Comparative analysis of bacterial communities in olive psyllids, Euphyllura straminea and Euphyllura pakistanica expose proteobacteria dominance

Insect–bacteria symbiont associations can be found in almost all insect orders which supply an array of physiological and ecological benefits to the host insect. Plant sap‐feeding insects, including psyllids, have developed symbiotic relationships with a variety of bacteria to compensate nitrogen‐poor diets. In this study, we determined the bacterial composition of two olive psyllid pest species, Euphyllura straminea and Euphyllura pakistanica (Hemiptera: Aphalaridae), using 16S rRNA gene sequence and qPCR analyses. Proteobacteria, including Alphaproteobacteria, Gammaproteobacteria and Betaproteobacteria, composed the primary bacterial structures of the psyllids. Alphaproteobacteria were the dominant bacterial group in E. straminea, while Gammaproteobacteria composed the main portion of the bacterial structure in E. pakistanica. The primary endosymbiont, Carsonella, was detected in both psyllids and Arsenophonus with high prevalence was the main secondary endosymbiont. Interestingly, we observed high titres of Wolbachia in E. straminea compared with E. pakistanica, which revealed a negative correlation between Wolbachia and Carsonella and Arsenophonus titres. Our results show that the limited core microbiota of olive psyllids is similar while the abundance of each bacterial symbiont can vary in different species. A possible role of Wolbachia in modulating bacterial composition in the psyllids was also suggested. There is the potential to utilize symbionts for developing pest control strategies by reducing the insect host's fitness such as targeting essential endosymbionts, transferring symbionts between species and parthenogenesis. Our results can be used as a basis for further research towards the implementation of novel strategies in symbiont‐based biological control of the olive psyllids.

As one of the most successful animal groups on earth, insects have occupied diverse ecological niches and possess primary (P-) and secondary (S-) endosymbionts (Baumann, 2005) which assist them in various life stages. The P-endosymbionts are intracellular, mostly live in special cells known as bacteriocytes and are obligate organisms necessary for the survival or reproduction of their host.
Because of their intracellular living, these endosymbionts have a reduced genome size with high AT content, are primarily vertically transmitted to the next generation and cannot survive out of their host body (Wernegreen, 2002). Maternally inheritable gene transfers throughout evolutionary times have been the primary causes of co-speciation between hosts and P-endosymbionts Kikuchi et al., 2009). Moreover, most insects harbour another bacterial endosymbiont, which is classified as facultative for their respective hosts (Pontes & Dale, 2006). These endosymbionts are classified as S-endosymbionts and are generally not essential for their host survival or reproduction (Dale & Moran, 2006). However, the S-endosymbionts can affect host reproduction, such as in the case of Wolbachia Guidolin & Cônsoli, 2013;Werren & Windsor, 2000), or can provide benefits for their host such as Arsenophonus (Ayoubi et al., 2020;Hansen et al., 2007).
There is a complex interaction between P-and S-endosymbionts within the host. In some cases, reduction in P-endosymbiont genome size can be sharp, because the presence of S-endosymbionts is necessary for complementation of their function in the host (Pérez-Brocal et al., 2006). The evolution of this relationship can lead to the elimination of P-endosymbionts and provide a more suitable environment for the S-endosymbionts. In these cases, the S-endosymbionts become obligate for normal growth of the host (Hall et al., 2016). Moreover, other research has indicated that the presence of Wolbachia can change the abundance of other endosymbionts in the host (Arbuthnott et al., 2016;Simhadri et al., 2017).
Among different orders of insects, blood and phloem-feeding species like psyllids are more dependent on their endosymbionts because of their restricted diet. The normal growth of these insects on this nutrient-poor diet is related to the P-endosymbiont synthesis of nutrients. Given their restricted diet, nearly all psyllids harbour Carsonella rudii, a P-endosymbiont, in special vesicles within bacteriocytes (Fukatsu & Nikoh, 1998;Spaulding & von Dohlen, 1998). This endosymbiont provides essential amino acids for their hosts and is vertically transmitted through generations (Nakabachi et al., 2006). Moreover, several S-endosymbionts have also been reported from different species of psyllid insects (Morrow et al., 2017;Thao et al., 2000). The presence of Proftella (Nakabachi et al., 2013), Arsenophonus (Hansen et al., 2007) and Wolbachia (Cooper et al., 2015) have also been published from psyllids in different studies. Moreover, some psyllids harbour other endosymbionts such as Liberibacter (Halbert & Manjunath, 2004) and Phytoplasma (Malagnini et al., 2010), which are considered among the most important plant pathogens in agriculture.
Olive psyllids, Euphyllura straminea, and Euphyllura pakistanica (Hemiptera: Aphalaridae), are among the most important pests of olive trees. Adults and juveniles feed on leaves and buds, shoots, flowers and young fruits, thus causing major damage within orchards with large economic losses (Asadi et al., 2009). Feeding by psyllids on plant sap and secretion of honeydew causes sooty mould, which reduces photosynthesis in the plants (Santos et al., 2013). These insects are also associated with a number of symbiotic bacteria that play important roles in their survival. Considering the diverse and important roles of the microbiome in insects, this study attempted to define the bacterial composition of the olive psyllids collected from Iran. This is a first attempt to identify bacterial communities in these insect pests to highlight the importance of microbial symbionts with the hopes of developing a symbiont-based control strategy.

| Insect sampling and DNA extraction and 16S rRNA gene amplicon sequencing
The adults of E. straminea were collected from Tehran (Es-Te) and Tarom (Es-Ta), while adults of E. pakistanica were collected from Shiraz (Ep-Sh), Iran. Immediately after collection, the insects were transferred to individual vials containing 1 ml acetone and preserved at room temperature until DNA extraction. Before DNA extraction all psyllids have dipped into alcohol 95% for surface sterilization. To explore the composition of the different bacterial endosymbionts in the psyllids, total DNA was extracted from 100 adults of psyllids in every three populations (every population from each town).

The concentration of DNA samples was measured by BioTek Epoch
Microplate Spectrophotometer, and the quality of the sample was cleared by running on a (1%) agarose gel.
To determine the bacterial composition, PCR was performed on the V3-V4 region of 16S rRNA of two species from Shiraz and Tarom using universal primer pairs

| PCR and Real-Time PCR (qPCR)
To evaluate the relative abundance of different bacterial endosymbionts in the three populations of male and female olive psyllids and also confirm the high throughput amplicon sequencing results, we performed qPCR using species-specific primers (16S rRNA of Carsonella rudii and ftsz of Wolbachia pipientis) and classspecific primers (Gammaproteobacteria, Alphaproteobacteria, Betaproteobacteria, Bacteroidia and Firmicutes). The 18S rRNA gene was used as a reference gene (Karamipour et al., 2016). The qPCR condition was 95°C for 15 min, followed by 40 cycles of 95°C for 15 s and 15 s at the annealing temperature and followed by the melting curve (68-95°C). The annealing temperature and primers list are shown in Table 1. The qPCRs were performed by using SYBR green (Ampliqon) with a micPCR instrument (Bio Molecular systems). The qPCR data were analysed using the ΔΔCt method (Livak & Schmittgen, 2001). Moreover, the presence of Arsenophonus was confirmed by PCR. The PCR reactions were conducted under a temperature profile of 95°C for 10 min, followed by 35 cycles of 95°C for 30 s, 55°C for 1 min and 72°C for 1 min and a final extension at 72°C for 10 min. Eventually, the PCR products were subjected to agarose (1%) electrophoresis.

| Phylogenetic analysis
To develop phylogenetic trees of the bacterial endosymbionts, phylogenetic analysis was performed using the annotated sequences obtained from the 16S rRNA gene amplicon analysis. To do this, the amplicon sequencing-derived were aligned to the 16S rRNA genes from the Ribosomal Database Project (RDP) using Blastn. Then, the most similar sequences from the database along with our sequences were used for multiple sequence alignments using ClustalX v.2 program (Larkin et al., 2007). Molecular phylogenetic analyses based on maximum likelihood and neighbour-joining algorithms were performed by using MEGA 6.06 (Tamura et al., 2013). GTR + G + I model was selected as the best-fit substitution model for the maximum likelihood trees. The Bootstrap values were calculated with 1000 replications for neighbour-joining methods and 100 replications for maximum likelihood methods.

| 16S rRNA gene sequencing analysis
Two species of the olive psyllids including, E. straminea and E. pakistanica, were collected from Tarom and Shiraz, respectively. They were used for bacterial community profiling using 16S rRNA gene amplicon sequencing ( Figure 1a).

| The olive psyllid bacterial microbiota analysis
The 16S  Our results also showed that the relative abundance of the microbiota of female psyllids was higher than males (Figure 3a,c,e).

| Phylogenetic analysis
The

| DISCUSS ION
The microbiota of insects plays diverse roles in populations, ranging from supplying essential nutrients to defence mechanisms (Engel & Moran, 2013), to evolution and speciation (Brucker & Bordenstein, 2012;Shropshire & Bordenstein, 2016). The diverse functions of symbionts in insects can provide promising and novel strategies for the biological control of insect pests and vectors.  (Fierer et al., 2005) Identification and functional characterization of microbial symbionts are the key steps towards harnessing these associations for developing symbiont-based pest control.

TA B L E 1 Primers used in this study
In the present study, the whole bacterial composition of the olive psyllids, E. straminea and E. pakistanica, was investigated. Rarefaction Carsonella is about 160 Kb (Nakabachi et al., 2006) and has lost genes that are responsible for amino acid synthesis (Sloan & Moran, 2012); therefore, the acquisition of other symbionts by psyllid hosts is necessary to compensate for this shortfall that may result in copresence of the P-and S-endosymbionts (Hall et al., 2016;Morrow et al., 2017;Thao et al., 2000). In addition to the P-symbiont, we also identified Arsenophonus, Wolbachia, Pseudomonas and other modification of dietary scope (Wagner et al., 2015), contributing to the performance and fitness of aphids (Ayoubi et al., 2020;Tian et al., 2019;Wulff & White, 2015) and providing essential B vitamins in whiteflies (Santos-Garcia et al., 2018); however, its specific role is not yet known in psyllids. It has been suggested that Arsenophonus may impose a defensive role against parasitism in the lerp psyllid, Glycaspis brimblecombei (Hansen et al., 2007), but this effect seems to be limited to this species (Wulff et al., 2013). Considering our results and previous studies, it appears that Arsenophonus is probably the most common S-symbiont in psyllids, providing benefits that are yet unknown.
We also detected Wolbachia in all the screened samples using 16S rRNA amplicon sequencing and qPCR with specific primers. The high prevalence of Wolbachia has been found in other psyllids such as the gall-forming psyllid, Trioza magnolia (Morrow et al., 2017), and the citrus psyllid, Diaphorina citri (Hosseinzadeh et al., 2019). Although, Wolbachia is primarily an intracellular bacterium that impacts insect reproduction, in some cases, it also functions as an obligate nutritional endosymbiont (Kaiwa et al., 2010;Nikoh et al., 2014), but this relationship is limited in insects. The role of Wolbachia in psyllids is not well understood, but, in the potato psyllid, Bactericera cockerelli the cytoplasmic incompatibility effect of Wolbachia has been reported. (Cooper et al., 2015) In our study, the abundance of Wolbachia in E. straminea (i.e. Es-Ta and Es-Te) was higher than in E. pakistanica Variation in the bacterial symbiont composition could affect different aspects of psyllid biology and ecology, including interactions with their parasites/predators (Hansen et al., 2007) and host plants.
There is also evidence for S-symbiont variation and replacement in close psyllid species to complement the nutritional function of Psymbionts (Morrow et al., 2017). Therefore, variation in the bacterial symbiont levels of the olive psyllids may modulate the fitness of these pest species and could be reflected in their damage level to olive trees, a topic worthy of further investigations.

| CON CLUS ION
We studied the bacterial composition of two close olive species pest management strategies and symbiotic-based pest control programmes such as targeting essential endosymbionts, transferring symbionts between species and parthenogenesis, which are promising approaches; however, these strategies require detailed information on microbial communities and their functions in the insect host. This study provides data on the bacterial microbiome in the economically important olive psyllids including primary and facultative bacterial symbionts which can be a basis for future research on developing symbiont-based control programmes for these pests.

This work was supported by Shahed University and Tarbiat Modares
University which is appriciated.

AUTH O R CO NTR I B UTI O N S
Experiments were designed and performed by T.F., N.K. and M.M.
Laboratory space, materials/supplies and reagents were provided by A.A., A.S. and M.M. All the authors analysed the data and wrote the paper.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data associated with this publication including workflow, bioinformatics analysis pipeline, 16S rRNA Fasta files can be accessed on zenodo (https://doi.org/10.5281/zenodo.5910758).

F I G U R E 5
Phylogenetic tree showing the relationship of bacteria identified in the olive psyllids, E. straminea and E. pakistanica as revealed by 16S rRNA gene phylogenetic analysis. Composition of the olive psyllids, E. straminea collected from Tarom and E. pakistanica collected from shiraz as revealed by 16S rRNA gene phylogenetic analysis. Maximum likelihood method based on GTR + G + I model with 100 replications. Bootstrap probabilities are displayed on nodes and sequence accession numbers are shown in parentheses.