Wolbachia (Rickettsiales: Alphaproteobacteria) Infection in the Leafhopper Vector of Sugarcane White Leaf Disease

Abstract

Wolbachia is a maternally inherited bacterium ubiquitous in insects that has attracted interest as a prospective insect pest-control agent. Here, we detected and characterized Wolbachia in the leafhoppers Matsumuratettix hiroglyphicus (Matsumura) (Cicadellidae: Hemiptera) and Yamatotettix flavovittatus Matsumura (Cicadellidae: Hemiptera), insect vectors of the phytoplasma that cause white leaf disease in sugarcane. The 16S rRNA and wsp gene markers revealed that Wolbachia was not present in the M. hiroglyphicus but naturally occurs in Y. flavovittatus. Additionally, the infection rates in adult leafhoppers ranged from 0 to 100% depending on geographic location. Moreover, Wolbachia was detected in the eggs and first- to fifth-instar nymphs of Y. flavovittatus. A phylogenic tree of Wolbachia indicated that it resided in the monophyletic supergroup B clade and clustered in the Ori subgroup. Furthermore, fluorescence in situ hybridization revealed that Wolbachia localized to the egg apices, randomly distributed in the egg cytoplasm, and was concentrated in the nymph and adult bacteriomes, as well as occasional detection in the thorax and abdomen. To the best of our knowledge, the present study is the first to demonstrate the prevalence of Wolbachia in the leafhopper Y. flavovittatus. The obtained results would provide useful information for the future development of Wolbachia as a biological control agent for the leafhopper vectors.

The genus Wolbachia is an intracellular, maternally inherited bacterium that naturally occurs in a wide range of arthropods and filarial nematodes (Werren 1997, Bandi et al. 1998). Several reports suggest that Wolbachia infection exists in 20–80% of all insect species, and it is especially prevalent in the orders Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Orthoptera (Werren and Windsor 2000, Hilgenboecker et al. 2008, Zug et al. 2012). The taxonomic classification of Wolbachia diversity is complex due to its high genetic diversity. Although based on the specific gene analyses, Wolbachia strains are clustered into ≥13 major clades known as supergroups (A–F; H–N; Lo et al. 2007, Augustinos et al. 2011). The supergroups A and B are the most widely distributed among insects (Zhou et al. 1998).

Several Wolbachia strains are reproductive parasites that cause alterations of their hosts via parthenogenesis, feminization, male killing, and cytoplasmic incompatibility (CI). These phenomena enable Wolbachia to spread and maintain the infection within the host populations (Stouthamer et al. 1999, Werren et al. 2008). Moreover, some associated strains exert benefits to the hosts such as protecting its hosts from viral infections (Hedges et al. 2008), or supplement its hosts with essential nutritious (Hosokawa et al. 2010).

Wolbachia serve as a novel potential biological control agent for vector and pest insects (Bourtzis et al. 2014, Hoffmann et al. 2015). Wolbachia from Drosophila melanogaster (wMel) transfected to Aedes aegypti shortened the mosquito–vector lifespan and reduced dengue virus transmission (McMeniman et al. 2009, Walker et al. 2011). The use of Wolbachia to control mosquito vectors has been extensively investigated (Bian et al. 2010, Hoffmann et al. 2011, Ye et al. 2015, Fraser et al. 2017). Moreover, Wolbachia has also been explored for plant insect–pest management, with its transfection from Rhagoletis cerasi (L.) to Ceratitis capitata (Wiedemann) (Tephritidae) inducing CI in the latter host (Zabalou et al. 2004).

The leafhoppers Matsumuratettix hiroglyphicus (Matsumura) and Yamatotettix flavovittatus Matsumura (subfamily Deltocephalinae; family Cicadellidae; suborder Auchenorrhyncha) are vectors of the phytoplasma that causes sugarcane white leaf (SCWL) disease (Hanboonsong et al. 2002, 2006, Thein et al. 2012, Youichi and Hanboonsong 2017). This disease causes substantial losses in sugarcane yield in southeast Asian countries, especially in Thailand, which is the second largest exporter of refined sugar worldwide (FAO 2017). Currently, there are no resistant sugarcane cultivars or effective means to control SCWL disease. Measures to limit the spread of the disease have focused on insect-vector control; however, the use of insecticides is unsustainable for large-scale commercial crops. Moreover, there have been no reports on the use of traditional insect pest-control methods or natural enemy insects against the leafhoppers M. hiroglyphicus and Y. flavovittatus.

Our preliminary data suggested that Wolbachia is present in some population of the leafhopper Y. flavovittatus (Tewaruxsa et al. 2017); however, more in-depth investigation of Wolbachia biology is needed. We attempted to obtain adequate information with potential to be use for future development of control methods of the vectors. Therefore, the aim of the present study was to increase our understanding of natural infections of Wolbachia in leafhopper vectors in order to elucidate Wolbachia distribution, inheritance, and localization in leafhopper vectors of SCWL disease.

Materials and Methods

Leafhopper Collection DNA Extraction

Adults of leafhoppers M. hiroglyphicus and Y. flavovittatus were collected from light traps in four different sugarcane fields during August and September of 2016 or 2017. These four sugarcane fields are located in different provinces of Thailand: Khon Kaen and Udon Thani located in the northeastern region, Lopburi located in the central region, and Kanchanaburi located in the western region. Some of the specimens were immediately immersed in absolute ethanol and stored at −20°C until DNA extraction, whereas others were maintained in sugarcane plant cages and transferred to the laboratory for mass rearing.

DNA Extraction

Insect DNA was extracted using the phenol-chloroform method (Ausubel et al. 2008). Briefly, each leafhopper was ground in DNA extraction buffer (200 mM Tris [pH 8.0], 250 mM NaCl, 25 mM ethylene diamine tetraacetic acid [EDTA], 0.5% [v/v] sodium dodecyl sulfate [SDS], and 0.1 mg/ml proteinase K [Invitrogen, Carlsbad, CA]) and incubated at 37°C for 24 h. The DNA was extracted with an equal volumetric ratio of phenol:chloroform:isoamyl alcohol (25:24:1), followed by 5-min centrifugation at 10,000g and 4°C. The supernatants were transferred to other vials, and an equal volumetric ratio of chloroform:isoamyl alcohol (24:1) was added. After a 5-min centrifugation at 10,000g and 4°C, the supernatants were precipitated with 3 M sodium acetate and isopropanol. The pellet was washed with 70% (v/v) absolute ethanol, air-dried, and resuspended in TE buffer (10 mM Tris and 1 mM EDTA) before being stored at −20°C until further use.

Wolbachia Detection

The leafhoppers were screened for Wolbachia by PCR using two Wolbachia-specific primers. The first primer pair was used to amplify 900 bp of the 16S rRNA gene using the forward primer 16SFV1 (5′-TTGTAGCCTGCTATGGTATAACT-3′) and the reverse primer 16SRV6 (5′-GAATAGGTATGATTTTCATGT-3′; O’Neill et al. 1992).

The wsp was used to amplify a 610-bp Wolbachia surface protein using the forward primer 81F (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and the reverse primer 961R (5′-AAAAATTAAACGCTACTCCA-3′; Zhou et al. 1998). The reaction for 16S rRNA was performed in a final volume of 25 µl comprising 2 µl of DNA templates, 1× PCR buffer, 2.5 mM MgCl₂, 0.2 µM primers, 0.2 mM dNTPs, and 1 U Taq DNA polymerase (Invitrogen). Cycling conditions consisted of an initial denaturation (95°C, 5 min), followed by 30 cycles of denaturation at 95°C (1 min), annealing at 55°C (1 min), extension at 72°C (1 min), and a final extension at 72°C (10 min). For wsp, the PCR was run in a final volume of20 µl comprising 2-µl DNA templates, 1× PCR buffer, 2.5 mM MgCl₂, 0.5 µM primers, 0.2 mM dNTPs, and 1 U Taq DNA polymerase (Invitrogen) and using a PCR program similar to that for 16S rRNA gene. The amplicons were analyzed by gel electrophoresis. The total specimens that were tested in this experiment included 200 males and 200 females of the leafhopper Y. flavovittatus and 100 males and 100 females of the leafhopper M. hiroglyphicus.

Wolbachia Detection in the Various Leafhopper Life Stages

Wolbachia distribution was determined for various life cycle stages of Y. flavovittatus and M. hiroglyphicus. Adult leafhoppers from Udon Thani province were maintained in sugarcane plant cages (10 males and 10 females per cage, with a total of three cages for each leafhopper species) and allowed to mate and produce a subsequent generation. The eggs and first- to fifth-instar nymphs were collected and subjected to DNA extraction using the aforementioned protocol. The individual samples (a total of 40 eggs and 200 nymphs for each leafhopper species) were tested for the presence of Wolbachia by PCR reaction as described.

Sequence and Phylogenetic Analyses

To validate the PCR products, certain Wolbachia-positive samples were selected for nucleotide sequencing. These included 16S rRNA and wsp fragments from adult males, adult females, and fifth instar nymphs of Y. flavovittatus. The PCR products were purified and cloned into a TOPO-TA vector (Invitrogen) according to manufacturer instructions. Five recombinant plasmid clones were randomly selected from each leafhopper DNA template. Sequencing was conducted by Macrogen, Inc. (Seoul, Korea), and the 30 sequences obtained were compared with those deposited in the National Center for Biotechnology Information (NCBI; Bethesda, MD) GenBank database using the basic local alignment search tool (BLAST).

Phylogenetic relationships were established between the sequences of each group and closely related and unrelated sequences retrieved from the GenBank database. Multiple alignment was performed with a MAFFT algorithm using the default parameters (Katoh and Standley 2013). Phylogenetic trees were constructed by the maximum likelihood method in the IQ-TREE web server (http://www.iqtree.org/; Nguyen et al. 2015, Trifinopoulos et al. 2016). The automatically selected best-fit substitution models for 16S rRNA and wsp were TPM2u+F+I and TPM3+F+G4, respectively. Ultrafast bootstrapping assigned branch-support values with 1,000 replicates (Minh et al. 2013). The phylogenetic tree was viewed and edited in FigTree (v.1.4.3; http://tree.bio.ed.ac.uk/software/figtree/).

Fluorescence In Situ Hybridization

Whole-mount in situ hybridization was performed on the eggs, nymphs, and adults of Y. flavovittatus to localize Wolbachia. We tested individual specimens in this experiment, including 50 eggs, 30 nymphs, and 20 females. The oligonucleotide probe for hybridization was W2 (5′-CTTCTGTGAGTACCGTCATTATC-3′), which aligned with positions 319 through 336 of Wolbachia 16S rRNA (Shi et al. 2016). This probe was 5′-labeled with Quasar 670 (excitation wavelength: 647 nm; emission wavelength: 670 nm). Fluorescence in situ hybridization (FISH) was conducted following previously reported methods (Sakurai et al. 2005, Gottlieb et al. 2006), with slight modifications. Briefly, fresh samples were collected and stored in acetone until further use. Nymphs and adults were immersed in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂KPO₄, and 2 mM KH₂PO₄), 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 in 6% (v/v) hydrogen peroxide in 100% ethanol at room temperature (28–35°C) for 2–3 wk to quench the autofluorescence of the insect tissues. After decolorization, the samples were soaked three times in 100% ethanol and PBS with Tween-20 [PBST; 0.2% (v/v)] for 10 min each time. Before hybridization, the samples were hydrated three times in hybridization buffer (20 mM Tris–HCl [pH 8], 0.9 M NaCl, 0.01% [v/v] SDS, and 30% [v/v] formamide] for 10 min each time.

For the hybridization reaction, buffers containing 100 nM probe was added to the samples, and the mixtures were incubated at 46°C in the dark overnight. Nonspecific binding probes were washed three times with PBST for 10 min each time. The samples were mounted on glass slides with ProLong antifade solution (Invitrogen) and covered with a coverslip. The slides were viewed under a confocal laser-scanning microscope (FV1000; Olympus, Tokyo, Japan) at the Center of Nanoimaging, Mahidol University, Thailand. Reactions without probes or RNase digestion before hybridization were used as negative controls.

Sequence Accession Numbers

Representative 16S rRNA and wsp gene of Wolbachia from Y. flavovittatus was submitted to the GenBank database under accession numbers MN509027 and MN631207, respectively.

Results

Wolbachia Detection by PCR

Wolbachia infection was tested in both leafhopper species with 16S rRNA and wsp specific primers. The results show that Wolbachia was absent (0/200) from M. hiroglyphicus collected at two locations (Table 1); however, it was identified from Y. flavovittatus. The infection rates varied among the four populations collected from different sugarcane fields (Table 1). Although the infection rates within the same location differed slightly depending on the effectiveness of the primers being used. The highest infection rates were obtained for leafhoppers collected in Udon Thani province, with levels of 100% (160/160) coverage for 16S rRNA and 95.62% (153/160) for wsp. For the population in Khon Kaen province, the average infection rates were 87.5% (70/80) for 16S rRNA and 77.5% (62/80) for wsp. The specimens collected from two other fields showed low infection rates of 18.75% (15/80) for 16S rRNA and 9.60% (12/80) for wsp (Lopburi province) and 2.5% (2/80) for 16S rRNA and undetected for wsp (Kanchanaburi province).

Table 1.

Wolbachia infection frequencies in natural Y. flavovittatus and M. hiroglyphicus leafhopper populations

Leafhopper species	Location province (yr)	Sex	No. tested	No. positive (%)
				16S rRNA	wsp
Y. flavovittatus	Udonthani (2016)	Male	40	40 (100)	39 (97.50)
		Female	40	40 (100)	39 (97.50)
	Udonthani (2017)	Male	40	40 (100)	37 (92.50)
		Female	40	40 (100)	38 (95.00)
	Khonkaen (2016)	Male	40	35 (87.50)	29 (72.50)
		Female	40	35 (87.50)	33 (82.50)
	Lopburi (2016)	Male	40	4 (10.00)	2 (5.00)
		Female	40	11 (27.50)	10 (25.00)
	Kanchanaburi	Male	40	1 (2.50)	0 (0)
	(2017)	Female	40	1 (2.50)	0 (0)
	Total	Male	200	120 (60.00)	107 (53.50)
		Female	200	127 (63.50)	120 (60.00)
M. hiroglyphicus	Khonkaen (2016)	Male	50	0 (0)	0 (0)
		Female	50	0 (0)	0 (0)
	Udonthani (2017)	Male	50	0 (0)	0 (0)
		Female	50	0 (0)	0 (0)
	Total	Male	100	0 (0)	0 (0)
		Female	100	0 (0)	0 (0)

Prevalence of Wolbachia in Different Leafhopper Life Stages

Wolbachia was detected at various growth stages of both leafhopper species. The samples tested in this study were the progeny of leafhoppers collected in Udon Thani province, where the Y. flavovittatus population displayed the highest measured incidence of Wolbachia. The results show that Wolbachia was found in the eggs and the five nymphal instar stages of Y. flavovittatus (Table 2). The 16S rRNA Wolbachia infection rates were 82.5% for the eggs and 92.5, 97.5, 90.0, 87.5, and 92.5% for the first through the fifth nymph stages, respectively. The rates of wsp infection were 60.0% for the eggs and 77.5, 90.0, 90.0, 90.0, and 87.5% for the first through the fifth nymph stages, respectively. Whereas no Wolbachia was found in the eggs and nymphal stages of the leafhopper M. hiroglyphicus.

Table 2.

Wolbachia infection rates in various developmental stages of Y. flavovittatus and M. hiroglyphicus

Leafhopper species	Stage	No. tested	No. positive (%)
			16S rRNA	wsp
Y. flavovittatus	Egg	40	33 (82.5)	24 (60.0)
	First nymph	40	37 (92.5)	31 (77.5)
	Second nymph	40	39 (97.5)	36 (90.0)
	Third nymph	40	36 (90.0)	36 (90.0)
	Fourth nymph	40	35 (87.5)	36 (90.0)
	Fifth nymph	40	37 (92.5)	35 (87.5)
M. hiroglyphicus	Egg	40	0 (0)	0 (0)
	First nymph	40	0 (0)	0 (0)
	Second nymph	40	0 (0)	0 (0)
	Third nymph	40	0 (0)	0 (0)
	Fourth nymph	40	0 (0)	0 (0)
	Fifth nymph	40	0 (0)	0 (0)

Nucleotides Analysis and Phylogenetic Analysis

To validate the PCR products, 16S rRNA and wsp amplicons of Wolbachia were cloned from infected males, females, and fifth nymphal instars of Y. flavovittatus. The results of nucleotide sequencing are presented in Table 3. Fifteen clones of 16S rRNA gene showed a sequence identity of 99.88% with Wolbachia of the citrus blackfly Aleurocanthus woglumi (GenBank accession no. JX281793; Pandey et al. 2013), with multiple alignments of these sequences revealing 100% identity.

Table 3.

BLAST search results for Wolbachia 16S rRNA and wsp genes sequences from Y. flavovittatus

Gene	Insect	No. clones	Closest match	Identity (%)
16S rRNA	Male	5	Wolbachia of Aleurocanthus woglumi (JX281793)	99.88
	Female	5	Wolbachia of Aleurocanthus woglumi (JX281793)	99.88
	Fifth nymph	5	Wolbachia of Aleurocanthus woglumi (JX281793)	99.88
wsp	Male	1	Wolbachia of Phaenoglyphis villosa (MG968806)	99.64
	Female	4	Wolbachia of Homoeocerus unipunctatus (AB109572)	99.63
		5	Wolbachia of Phaenoglyphis villosa (MG968806)	99.64
	Fifth nymph	2	Wolbachia of Homoeocerus unipunctatus (AB109572)	99.63
		3	Wolbachia of Phaenoglyphis villosa (MG968806)	99.64

A BLAST search for wsp gene disclosed nine samples showing a sequence identity of 99.64% with Wolbachia found in parasitoid wasp Phaenoglyphis villosa (GenBank accession no. MG968806; Ferrer-Suay et al. 2018). The other six samples showed 99.63% identity with Wolbachia found in the true bug Homoeocerus unipunctatus (GenBank accession no. AB109572; Kikuchi and Fukatsu 2003). Multiple sequence alignments for wsp revealed 99–100% identity.

The phylogenetic trees were constructed to confirm systematic affinity of Wolbachia from the leafhopper Y. flavovittatus. The 16S rRNA gene sequence from this study was aligned with representative sequences of the Wolbachia from supergroups A and B. A tree analysis of 16S rRNA gene revealed that the Wolbachia from Y. flavovittatus was positioned in the monophyletic clade of supergroup B (100% support value), with the most closely related Wolbachia occurring in the whitefly Bemisia tabaci (GenBank accession no. AY850932; Fig. 1).

Fig. 1.

Phylogenetic tree of Wolbachia in leafhopper Y. flavovittatus (MN509027) and other insect hosts based on 16S rRNA gene sequences. Groups A and B of Wolbachia are listed on the right side. The numbers at each node indicate clade support based on 1,000 bootstrap replications. The scale bar represents the number of substitutions per site.

For phylogenetic analysis of wsp gene, the sequences of Wolbachia from the leafhopper Y. flavovittatus was aligned with 24 wsp gene sequences that belonging to various subgroups under supergroups A and B (Zhou et al. 1998). The wsp tree confirmed that Wolbachia from the Y. flavovittatus was clustered in the monophyletic group of supergroup B (100% support value) and placed in the clade containing Ori subgroups with 99% statistical support. However, the wsp gene sequence in present study formed a distinct evolutionary lineage from other Wolbachia strains (Fig. 2).

Fig. 2.

Phylogenetic tree of Wolbachia in Y. flavovittatus (MN631207) and other hosts based on wsp gene sequences. Wolbachia strains are indicated after accession numbers; groups and subgroups are listed on the right side. The numbers at each node indicate clade support based on 1,000 bootstrap replications. The scale bar represents the number of substitutions per site.

Localization of Wolbachia by FISH Assay

To localize the Wolbachia, a FISH assay was conducted on whole mounts of Y. flavovittatus eggs, nymphs, and adults. Wolbachia was detected at all developmental stages, with the signals indicating the presence of Wolbachia in the eggs (Fig. 3A–D). In eggs that were 2–3 d old, the Wolbachia signals concentrated around the apices (Fig. 3A and B), whereas in eggs that were 5–6 d old, the signals were randomly distributed (Fig. 3C and D). The whole mounts of eggs without probes or RNase digestion revealed no signals.

Fig. 3.

FISH visualization of Wolbachia-specific signal probes (red) in Y. flavovittatus eggs. (A, B) Eggs at 2–3 d. (C, D) Eggs at 5–6 d. Scale bar = 0.5 mm.

For the Y. flavovittatus nymphs and adults, the Wolbachia signals were concentrated in the bacteriomes along the lateral margins of the anterior abdomen (Fig. 4A–D), with the highest Wolbachia concentrations detected in the adult bacteriomes (Fig. 4D). Moreover, the Wolbachia signals were more highly concentrated in the third instar (Fig. 4C) than the first or second instar (Fig. 4A and B). Additionally, the Wolbachia signals were observed in the thorax (Fig. 4B and D) and the abdomen (Fig. 4E and F). The whole mounts of adults and nymphs without probes or RNase digestion revealed no signals.

Fig. 4.

FISH visualization of Wolbachia-specific signal probes (red) in nymph and adult Y. flavovittatus. (A–C) First, second, and third instars, respectively. (D) Adult stage. (E, F) Nymph abdomen. Scale bar = 1 mm. B, bacteriome; T, thorax; A, abdomen.

Discussion

In recent years, there has been an upsurge in the investigations on Wolbachia infection among different insect hosts due to several important reasons such as its effect on reproductive systems, influence on fitness and development traits as well as the application as an agent for controlling insect pests/vector diseases (Werren et al. 2008, Hoffmann et al. 2015). These interesting properties encouraged us to study the Wolbachia infection in the leafhopper vectors of SCWL disease in order to determine the potential for future development of sustainable vector control. To the best of our knowledge, this report is the first to disclose the presence of Wolbachia in Y. flavovittatus leafhopper.

Wolbachia infection frequency in Y. flavovittatus ranged from 0 to 100% depending on the geographic location. Variations in Wolbachia infection rate commonly occur within separate subpopulations of the same insect host, with infection frequency for the same species either very high (>90%) or very low (<10%; Hilgenboecker et al. 2008). This phenomenon was also reported for the whitefly (Bemisia tabaci) (Nirgianaki et al. 2003), aphid (Sitobion miscanthi) (Wang et al. 2009), rice leafroller (Cnaphalocrosis medinalis) (Chai et al. 2011), and bed bug (Cimex lectularius) (Akhoundi et al. 2016). The nature of the Wolbachia infection in different populations differed. There was a lack of clarity in the reason for the different rates between the different geographic regions from which the insects were collected. Several factors might have contributed to these differences, such as genetic variations, migration, natural enemies, and the host plants of infected insects. Moreover, Wiwatanaratanabutr (2015) suggested that the Wolbachia dynamics of leafhoppers and planthoppers might be affected by ecological factors.

The 16S rRNA and the wsp genes used to establish the Wolbachia infection rates did not generate results of similar accuracy, even when they were applied to the same specimens across all developmental stages (Tables 1 and 2). These primers might not be equally sensitive to Wolbachia DNA detection in the leafhopper Y. flavovittatus. Wolbachia-specific primers, such as 16S rRNA, wsp, and ftsZ, differ in their effectiveness for Wolbachia detection in various hosts (Hong and Gotoh 2002). In addition to primer effectiveness, PCR sensitivity in the present study might have been affected by the use of manual DNA extraction and the small sizes of the egg and early nymph specimens. Very little genetic material was obtained from them, and their templates contained relatively low amounts of Wolbachia. Consequently, the PCR assays occasionally failed to reveal Wolbachia DNA and generated false negatives. Apart from 16S rRNA and wsp, other primers, such as ftsZ, gltA, and groEL, have been used for gene detection and analysis of the phylogenetic relationships of Wolbachia (Wang et al. 2014). Therefore, more accurate data can be acquired with additional analyses using the appropriate primers to enable the detection of other genes.

Phylogenetic analyses of partial 16S rRNA and wsp sequences were conducted with Wolbachia strains from other arthropod hosts. The results were well-structured trees as shown by the bootstrap values and the retrieving sequences consistent with reported groups and strains of Wolbachia. Both phylogenetic trees indicated that Wolbachia from the leafhopper Y. flavovittatus belongs to supergroup B, which is commonly distributed in insects (Zhou et al. 1998). Our wsp gene sequence fell into the clade of Ori subgroup, though it revealed a new lineage of Wolbachia strains. Thus, based on classification and the nomenclature system (Zhou et al. 1998), we propose the designation wYfla as the specific strain for Wolbachia from the leafhopper Y. flavovittatus.

However, different patterns of supergroup infection in insect hosts have been reported (Kikuchi and Fukatsu 2003, Ahmed et al. 2010, Wang et al. 2014). In the present study, the number of clones sequencing were quite low; therefore, the obtained data could not support clear inferences of infection by either a single supergroup or other supergroups that remain undetected in the leafhopper Y. flavovittatus. Recently, the analysis of the housekeeping genes (gatB, coxA, hcpA, fbpA, and ftsZ) by multilocus sequence typing (MLST; Baldo et al. 2006) has been widely employed to characterize the genetic diversity of Wolbachia. In future research, Wolbachia strain genotyping will be performed by means of MLST using a larger number of specimens collected from various locations.

Wolbachia was detected in the egg and nymph stages of Y. flavovittatus samples collected from Udon Thani province, where there was a high incidence of Wolbachia infection in adult Y. flavovittatus. Wolbachia was observed in all developmental stages of this leafhopper species. Our data confirmed that Wolbachia is likely vertically transmitted in Y. flavovittatus, as previously reported (Gottlieb et al. 2008, Bing et al. 2014, Guo et al. 2018). Evidence for Wolbachia infection and vertical transmission in Y. flavovittatus was acquired by FISH assays on the eggs and nymphs; however, we did not establish the presence of Wolbachia in the female adult ovaries. FISH analysis of nymph and adult Y. flavovittatus revealed that Wolbachia localized primarily in the bacteriomes on both sides of the abdomen (Fig. 4), with negligible detection in the thorax and abdomen. The bacteriomes of Y. flavovittatus harbor the primary symbionts Candidatus Sulcia muelleri and Candidatus Yamatotia cicadellidicola (Wangkeeree et al. 2019). The coexistence of Wolbachia and primary symbionts in bacteriomes has been reported for the aphid Cinara cedri (Gomez-Valero et al. 2004), the bed bug Cimex lectularius (Hosokawa et al. 2010), the whitefly B. tabaci (Shi et al. 2016), and the psyllid Diaphorina citri (Ren et al. 2018). Wolbachia localization and density in different tissues might depend on Wolbachia–host interactions according to coevolution, routes of transmission and functions or effects of Wolbachia (Pietri et al. 2016).

This study of Wolbachia infection in Y. flavovittatus demonstrated that Wolbachia might represent a novel strategy for noninsecticide-based control of economically important plant pathogen vectors. This research is the first step toward providing sufficient information for potential future development; however, further research is required, including elucidation of the effects of Wolbachia on leafhopper reproduction and development, as well as Wolbachia genetic diversity and relationship with pathogen transmission. We found that Wolbachia was not detected in the leafhopper M. hiroglyphicus. One explanation for this might be the coevolutionary history of Wolbachia–host interactions or the host genetic background. Additionally, it is possible that the low infection prevalence and density of Wolbachia played a role. To resolve this or confirm the naturally uninfected status, future research will aim to increase the detection sensitivity through the use of quantitative PCR, and a larger number of specimens from various geographic locations will be processed.

Future development of Wolbachia as a biological control agent for the leafhopper M. hiroglyphicus can be potentially achieved by transinfection with virulent Wolbachia strains.