Probing behavior of the leafhopper analyzed through DC electropenetrography and microscopy.

Yamatotettix flavovittatus Matsumara is a new leafhopper species vector of sugarcane white leaf (SCWL) phytoplasma that causes sugarcane chlorosis symptoms. The effects of probing behavior of Y. flavovittatus on sugarcane and its implication for SCWL phytoplasma transmission are yet to be studied. In this research, we used DC electropenetrography (EPG) to define waveforms produced by adult and fifth-instar nymphal Y. flavovittatus on sugarcane and correlated them with salivary sheath termini (likely stylet tip locations) via light and scanning electron microscopy. The following six waveforms and associated activities are described: (NP) non-probing, (Yf1) stylet probing into epidermal cells, (Yf2) stylet probing through mesophyll/parenchyma, (Yf3) stylet contact with phloem and likely watery salivation, (Yf4) active ingestion of sap from phloem, probably sieve elements, and (Yf5) unknown stylet activity in multiple cell types. Study findings reveal that the Y. flavovittatus vector ingests sieve tube element more frequently and for longer durations than any other cell type, supporting that Y. flavovittatus is primarily a phloem feeder. Adult Y. flavovittatus show a longer total probing duration and produces a high density of puncture holes on sugarcane leaves. Moreover, probing behaviors revealed that adults typically ingest phloem sap more frequently and for longer durations than fifth-instar nymphs, enhancing sap ingestion. Furthermore, we propose that adults are more likely to acquire (during Yf4) and inoculate (during Yf3) higher amounts of phytoplasma than fifth-instar nymphs. This information on the penetration behavior of leafhopper Y. flavovittatus serves as a basis for advanced studies on the transmission mechanism of SCWL phytoplasma.

Selective Elimination of Wolbachia from the Leafhopper Yamatotettix flavovittatus Matsumura.

Wolbachia infections affect the reproductive system and various biological traits of the host insect. There is a high frequency of Wolbachia infection in the leafhopper Yamatotettix flavovittatus Matsumura. To investigate the potential roles of Wolbachia in the host, it is important to generate a non-Wolbachia-infected line. The efficacy of antibiotics in eliminating Wolbachia from Y. flavovittatus remains unknown. This leafhopper harbors the mutualistic bacterium Candidatus Sulcia muelleri, which has an important function in the biological traits. The presence of Ca. S. muelleri raises a major concern regarding the use of antibiotics. We selectively eliminated Wolbachia, considering the influence of antibiotics on leafhopper survival and Ca. S. muelleri prevalence. The effect of artificial diets containing different doses of tetracycline and rifampicin on survival was optimized; high dose (0.5 mg/ml) of antibiotics induces a high mortality. A concentration of 0.2 mg/ml was chosen for the subsequent experiments. Antibiotic treatments significantly reduced the Wolbachia infection, and the Wolbachia density in the treated leafhoppers sharply declined. Wolbachia recurred in tetracycline-treated offspring, regardless of antibiotic exposure. However, Wolbachia is unable to be transmitted and restored in rifampicin-treated offspring. The dose and treatment duration had no significant effect on the infection and density of Ca. S. muelleri in the antibiotic-treated offspring. In conclusion, Wolbachia in Y. flavovittatus was stably eliminated using rifampicin, and the Wolbachia-free line was generated at least two generations after treatment. This report provides additional experimental procedures for removing Wolbachia from insects, particularly in host species with the coexistence of Ca. S. muelleri.

Population Dynamics of Wolbachia in the Leafhopper Vector Yamatotettix flavovittatus (Hemiptera: Cicadellidae).

Wolbachia (Rickettsiales: Alphaproteobacteria) infections induce abnormalities in the reproductive system and affect various biological traits of the host insects. The density of Wolbachia is one of the major parameters that influence induced phenotypes and interactions with the hosts. Wolbachia occurs naturally in populations of the leafhopper Yamatotettix flavovittatus Matsumura (Hemiptera: Cicadellidae), which transmits phytoplasma that cause white leaf disease in sugarcane. However, the quantity and dynamics of Wolbachia in this leafhopper are not well understood. In the current study, we estimated the number of Wolbachia by absolute quantification of the copy number of wsp, which encodes the outer surface protein, using real-time quantitative polymerase chain reaction (PCR). This investigation was performed using natural populations and laboratory colonies from three lineages of leafhoppers (designated as UD, KK, and SK). There was no significant difference in the number of wsp copies in most of field-collected adults. During the immature developmental stages, there were differences in the dynamics of Wolbachia infection between the UD lineage and the other two lineages. However, the number of wsp copies increased in the early instar and plateaued in the later nymphal instars. Sex had no influence on the number of Wolbachia within the same lineages. The number of Wolbachia was relatively constant during the adult stage in the UD lineage but fluctuated in the other two lineages. In conclusion, the present data provide a framework for exploring the relationship between Wolbachia and the leafhopper and could facilitate future research into management strategies using Wolbachia.

Phylogeny and Strain Typing of Wolbachia from Yamatotettix flavovittatus Matsumura Leafhoppers.

Wolbachia is a maternally inherited bacterium of insects that can alter the reproduction, biology, and fitness of the hosts. It was detected in natural populations of the Yamatotettix flavovittatus Matsumura leafhoppers, the vector of phytoplasma, which is responsible for sugarcane white leaf disease. Wolbachia infection prolongs the longevity of female leafhoppers and promotes a strong reproductive incompatibility; importantly, highly maternal transmission rate was observed. However, limited data on the diversity or strain typing of Wolbachia in Y. flavovittatus are available. We aimed here to detect the presence of Wolbachia in different populations by amplification of the wsp gene, which was then sequenced. Multilocus sequence typing (MLST) was also performed to explore the diversity of the Wolbachia strains. Based on the wsp sequences, Wolbachia in the Y. flavovittatus leafhoppers belonged to supergroup B, and formed a distinct evolutionary lineage; therefore, we designated this new specific strain as wYfla. The MLST profiles revealed ten potential new sequence types (STs) in different leafhopper populations. Multiple STs were detected in individual leafhoppers, among which the ST-wYfla1 strain was predominant. Furthermore, we obtained congruent results for the phylogenetic analyses using the wsp gene and MLST loci. To the best of our knowledge, this is the first study characterizing Wolbachia strains in Y. flavovittatus. Our results reveal a novel strain and multiple STs of Wolbachia, and these data may prove useful in the exploitation of Wolbachia as a biological Y. flavovittatus control agent.

Effect of Wolbachia infection states on the life history and reproductive traits of the leafhopper Yamatotettix flavovittatus Matsumura.

Wolbachia is a maternally inherited bacterium of insects that can affect host reproduction and fitness. We examined the effect of Wolbachia infection on the life history and reproductive traits of the leafhopper Yamatotettix flavovittatus, which is a vector of the phytoplasma that causes white leaf disease in sugarcane. This investigation was performed using Wolbachia-infected and uninfected leafhopper lineages. Results revealed that Wolbachia infection did not significantly affect the survival of nymphal stages, male longevity, and sex ratio. However, Wolbachia-infected lineages had prolonged immature development periods and female longevity. In intrapopulation crosses, Wolbachia infection had no significant effects on occupation success, number of eggs laid, and female offspring, but the effect on egg-hatching varied. In interpopulation crosses, Wolbachia infection had no significant effect on occupation success and female offspring, but it did affect the number of eggs laid and egg-hatching rates. Assortative pairings regarding infection status resulted in normal egg deposition and hatching, whereas disassortative pairings resulted in lower egg deposition and no hatching. Wolbachia was thus shown to be highly vertically transmitted (>98% of the tested individuals). Our findings provide additional data on the interactions between Wolbachia in insect hosts. This evidence of perfect maternal transmission and strong reproductive incompatibility highlights the importance of further studies on the use of Wolbachia as a biological control agent for the leafhopper vector.

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

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.

Candidates for symbiotic control of sugarcane white leaf disease.

The leafhopper Matsumuratettix hiroglyphicus (Matsumura) is the most important vector of a phytoplasma pathogen causing sugarcane white leaf (SCWL) disease. The purpose of this study was to evaluate candidate bacterial symbionts for possible use as vehicles in the control of the disease. 16S rRNA bacterial genes were amplified from whole bodies of M. hiroglyphicus leafhoppers and analyzed by cloning and sequencing. Two dominant groups were found: one belonged to the Betaproteobacteria that did not closely match any sequences in the database and was named bacterium associated with M. hiroglyphicus (BAMH). Another one found to be abundant in this leafhopper is "Candidatus Sulcia muelleri" in the order Bacteroidetes, which was previously reported in the insect members of the Auchenorrhyncha. Most M. hiroglyphicus leafhoppers carry both BAMH and "Ca. Sulcia muelleri." Fluorescent in situ hybridization showed that BAMH and "Ca. Sulcia muelleri" colocalized in the same bacteriomes. BAMH was present in the midgut and ovaries of the leafhopper and was found in all developmental stages, including eggs, nymphs, and adults. Because BAMH appears to be specific for the SCWL vector, we evaluated it as a candidate for symbiotic control of sugarcane white leaf disease.