Localization and dynamics of Wolbachia infection in Asian citrus psyllid Diaphorina citri, the insect vector of the causal pathogens of Huanglongbing

Abstract Wolbachia is a group of intracellular bacteria that infect a wide range of arthropods including the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. This insect is the vector of Candidatus Liberibacter asiaticus (CLas), the causal pathogen of Huanglongbing or citrus greening disease. Here, we investigated the localization pattern and infection dynamics of Wolbachia in different developmental stages of ACP. Results revealed that all developmental stages of ACP including egg, 1st–5th instar nymphs, and adults of both gender were infected with Wolbachia. FISH visualization of an ACP egg showed that Wolbachia moved from the egg stalk of newly laid eggs to a randomly distributed pattern throughout the egg prior to hatching. The infection rate varied between nymphal instars. The titers of Wolbachia in fourth and fifth instar nymphs were significantly higher than those in the first and second instar nymphs. Wolbachia were scattered in all nymphal stages, but with highest intensity in the U‐shaped bacteriome located in the abdomen of the nymph. Wolbachia was confined to two symmetrical organizations in the abdomen of newly emerged female and male adults. The potential mechanisms of Wolbachia infection dynamics are discussed.

In addition, feeding and honeydew production of D. citri can result in reduced photosynthesis, growth of sooty mold and the death of young foliage at high population densities (Gottwald, 2010;Halbert & Manjunath, 2004). CLas is a phloem-limited fastidious bacterium, which has not yet been cultured in vitro (Duan et al., 2009;Halbert & Manjunath, 2004). Typical symptoms of HLB include small and bitter fruits; chorotic shoots, blotchy mottle or variegated type of chlorosis, poor root growth, twig dieback and ultimately plant death (Bove, 2006;Gottwald, 2010;Yang et al., 2006). Two distinct intracellular symbionts are harbored in the yellow and bilobed bacteriome located in the psyllid abdomen. The primary endosymbiont, Candidatus Carsonella ruddii, is located in uninucleate bacteriocytes on the surface of the bacteriome, while Candidatus Profftella armatura is found in syncytial cytoplasm at the center of the bacteriome (Nakabachi et al., 2013). Besides these primary symbionts, citrus psyllids also harbor secondary symbionts including Wolbachia and Arsenophonus (Chu, Gill, Hoffmann, & Pelz-Stelinski, 2016;Saha et al., 2012).
Thus, there is an urgent need for effective means to manage the insect vector in order to reduce the incidence of this disease. Symbionts have been considered as a potential approach for control of many insect pests (Benlarbi & Ready, 2003;Mcmeniman et al., 2009;Moreira et al., 2009;Zabalou et al., 2004). Among the secondary endosymbionts, Wolbachia is the most abundant in arthropods (Weinert et al., 2015). It can induce reproductive disorders, cytoplasmic incompatibility (CI), parthenogenesis, male feminization and death; all of which warrant their manipulation as potential control agents (O'Neill et al., 1997;Werren, 1997;Werren, Baldo, & Clark, 2008) with cytoplasmic incompatibility being the most promising. This favors a particular Wolbachia strain that induces early embryonic death to egg and sperm combinations that are not both infected with the same strain. The potential use of this mechanism to control mosquitos has been explored in several studies including Xi and Dobson (2005), Kambris, Cook, Phuc, and Sinkins (2009), Moreira et al. (2009), Bian, Xu, Lu, Xie, andXi (2010) and Walker et al. (2011). In addition, Wolbachia strains such as wMel, wAlbB have been used to suppress transmission of human pathogens in Anopheles gambiae, A. stephensi and Aedes albopictus, respectively (Bian et al., 2013;Blagrove, Arias-Goeta, Failloux, & Sinkins, 2012;Hughes, Koga, Xue, Fukatsu, & Rasgon, 2011). It is therefore likely that endosymbionts, such as Wolbachia could be used to manipulate reproduction of ACP through cytoplasmic incompatibility and so suppress transmission of CLas to citrus plants (Hoffmann, Coy, Gibbard, & Pelz-Stelinski, 2014). However, to achieve this goal it is essential to understand the infection biology of Wolbachia in ACP, including determining the identity of the strains, their infection level and localization patterns (Chu et al., 2016;Kruse et al., 2017).
In this study, we used PCR, qPCR, and whole-mount fluorescence in situ hybridization (wFISH) to firstly detect the infection prevalence of Wolbachia, and secondly, determine the localization pattern of this endosymbiont in all life stages of ACP.

| Insects
The Asian citrus psyllid population used in this study was collected in

| DNA extraction from ACP
To extract the DNA, eggs, nymphs, and adults of both genders were collected from M. exotica plants, washed with 70% ethanol and then dried at room temperature. Nymphs were separated by instar based on their morphological characteristics (Tsai & Liu, 2000). DNA extractions were conducted by two methods. In the first method, individual psyllids were first washed with double distilled water to remove all alcohol. The sample containing either one individual of each nymphal instar, a male or female adult, or 10 eggs together as one unit due to their small size was homogenized in 2μl STE (10 mmol/L Tris-HCl, pH 8.0, 25 mmol/L NaCl, 25 mmol/L EDTA, 1% SDS, proteinase K 200 mg/ml) in a 0.5 ml microcentrifuge tube. The mixture for each sample was finally complemented with 15 μl STE in the 0.5 ml microcentrifuge tube. The homogenate was incubated at 56°C for 2-3 hr and then placed in 95°C water for 10 min to inactivate the proteinase K. After incubation, the samples were centrifuged for a short time and then used for PCR detection of Wolbachia.
In the second method, total DNA was extracted from groups of 40-50 ACP eggs, 1-2 instar nymphs or 10-20 individuals of 3-5 instar nymphs, male/female adults for qPCR using the TIANamp genomic DNA kit (TIANGEN Biotech, Beijing, China) with minor modifications for preparation of DNA from animal tissues. To assess DNA integrity, each sample was separated by electrophoresis on a 1% agarose gel (1%, 0.05μl/ml GoldView, TRIS-EDTA-Buffer) at 5 V/cm, and visualized on a UV transilluminator and then photographed via the gel imager. Additionally, quality and quantity of total DNA was measured on a NanoDrop 2,000 spectrophotometer to ensure uniformity among all samples for qPCR (Dossi, Da Silva, & Consoli, 2014;Tiwari, Gondhalekar, Mann, Scharf, & Stelinski, 2011). 5′-AAAAATT AAACGCTACTCCA-3′, which are specific to the Wolbachia endosymbiont (Braig, Zhou, Dobson, & O'Neill, 1998). The PCR procedure was: pre-denatured at 94°C for 3 min, followed by 35 cycles at 94°C for 35 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min. PCR amplified products were visualized on a 1% agarose gel containing GoldView colorant. When bands with the expected size were visible in the gels, 20 μl PCR products were sent for sequencing.

| PCR detection of Wolbachia in ACP
As mentioned above, ten eggs together as a unit, one individual of 1st-5th instar nymph, or one adult of each gender were treated as one replicate. In total 30 replicates were tested (10 replicates in one repeat ×3) in each experiment. Each PCR reaction included a positive (primary endosymbiont, Carsonella) and negative (ddH 2 O) control to verify DNA quality.

| Distribution of Wolbachia in different life stages of ACP
Eggs, and nymphs of each instar stage along with newly eclosed adults of ACP were collected from healthy M. exotica shoots with a camelhair brush. Fluorescence in situ hybridization (FISH) analysis of different psyllids stages and gender was performed as described by Gottlieb et al. (2006) with the probe W2-Cy3 (5′-Cy3-CTTCTGTGAGTACCGT

| Statistical analysis
Differences among nymphal stages and between male and female adult ACP in incidence and titer of Wolbachia were analyzed using one-way ANOVA (SPSS 17.0 software, SPSS Inc., Chicago, IL, USA).
Fisher's protected Duncan test was used for mean separation contingent on a significant treatment F value.
However, these differences were not significantly different (Table 1).

| Quantification of Wolbachia titer in different stages of ACP
Taking the psyllid actin gene as the baseline, the titer of Wolbachia increased with successive nymphal instars (Figure 2), for example, Wolbachia titers in the 4th-5th instar nymphs were significantly higher than those in the 1st-3rd instar nymphs (F = 45.37, p < .0001).
The Wolbachia titer of 5th instar ACP nymph was even higher than that of the ACP male and female adults, but no significant differences were found between the nymph and adults. One interesting finding was that, the titer of Wolbachia in ACP eggs was higher than that of the first instar nymph. The titer of Wolbachia did not differ significantly between adult genders but was relatively higher in males than in females (F = 0.51, p = .5007, Figure 3).

| Distribution of Wolbachia in different life stages of ACP using Fluorescence in situ hybridization
Distribution of Wolbachia varied over the course of egg development.
Wolbachia was most concentrated in the bacteriome at the basal pedicel end of newly laid eggs, although a more diffuse concentration could also be seen around the apex (Figure 4a and b). Later on, Wolbachia gradually spread out from the two poles to give a more uniform distribution (Figure 4c and f). In older eggs, Wolbachia were more random in distribution (Figure 4g and  of Wolbachia is mostly due to their reproductive parasitism but also mutualistic effects such as increased host fecundity and protection against pathogens (Zug & Hammerstein, 2015b). In the current study, detection frequencies of Wolbachia in ACP varied among different life stages and between gender from 100% in both the second instar nymphs and adult males to 90.0% in eggs and first instar nymphs. Guidolin and Consoli (2013) reported 100% incidence of Wolbachia in ACP specimens tested in Brazil. Subandiyah, Nikoh, Tsuyumu, Somowiyarjo, and Fukatsu (2000) found  related host immunity (Douglas, Bouvaine, & Russell, 2011;Gorman, Kankanala, & Kanost, 2004;Nishikori, Morioka, Kubo, & Morioka, 2009). However, regardless of both possibilities, the causation of the mechanism of infection warrants further study. Other studies have shown that environmental changes, such as insecticide exposure, temperature, host genotype diversity and Wolbachia strain can also influence their titer (Hurst, Jiggins, & Robinson, 2001;Weeks, Reynolds, Hoffmann, & Mann, 2002).
Our finding that more Wolbachia is present in males than females is consistent with previous related work with ACP (Hoffmann et al., 2014) as well as the pattern of wAlbB infection in Aedes albopictus (Tortosa et al., 2010). In many other insect species, the titer of Wolbachia is usually higher in adult females than in males (Correa & Ballard, 2012;Mouton et al., 2004;Tortosa et al., 2010). However, the reasons for the higher titers of Wolbachia in male compared to female ACP are still not clear. Rio, Wu, Filardo, and Aksoy (2006) found that the Wolbachia density in male tsetse fly was much higher than that of female, with males also showing a broader tissue distribution of Wolbachia com- eggs (Gottlieb et al., 2006). Localization of Wolbachia in different parts of the egg appears to be related to diversion of the cytoskeleton which is known to play an essential role in repartition of organelles in cells (Sicard, Dittmer, Greve, Bouchon, & Braquart-Varnier, 2014).
In nymphs, we found the highest concentration of Wolbachia in the U-shaped bacteriome located in the abdomen, with lower concentrations in the thorax, and occasional presence in the head. The ACP bacteriome is known to harbor three symbionts: Carsonella, Profftella (Nakabachi et al., 2013), and now Wolbachia. This result suggests a specific immune profile for Wolbachia allowing the host to maintain and control the symbiosis (Anselme, Vallier, Balmand, Fauvarque, & Heddi, 2006;Heddi et al., 2005). The distribution of Wolbachia in late fifth instar nymphs is quite similar to that in ACP adults; reflecting the transition from nymph to adult. Using FISH methodology, Kruse et al. (2017) found that Wolbachia has a widespread distribution throughout the ACP gut tissue, including the midgut, filter chamber and Malpighian tubules. They also determined that Wolbachia and CLas are capable of residing in the same ACP gut cells, but that they do not have a high degree of co-localization within cells.
The localization of Wolbachia has also been studied in other insects.
In the bedbug Cimex lectularius, Wolbachia symbiont was specifically localized in the bacteriomes and vertically transmitted via the somatic stem cell niche of germalia to oocytes. Here, it infected the incipient symbiotic organ at an early stage of the embryogenesis in adults. In the males, Wolbachia was restricted to the testis-associated bacteriomes, whereas in the females, it was found in the bacteriomes and the ovaries (Dobson et al., 1999). In Drosophila melanogaster, Karr (2002, 2003)  The potential of Wolbachia to control disease vectors, and interfere with the ability of mosquitos to vector malaria and dengue has been demonstrated (Bian et al., 2013;Bourtzis et al., 2014;Guidolin & Consoli, 2013). As mentioned above, Fagen et al. (2012) and Kolora et al. (2015) reported that Wolbachia has a positive association with the CLas, while Chu et al. (2016) revealed that both the densities of primary Carsonella and facultative Wolbachia were significantly higher in CLas-negative ACP compared to CLas-positive ACP. Whichever reflect the true infection status in the field, the interactions of Wolbachia-CLas can be further explored as a novel strategy to potentially control HLB through artificial manipulation of insect symbionts. Moreover, our molecular phylogenetic study has indicated that the Wolbachia of ACP from South China belongs to the Con strain in the Wolbachia B supergroup. The potential strategy of using Wolbachia to reduce ACP populations in the field may be practical by releasing a male ACP population with another strain of Wolbachia (single strain strategy, to realize this work we can first eliminate the original strain of Wolbachia and infect the ACP with a new strain by artificial micro-infection), or overlay with another strain with this Con strain (double strain strategy). Therefore, cytoplasmic incompatibility may occur when these two types of male adults mate with wild female ACP adults.
In summary, considering the potential use of Wolbachia for vector and disease management, studies on the ecological factors that affect the interactions between Wolbachia and its ACP host may be beneficial in developing novel strategies for ACP and HLB management. The current study moves toward this final goal.

ACKNOWLEDGMENTS
The authors thank Andrew G. S. Cuthbertson (York, UK) for his critical review on an earlier version of the manuscript.

CONFLICT OF INTEREST
None declared.