The Detection of Wolbachia in Tea Green Leafhopper ( Empoasca onukii Matsuda) and Its Inﬂuence on the Host

: Tea green leafhopper ( Empoasca onukii Matsuda) is a critical pest in tea production. Wolbachia has attracted much attention as a new direction of pest biological control for its ability of manipulating the hosts’ reproductive biology. In this work, we focused on the detection of Wolbachia in tea green leafhopper and its effect on host reproduction and development. Polymerase chain reaction (PCR), real-time PCR, and ﬂuorescence in situ hybridization (FISH) techniques were used to detect the distribution of Wolbachia in tea green leafhopper. Wolbachia infection levels were different in different organs of hosts in different insect stages. In addition, comparison between the infected populations and cured population (treated by tetracyclines) revealed that presence of Wolbachia apparently inﬂuenced the growth, life cycle, and other reproductive factors of tea green leafhopper, caused, for example, by cytoplasmic incompatibility (CI), thereby reducing number of offspring, shortening lifespan, and causing female-biased sex ratio. This research conﬁrmed that the bacteria Wolbachia was of high incidence in tea leafhoppers and could signiﬁcantly affect the hosts’ reproductive development and evolution. The standard curve was generated according to the relation between the wsp gene concentration in total DNA and cycle threshold (CT) (Figure 2). The regression equation was established as y = − 2.864x + 32.51, where R 2 = 0.9816 indicates that the concentration of wsp gene in the diluted sample had a good linear relationship. Therefore, the content of Wolbachia in different samples could be calculated by measuring CT values. (Meitan), in


Introduction
The tea green leafhopper, (Empoasca onukii Matsuda) (Hemiptera: Cicadellidae), is the major pest affecting tea production in China [1]. The tea green leafhopper, because of its small size and high mobility, is usually unnoticeable until it proliferates in large numbers. It has hemimetabolous development and has 9 to 11 generations a year in the tea producing areas [2,3]. A tea green leafhopper pierces and sucks the leaf sap and causes tea seedlings to wither and become brown [4,5] and the female adults lay eggs in the young tea shoots, which ultimately leads to serious loss on the quality and yield of tea production [6][7][8][9]. The controlling method of this pest mainly relies on frequent use of pesticides, which might lead to pesticide residues in tea, resistance of insects, pollution of the environment, and destruction of natural enemies [4,10,11]. Since the diversity and distribution of symbiotic bacteria Wolbachia has been studied in many groups of arthropods including leafhoppers [12][13][14], it is expected to be an eco-friendly alternative management method to control tea leafhopper by inhibiting or antagonizing symbiotic bacteria and realize the green and sustainable development of tea production.
An endosymbiont lives within the body or cells of another insect forming an endosymbiosis and interacting with the host in a symbiotic or parasitic way [15][16][17][18]. Some examples of endosymbiosis are bacteria [17][18][19], microsporidia [20], and RNA viruses [21], which can manipulate reproduction of the host. The well-studied endosymbiont Wolbachia, a genus of intracellular bacteria, can infect arthropod species, including a high proportion of insects [22]. Studies estimate that approximately 40% of terrestrial arthropod species [23] and more than 16% of insects in the world are potential hosts of Wolbachia [24]. Its interactions with its hosts are often complex, and in some cases have evolved to be mutualistic rather than parasitic [22]. Wolbachia is usually responsible for three types of reproductive manipulation of host, namely male killing, feminization, and cytoplasmic incompatibility (CI) [22,[25][26][27]. Some Wolbachia strains have a negative effect on hosts by changing the sex ratio or resulting in a shorter insect life [22,26]. CI occurs when a Wolbachia-infected male mates with an uninfected female or when mating occurs between males and females infected with different Wolbachia strains, giving birth to progeny that die during very early embryogenesis and reducing the number of offspring [28,29]. So, Wolbachia is of considerable interest nowadays and has potential for use as a biocontrol agent due to its ubiquitous distribution and various evolutionary functions [30][31][32][33][34].
Antibiotics application could change or cure Wolbachia infection which in turn could influence the offspring production [35][36][37][38][39]. For example, antibiotic treatment cured Wolbachia infection in Encarsia Formosa and affected hosts' reproductive development [35]. These reports concluded that the symbiotic bacteria Wolbachia has impact on the reproductive development of host and can be cured or regulated by antibiotics.
However, information regarding Wolbachia infection and influence on tea leafhopper in tea agroecosystems has not been investigated, except in a survey about different symbiotic bacterial infections in tea garden insects [40]. Here, we revealed the Wolbachia infection status of tea green leafhoppers from different geographical tea gardens and variation in infection rates of Wolbachia in different insect stages and followed the potential influence on the development and reproduction of tea green leafhopper by using polymerase chain reaction (PCR)-based detection, fluorescence in situ hybridization (FISH), and tetracycline treatment comparison.

Insect Collection
Sample Collection: The adults of tea green leafhopper were collected from four counties in Guizhou province, at an average height of about 1000 m, at geographical coordinates of 106-107 degrees north(N) and 25 In addition, tea green leafhopper's eggs that were adhered to branches were collected in Jiu'an and hatched in a climate-controlled room (26 ± 1 • C, 65 ± 5%) of relative humidity (RH), 16 h:8 h light(L)/dark(D). Fresh tea branches were provided to feed the insects. The tea branches were cultured with nutrient solution [41], which was replaced every 5 days to keep the tea branches fresh.
This insect rearing method was applied for tea green leafhopper and its offspring in the study of Wolbachia's effects on insect reproductive development.

DNA Preparation
Genomic DNA from leafhoppers was extracted and examined for the presence of Wolbachia using Wolbachia-specific surface protein (wsp) primers [42]. A Biospin Insect Genomic DNA Extraction Kit (Biomars, Beijing, China) was used for total DNA extraction.
The whole body of tea leafhopper (five insects in a group) was ground using a sterile plastic pestle in a 1.5 mL centrifuge tube containing 200 µL ddH 2 O, 20 µL proteinase K, and 200 µL Buffer gA. The homogenate was incubated at 56 • C for 1 h and transferred to a spin column and centrifuged at 12,000× g for 1 min. Extracted DNA in column was washed with 500 µL Buffer PW twice and then 55 µL TE buffer was applied to elute and obtain the DNA solution, which was then stored at −20 • C for future use. The concentration of elution was determined by a Nanodrop (IMPLEN). 1 µL of the solution was used as template DNA subjected to PCR-screen for examining Wolbachia infection.

PCR Amplification
The wsp1 (81F: TGGTCCAATAAGTGATGAAGAAAC; 691R: AAAAATTAAACGC-TACTCCA), 16S rRNA (F: CGGGGGAAAATTTATTGCT; R: AGCTGTAATACAGAAAG-GAAATCGCCA) and FtsZ (F: ATYATGGARCATATAAARGATAG; R: TCRAGYAATG-GATTRGATA) [42,43] were used for amplification. The PCR reaction mixture contained 1.0 µL of genomic DNA template, 5 µL of 10× buffer, 4 µL of dNTP's (2.5 mM), 2.0 µL of each primer (10 nM of Primer F and Primer R), 0.5 µL of Pyrobest DNA Polymerase (5 U/µL). ddH 2 O was added to the mixture to make a final 50 µL reaction solution. The PCR program was initially started by 32 cycles of amplification at 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 60 s, followed by one cycle of amplification at 94 • C for 30 s, 52 • C for 30 s, and 72 • C for 60 s, and a final extension at 72 • C for 60 min. PCR was performed using a 2720 Thermal Cycler (Applied Biosystems, USA). The PCR products were separated on a 1% (w/v) agarose gel stained with ethidium bromide (EB) and photographed by a gel imaging system.

Real-Time PCR
Symbiotic bacterial infection was detected on real-time PCR by using the wsp2 (F: TG-GAACCCGCTGTGAATGAT; R: GCACCATAAGAACCGAAATAACG). The PCR reaction mixture was prepared as follows formula: 5 µL of 2× TSINGKE' Master real-time PCR Mix-SYBR, 0.5 µL of Primer F and Primer R, 1 µL of DNA template, and proper ddH 2 O added to achieve 10 µL final reaction solution. The reaction mixture was initially denatured at 95 • C for 600 s; followed by 40 cycles of amplification at 95 • C for 10 s, 60 • C for 10 s, and 72 • C for 10 s using a Light Cycler ® 96 (Roche, Basel, Switzerland).

Fluorescence In Situ Hybridization (FISH)
Hybridization nucleotide probe W2(5 -CTTCTGTGAGTACGTCATTATC-3 ) in FISH was applied to the nymphs and adults of tea green leafhopper collected from Jiu'an. The probe was a 5'-labeled quasar 670 (excitation wavelength: 647 nm; emission wavelength: 670 nm) according to Jureemart Wangkeeree's method [44]. Fresh samples were collected and stored in acetone until further use. Nymphs and adults were immersed in phosphatebuffered saline (PBS; 137 mM NaCl, 2.7 Mm KCl, 10 mM Na 2 KPO 4 , and 2 mM KH 2 PO 4 ), and cuticles were pricked with a needle in two or three places under a microscope to facilitate reagent infiltration. All samples were fixed overnight in Carnoy's solution (ethanol: chloroform: acetic acid, 6:3:1) at room temperature (28-35 • C) in a shaker. The samples were decolorized by immersion and 6% (v/v) hydrogen peroxide in ethanol at room temperature (28-35 • C) for 2-3 weeks to quench the autofluorescence of the insect tissues. For the hybridization reaction, buffers containing 100 nM probe were added to the samples, and the mixtures were incubated overnight at 46 • C in the dark. Non-specifically bound probes were washed three times with PBS for 10 min each time. Samples were finally mounted on a slide with a lid using a slide imaging confocal laser scanning microscope at Abace Biology. Reactions without probes or RNase digestion before hybridization were used as negative controls [45].

Antibiotic (Tetracycline) Treatment on Wolbachia Hosted Tea Green Leafhopper
As reported [37], tetracycline can eliminate the symbiotic bacteria Wolbachia in insects. According to the requirements of breeding and experiment, we designed tetracycline solutions with different concentrations (1 mg/mL, 5 mg/mL, and 10 mg/mL). Different concentrations of tetracycline solution were smeared on the tea tree twigs and leaves, which then were dried slowly. Subsequently, 20 newly hatched tea green leafhopper nymphs in each group were transferred into the fresh tea leaves in a container. Tetracycline was applied to the leaves once a day, and water served as control.
The tea green leafhoppers were treated with different concentrations of tetracycline and dynamically observed for 35 days every 5 days.

The Effect of Wolbachia on the Reproductive Development of the Tea Green Leafhopper
The naturally Wolbachia-infected populations and uninfected (tetracycline-treated populations) were compared for their longevity in different stages.
Newly emerged adults (two pairs) were randomly selected and raised in self-made vials, at 25 ± 1 • C, L/D = 16:8, and RH 65 ± 5%. Then, 15 replicates were set for each treatment. The fresh tea buds were provided and replaced every day. The eggs of tea green leafhopper attached to tea buds were collected and the number was recorded. Those eggs were then gathered in an artificial climate box and moisturized with wet filter paper until they hatched. After hatching, the newly emerged nymphs were reared with fresh tea buds and observed every 12 h for their reproductive parameters, such as the time span of each stage (spawning, egg, nymph, and adult stages), until their death.

Reproductive Regulation of Wolbachia in Tea Green Leafhopper
The populations (tea green leafhopper collected from Jiu'an) that were naturally infected with Wolbachia were marked as "W". "W−" represented tetracycline-cured populations, which were Wolbachia-free (uninfected). The mating design was set as follows: W−♀×W♂, W♀×W♂, W♀×W−♂, W−♀×W−♂(♀female, ♂male). One male and one female were matched as a crossing pair and mated in the same enclosed environment. The sex ratio of their offspring was recorded.
The data of each population was analyzed by TWOSEX-MS Chart software, and the survival rate (H), fecundity (mx), and other parameters of each population were obtained. Data were expressed as the mean ± standard error, and the data were subjected to one-way analysis of variance (ANOVA)(p < 0.05) followed by a significant difference test (LSD) using SPSS statistics 19.0 (IBM, USA) and using Sigmaplot 14.0 (Systat International Software, USA) to plot.

Wsp Content in Tea Green Leafhopper by Real-Ti
The standard curve was generated accordin concentration in total DNA and cycle threshold ( was established as y = −2.864x + 32.51, where R 2 of wsp gene in the diluted sample had a good line Wolbachia in different samples could be calculated

Wsp Content in Tea Green Leafhopper by Real-Time PCR
The standard curve was generated according to the relation between the wsp gene concentration in total DNA and cycle threshold (CT) (Figure 2). The regression equation was established as y = −2.864x + 32.51, where R 2 = 0.9816 indicates that the concentration of wsp gene in the diluted sample had a good linear relationship. Therefore, the content of Wolbachia in different samples could be calculated by measuring CT values.
hoppers from those two locations. However, no bands were det and Ftsz genes.

Wsp Content in Tea Green Leafhopper by Real-Time PCR
The standard curve was generated according to the relation concentration in total DNA and cycle threshold (CT) (Figure 2). T was established as y = −2.864x + 32.51, where R 2 = 0.9816 indicate of wsp gene in the diluted sample had a good linear relationship. T Wolbachia in different samples could be calculated by measuring C The relative amounts of wsp gene to total DNA (wsp gene co samples, including four different geographic locations, different ages, and from both sexes of tea green leafhopper were determine wsp gene concentration in total DNA could be calculated using th tion from Figure 2 and obtained content of Wolbachia in different s The relative amounts of wsp gene to total DNA (wsp gene concentration) in various samples, including four different geographic locations, different insect body parts and ages, and from both sexes of tea green leafhopper were determined by real-time PCR. The wsp gene concentration in total DNA could be calculated using the standard curve equation from Figure 2 and obtained content of Wolbachia in different samples ( Figure 3).
As shown in Figure 3A, there was no significant difference for the content of Wolbachia in samples from three (Fenggang, Jiu'an, and Meitan) of the four involved locations, with around 3.3-3.7 copies/µL of wsp gene copy, while Wolbachia could not be detected in the samples from Pingtang. Wolbachia levels in different body parts of the female insect ( Figure 3B) fit the order of abdomen > thorax > head, with a statistically significant difference observed. In addition, a female tea green leafhopper had a much higher Wolbachia amount (4.4 copies/µL) than that of a male (3.1 copies/µL) ( Figure 3C). Moreover, Wolbachia amount was found to increase from 2nd instar to 5th instar and arrived at the max at 5th instar in the nymph stage and kept increasing in the adult stages ( Figure 3D).  As shown in Figure 3A, there was no significant difference for the content of Wolbachia in samples from three (Fenggang, Jiu'an, and Meitan) of the four involved locations, with around 3.3-3.7 copies/μL of wsp gene copy, while Wolbachia could not be detected in the samples from Pingtang. Wolbachia levels in different body parts of the female insect ( Figure 3B) fit the order of abdomen > thorax > head, with a statistically significant difference observed. In addition, a female tea green leafhopper had a much higher Wolbachia amount (4.4 copies/μL) than that of a male (3.1 copies/μL) ( Figure 3C). Moreover, Wolbachia amount was found to increase from 2nd instar to 5th instar and arrived at the max at 5th instar in the nymph stage and kept increasing in the adult stages ( Figure  3D).

Localization of Wolbachia by FISH
The nymphs and adults of the tea green leafhopper in the natural population of Jiu'an were in situ hybridized to localize Wolbachia ( Figure 4A-D). It was obvious that the fluorescence intensity in the figure intensified from diagram A to diagram D, which indicated the Wolbachia content in tea green leafhopper increased from 2nd instar to adult stage.

Localization of Wolbachia by FISH
The nymphs and adults of the tea green leafhopper in the natural population of Jiu'an were in situ hybridized to localize Wolbachia ( Figure 4A-D). It was obvious that the fluorescence intensity in the figure intensified from diagram A to diagram D, which indicated the Wolbachia content in tea green leafhopper increased from 2nd instar to adult stage.

Elimination of Wolbachia in Tea Green Leafhopper
The cure effect of tetracycline (1, 5, and 10 mg/mL) on Wolbachia infection in tea green leafhopper is shown in Figure 5. Wolbachia infection continued to decline during observation, and the infection rate in groups 5 and 10 mg/mL tetracycline were completely extinguished by day 25 and day 20, respectively. Meanwhile, the infection rate in the 1 mg/mL

Elimination of Wolbachia in Tea Green Leafhopper
The cure effect of tetracycline (1, 5, and 10 mg/mL) on Wolbachia infection in tea green leafhopper is shown in Figure 5. Wolbachia infection continued to decline during observation, and the infection rate in groups 5 and 10 mg/mL tetracycline were completely extinguished by day 25 and day 20, respectively. Meanwhile, the infection rate in the 1 mg/mL treatment group fell to 60% by day 25 and maintained a gradual decline until day 35 or death of the adult. This indicated that tetracycline solution can eliminate Wolbachia, and 5 mg/mL is properly effective.

Elimination of Wolbachia in Tea Green Leafhopper
The cure effect of tetracycline (1, 5, and 10 mg/mL) on Wolbachia infection in tea green leafhopper is shown in Figure 5. Wolbachia infection continued to decline during observation, and the infection rate in groups 5 and 10 mg/mL tetracycline were completely extinguished by day 25 and day 20, respectively. Meanwhile, the infection rate in the 1 mg/mL treatment group fell to 60% by day 25 and maintained a gradual decline until day 35 or death of the adult. This indicated that tetracycline solution can eliminate Wolbachia, and 5 mg/mL is properly effective.

Effects of Symbiotic Bacteria on the Reproductive Development of Tea Green Leafhopper
The influence of Wolbachia on the insect stage development and total life span of tea green leafhopper was investigated by comparison between natural population and cured population ( Figure 6).
As can be seen, stage period was generally prolonged in the cured populations group ( Figure 6A,B,D) and hence the total life span was also prolonged ( Figure 6E). This led to the total lifespan difference between the natural group and the cured group. Since tea green leafhopper would differentiate into male and female individuals when they emerged to the adult stage, their lifespans differed and apparently females live longer, with 31 days for female and 28 days for male in natural populations and 38 days for female and 34 days for males in cured populations. In sum, the life span of the whole generation of cured populations was prolonged for about 6-7 days longer than the natural population ( Figure 6E). Therefore, it could be assumed that the symbiotic bacterium Wolbachia had an influence on the growth and development of tea green leafhopper, shortening its generation cycles.

Effects of Symbiotic Bacteria on the Reproductive Development of Tea Green Leafhopper
The influence of Wolbachia on the insect stage development and total life span of tea green leafhopper was investigated by comparison between natural population and cured population ( Figure 6). As can be seen, stage period was generally prolonged in the cured populations group ( Figure 6A,B,D) and hence the total life span was also prolonged ( Figure 6E). This led to the total lifespan difference between the natural group and the cured group. Since tea green leafhopper would differentiate into male and female individuals when they emerged to the adult stage, their lifespans differed and apparently females live longer, with 31 days for female and 28 days for male in natural populations and 38 days for female and 34 days for males in cured populations. In sum, the life span of the whole generation of cured populations was prolonged for about 6-7 days longer than the natural population ( Figure 6E). Therefore, it could be assumed that the symbiotic bacterium Wolbachia had an

Mating Result between the Wolbachia-Infected Host and Uninfected Tea Green Leafhopper
This combination of partners and their progeny's life information were investigated ( Table 1). The leafhoppers' mating was designed as the following four types: W−♀×W♂, W♀×W♂, W♀×W−♂, W−♀×W−♂(♀female, ♂male). "W" represented naturally infected populations and "W−" represented Wolbachia-free (uninfected) populations that were cured by tetracycline. The numbers of the offspring derived from different crossing pairs W−♀×W♂, W♀×W♂, W♀×W−♂, W−♀×W−♂, were 16, 18, 20, and 25, respectively, with significant deference. Obviously, the mates with cured male W−♂produced relatively more offspring than the mates with naturally infected male W♂. We also calculated the ratio between numbers of female and male offspring by sex differentiation at adult stage. The general population deviated from equilibrium by favoring female at 1.7, 1.6, 1.5, and 1.3, respectively. The mate (W−♀×W♂) produced the biggest disequilibrium and was obviously skewed towards dominance of females (1.7:1). For the mates of cured populations (W−♀×W−♂), the number of female and male offspring were close, at 14 female insects and 11 male insects, and sex ratios was less biased (1.27:1, close to 1).
The durations of the adult period for female and male offspring were also observed and recorded (Table 1). As shown, the adult period ranged from 9.5 to 12.7 days for males and 9.8-15.4 days for females. The offspring from mate W♀× W♂retained the shortest adult lifetime, at 9.5 days for males and 9.8 for females. It is quite clear that the female offspring had relatively longer lifetimes than the males. Also, new generations from mates with Wolbachia-infected males (W−♀×W♂and W♀×W♂) lived relatively short adult periods.

Discussion
In this study, the symbiotic bacterium Wolbachia was examined for its existence and effects on the host tea green leafhopper, based on wsp gene (which evolves quickly and is suitable for grouping Wolbachia strains [47,48]). According to PCR amplification, Wolbachia was present or hosted in the tea leafhopper collected from three tea-production locations (Jiu'an, Meitan and Fenggang) but absent from samples from Pingtang. The result might be caused by different altitude, climate, or ultraviolet intensity in different locations [49,50]. On the one hand, possible extremely low Wolbachia-infection rate among populations made it impossible to detect genes using PCR molecular detection. Another reason might be the random samples collection falling outside the area of highly infected clusters [51]. Also, assured by real-time PCR, wsp gene content differed among those hosts and increased with the evolution of tea leafhoppers. The FISH study verified the result and was consistent with the real-time PCR detection in terms of insect stage development comparison. Later, we used a double-negative control (no-probe and RNAse-digested control) as suggested by Nugnes et al. [45] and did not find any signal (data was not provided).
Wolbachia could be removed or cleared from insects by tetracycline and rifampicin antibiotics [52,53]. Some studies explored the effect of antibiotics on the removal of Wolbachia in different insects [54,55]. This process might not only involve bacterial clearance but also other changes in the microenvironment of the host [56]. Many studies reported that Wolbachia had obvious effects on the host's reproductive development through multiple mechanisms, mainly including CI, male killing, parthenogenesis, feminization, and enhancement of female reproduction and male fertility, etc. [25][26][27][57][58][59].
CI meant some hosts infected with Wolbachia strains showed early embryogenesis death when Wolbachia-infected males mated with uninfected females [28,29]. In this study, mating of infected male and uninfected females resulted in a tendency of CI, with the highest being 25 offspring from the one pair uninfected mate W−♀×W−♂and the lowest being 16, derived from the crossing pair of W−♀×W♂. Also, Wolbachia infection caused a female-biased sex ratio.
The inherited symbiotic bacteria play an important role in manipulating the reproductive biology of their hosts. Therefore, the use of insect symbiotic bacteria for developing new pest control technologies has important application prospects [60].

Conclusions
In this study, the presence and influence of symbiotic bacterium Wolbachia was investigated in tea green leafhoppers, the main pest in tea gardens. Wolbachia presence could manipulate evolution of tea green leafhopper hosts by inducing changes in host reproductive and sex-determination systems. The frequent incidence of Wolbachia and their influence has led to the proposal of their use as potential agents in the evolution and control of tea pests.