands and saliva of several herbivore insect species can help overcome defensive responses of host plants^{[5, 6]}. The occurrence of bacteria in the salivary glands of different insects (e.g.,fruit flies,mosquitos and leafhoppers) has been suggested to serve as a route for their horizontal transmission to plants or other intermediate hosts^{[29, 30]}. Herein,the seven bacteria found in the salivary glands of H.maculaticollis may imply that these bacteria can overcome the barrier and adapt the special microenvironment of salivary glands to live. However,more studies are required to clarify the bacterial community and their exact functions in the salivary glands of the Cicadidae.

Members of Enterobacter are common components of insect gut microbiota,which have been found in many insects^{[31, 32, 33]}. For example,some bacteria of Enterobacter were found to be the most prominent microorganisms in the gut of mosquitoes Aedes triseriatus (Say),Culex pipiens L.,and Psorophora columbiae (Dyar & Knab)^[34]. Several species of this bacterial genus have been recognized as pathogens^[35]. However,some species of Enterobacter residing in insects may be valuable to their hosts. For example,the dominant presence of the enterobacterial community in the gut of medfly Ceratitis capitata (Wiedemann) contributes to its longevity and fitness^[36],and they are also capable of contributing to the nitrogen and carbon metabolism,copulatory success and development of their hosts^{[37, 38]}. Davidson et al^[39] showed that Enterobacter cloace is mildly pathogenic and penetrates the insect gut cells on ingestion. Enterobacter also aids in the insecticidal activity of Bacillus thuringenesis in the gypsy moth^[40]. In addition,according to studies on various insects such as aphids,whiteflies,parasitic wasps and tsetse flies,the Enterobacteriaceae has generated numerous facultative symbionts^{[41, 42]}. Facultative symbionts,especially those from Enterobacteriaceae,appear to be particularly common in hosts that possess bacteriome-associated obligate symbionts,including aphids,whiteflies,tsetse flies,and mealybugs^[43]. Herein,we infer that the predominant bacterium Enterobacter sp. (EU545406.1) harbored in the salivary glands of H. maculaticollis might play a beneficial role to the cicada host,and it would be interesting in a future study to verify whether this bacterium has been evolved into a f acultative symbiont in cicadas.

Pseudomonas aeruginosa is the other dominant bacterium isolated from salivary glands of H. maculaticollis. This bacterium is usually a common opportunistic pathogen that occupies a wide variety of environmental niches^{[44, 45]}. It has been reported in the guts of several insects,such as ants,beetles,mosquitoes,and termites,etc.^{[34, 36, 37, 46, 47]}. He et al^[48] detected high levels of P. aeruginosa in the crop,midgut and hindgut of the ant Camponotus fragilis (Pergande) from the lab-raised colonies,but it was absent in the field-collected colonies. They inferred that the infection of P. aeruginosa is a laboratory artifact and may be harmful to laboratory colonies^[48]. However,P. aeruginosa can play a positive effect to its host insects,e.g.,preventing parasite establishment in the midgut of mosquitoes^[49],and having a detoxification function in Paederus beetles^[50]. Indiragandhi et al^[51] indicated that Pseudomonas sp. may have antagonistic activity towards entomopathogenic fungi in the diamondback moth. Though we revealed that P. aeruginosa was one of the most abundant species in the salivary glands of H.maculaticollis,further work is needed to clarify the effect of the infection of thi s bacterium in the salivary glands of H.maculaticollis,and to investigate if it is ubiquitous in cicadas.

Other five bacterial species occurred in very small amount. The endosymbiont Candidatus Sulcia muelleri were not detected in the salivary glands of cicada H. maculaticollis,which has been reported generally distributed in the bacteriomes of cicadas and can supply the host with nutrients limiting in their diet^[22]. This implies that this endosymbiont probably only distributes in the bacteriomes of H. maculaticollis.