Unveiling metabolic integration in psyllids and their nutritional endosymbionts through comparative transcriptomics analysis

Summary Psyllids, a group of insects that feed on plant sap, have a symbiotic relationship with an endosymbiont called Carsonella. Carsonella synthesizes essential amino acids and vitamins for its psyllid host, but lacks certain genes required for this process, suggesting a compensatory role of psyllid host genes. To investigate this, gene expression was compared between two psyllid species, Bactericera cockerelli and Diaphorina citri, in specialized cells where Carsonella resides (bacteriomes). Collaborative psyllid genes, including horizontally transferred genes, showed patterns of conserved gene expression; however, species-specific patterns were also observed, suggesting differences in the nutritional metabolism between psyllid species. Also, the recycling of nitrogen in bacteriomes may primarily rely on glutamate dehydrogenase (GDH). Additionally, lineage-specific gene clusters were differentially expressed in B. cockerelli and D. citri bacteriomes and are highlighted here. These findings shed light on potential host adaptations for the regulation of this symbiosis due to host, microbiome, and environmental differences.

In this study, we present bacteriome and body transcriptomic data for two additional psyllid species D. citri and B. cockerelli.Furthermore, we conduct inter-species comparative transcriptomic analyses, aiming to enhance our understanding of the evolutionary aspects of psyllid host regulation.In comparison to B. cockerelli, D. citri hosts an additional obligate bacterial endosymbiont known as Candidatus Profftella armatura (hereafter referred to as Profftella), which resides in the syncytial region of the bacteriome. 23Profftella is presumed to primarily serve as a defensive symbiont for the psyllid by producing a polyketide toxin called diaphorin, but its genome also contains genes related to the biosynthesis of cysteine, hemolysin, riboflavin, biotin, and carotenoids, which may contribute to D. citri's nutritional symbiosis. 23,249][30][31] The impact of Wolbachia on the nutritional metabolism of the psyllid host-symbiont relationship is also currently unknown.Therefore, It is of significant interest to investigate the following questions: 1) To what extent are gene expression patterns conserved between D. citri and B. cockerelli bacteriomes for psyllid homologs that collaborate with Carsonella?, 2) Are there species-specific gene expression patterns within D. citri and B. cockerelli bacteriomes, suggesting divergence in their nutritional symbioses?, and 3) Do psyllid bacteriomes exhibit distinct gene expression patterns for homologs with multiple gene copies, such as the horizontally transferred genes (HTGs) predicted to collaborate with Carsonella?By examining these questions, we can gain deeper insights into the evolution of psyllid host regulation and the intricate dynamics of their nutritional symbioses.

Global differential gene expression in bacteriomes of D. citri and B. cockerelli
To investigate conserved and lineage-specific gene expression patterns related to the nutritional symbiosis in two psyllid species, D. citri and B. cockerelli, we pooled 60 bacteriomes and the rest of the body tissues with three biological replicates each for each species (N = 12 samples; Figure S1; see STAR methods for more detail).For D. citri, we obtained an average of 24,621,735 and 24,442,844 high-quality trimmed reads from three biological replicates of bacteriomes and body tissues, respectively, with an average of 75% and 89% of these reads mapping to the D. citri genome, respectively (Table S1).For B. cockerelli, we obtained an average of 26,365,738 and 27,932,791 trimmed reads from three biological replicates of bacteriomes and body tissues, respectively, with an average of 80% and 76% of these reads mapping to the B. cockerelli genome, respectively (Table S1).

Inter-species comparison of one-to-one orthologs
When comparing one-to-one orthologs (N = 5,555) between both psyllid species (Table S4), we identified an overlap of 852 orthologs (15%) that were significantly up-regulated in bacteriomes compared to body for both psyllid species, while 511 and 1,002 orthologs were significantly up-regulated in only D. citri or B. cockerelli, respectively (Figure 1; Table S4).The most represented GO-terms for shared up-regulated one-to-one orthologs include metal ion binding, ubiquitin-dependent protein catabolic process, protein transport, and transmembrane transport.The most represented GO-terms for one-to-one orthologs with species-specific up-regulation patterns include trehalose transport and vesicle-mediated transport for D. citri, and regulation of transcription, RNA splicing and viral processes for B. cockerelli (Table S4).For significantly down-regulated genes in bacteriomes compared to body for both psyllid species there was an overlap of 1,076 orthologs (19%), whereas 364 and 776 orthologs were significantly down-regulated in only D. citri or B. cockerelli, respectively (Figure 1; Table S4).The most represented GO-terms for shared down-regulated one-to-one orthologs include signal transduction, visual perception, integral component of membrane and multicellular organism development.The most represented GO-terms for one-to-one orthologs with species-specific down-regulation include zinc ion binding, response to oxidative stress and Wnt signaling pathway for D. citri and translation, ventral cord development and cell adhesion for B. cockerelli (Table S4).
To further explore the similarities and differences in gene expression between D. citri and B. cockerelli, we conducted Principal Component Analyses (PCA) on the relative expression of one-to-one orthologs similar to Argandona et al. (2023), 32 Georgiadou et al. (2022)  33

Korb et al. (2021)
. 34 Approximately 39% of one-to-one orthologs were significantly differentially expressed in bacteriomes compared to the body tissues for both psyllid species and these genes were examined further using PCA analysis where axis 1 and 2 explained $51% and 48% of the variance in the data, respectively.Out of the top 100 orthologs that displayed the highest positive and negative correlations with principal components 1 and 2, 67 of these genes were non-redundant and examined further.These orthologs consist of 43 up-regulated and 24 down-regulated genes in B. cockerelli and 41 up-regulated and 26 down-regulated genes in D. citri (Figure S2; Table S5).
Out of the 67 orthologs, those that are involved in a horizontal gene transfer event and symbiosis include the rRNA methylation gene (RSMJ) which was up-regulated in both species in bacteriomes compared to the body tissues (Tables 2 and S5).Moreover, up-regulated orthologs appear to have two distinct expression profiles with a subset of orthologs having greater (or lower) FC magnitudes in either D. citri or B. cockerelli (Figure S2).For instance, orthologs that were associated with the sulfur compound metabolism and ubiquitin binding showed higher up-regulation profiles in D. citri, while orthologs that were associated with rRNA-methyltransferase activity, trigeminal ganglion development exhibited higher up-regulation profiles in B. cockerelli (Figure S2; Table S5).Down-regulated genes in both species include those associated with thermotaxis, bursicon, and the regulation of sleep (protein quiver/sleepless) (Figure S2; Table S5).
In contrast, the ortholog ornithine aminotransferase (OAT, EC 2.6.1.13),which provides an alternative pathway to synthesize ornithine via P5CS in the arginine pathway, was significantly up-regulated in D. citri and significantly down-regulated in B. cockerelli (Table 1; Figure 2).Species-specific expression patterns were also observed for other collaborative psyllid homologs in the phenylalanine, methionine, and branched-chain amino acid biosynthesis pathways (e.g., leucine, valine, and isoleucine), suggesting distinct strategies in collaborating with Carsonella to synthesize essential amino acids.For instance, aspartate aminotransferase (AAT, EC 2.6.1.1)was significantly up-regulated in bacteriomes compared to body tissues only in B. cockerelli for phenylalanine synthesis.Cysteine-S-conjugate beta-lyase (CBL, EC 4.4.1.13)was only significantly up-regulated in bacteriomes compared to body tissues in D. citri for the methionine pathway, and branched-chain aminotransferase (BCAT, EC 2.6.1.42)was significantly up-and down-regulated in bacteriomes compared to body tissues in D. citri and B. cockerelli, respectively, for the biosynthesis of leucine, valine, and isoleucine (Table 1; Figure 2).

Expression patterns of psyllid genes in the non-essential amino acid metabolism
The two enzymes, glutamine synthetase (GS, EC 6.3.1.2) and glutamine oxoglutarate aminotransferase (GOGAT, EC 1.4.1.13),are hypothesized to work together in recycling waste ammonia (NH 3 ) in aphid bacteriocytes into glutamine and glutamate, which are used as amino donors for amino acid biosynthesis. 2Recycling of ammonia is crucial for sap-feeding insects due to the limited availability of nitrogen in their diets. 35Here in both B. cockerelli and D. citri, we found all GS orthologs were up-regulated in bacteriomes compared to the body tissues, while species-specific expression patterns for the GOGAT ortholog was observed (Table 1; Figure 2).The GOGAT ortholog in B. cockerelli was significantly down-regulated in bacteriomes compared to the body tissues whereas this ortholog was not significantly differentially expressed in D. citri (Table 1; Figure 2).Interestingly, orthologs for glutamate dehydrogenase (GDH, EC 1.4.1.3),an alternative to the GS/GOGAT pathway for recycling ammonia into glutamate, were significantly up-regulated in bacteriomes compared to body tissues for both psyllid species (Table 1; Figure 2).The activity of asparaginase (ASPG, EC 3.5.1.1),that converts asparagine to aspartate, has been hypothesized to be a primary source of ammonia for the GS/GOGAT cycle. 6,7,36Here, we found that the ASPG ortholog in both psyllid species were significantly down-regulated in bacteriomes compared to the body tissues (Table 1; Figure 2).However, there were species-specific expression patterns for the biosynthesis of asparagine via asparagine synthase (ASNS, EC 6.3.5.4),where all three homologs in B. cockerelli were significantly up-regulated in bacteriomes compared to the body tissues, however, the two ASNS genes identified in D. citri were not significantly differentially expressed (Table 1; Figure 2).

Horizontal gene transfer events in psyllid genomes may support gene losses in Carsonella
Expression levels of HTGs were examined for differential gene expression in bacteriomes compared to body for both psyllid species B. cockerelli and D. citri (Table 2).Two of these HTGs are predicted to collaborate with Carsonella for the biosynthesis of the essential amino acids, arginine and phenylalanine. 7,22These genes include argininosuccinate lyase (ASL, EC 4.3.2.1), which catalyzes the terminal step in the arginine biosynthesis pathway, and chorismate mutase (CM, EC 5.4.99.5), which is responsible for the conversion of chorismate into an intermediate precursor in the phenylalanine pathway (Figure 2).Phylogenetic analyses carried out in this study reveal distinct patterns within the genomes of B. cockerelli and D. citri.Specifically, we observed the presence of three distinct gene copies of ASL and CM in the genome of B. cockerelli, whereas D. citri's genome possesses two gene copies of ASL and four gene copies of CM (Figure 3).All copies of ASL and CM in both psyllid species were significantly up-regulated in bacteriomes compared to the body tissues (Table 2; Figure 3), suggesting that all gene copies play important roles in mediating the symbiosis with Carsonella for arginine and phenylalanine biosynthesis.
For the other HTGs in the psyllid genomes, six out of eight HTGs share the same expression pattern for both D. citri and B. cockerelli.These HTGs exhibit significant up-regulation in the bacteriomes compared to the body tissues, indicating their involvement in vital cellular processes.For instance, five HTGs were significantly up-regulated in bacteriomes compared to the body tissues (RIBC, RSMJ, ORF-1, -2, and -3a) for both psyllid species and are predicted to be involved in riboflavin biosynthesis (RIBC), rRNA methylation (RSMJ) and diverse cellular activities (ORFs, AAA-ATPase).One ortholog was significantly down-regulated (MUTY) in bacteriomes compared to body tissues for both psyllid species (Table 2) and is predicted to encode an A/G specific mismatch repair enzyme.Gene expression patterns here indicate that this latter enzyme may play a more important role in body tissues compared to the bacteriome for both psyllid species (Table 2).The HTG YDCJ, a conserved bacterial gene which belongs to the vicinal oxygen chelate (VOC) family, showed species-specific expression where YDCJ was significantly only up-regulated in D. citri bacteriomes (Table 2).

Lineage-specific gene clusters differentially expressed in bacteriomes
To explore the evolutionary divergence and functional specialization of bacteriome expression in D. citri and B. cockerelli, we investigated lineage-specific gene clusters in both psyllid species.These gene clusters are unique to either D. citri or B. cockerelli, encompassing a total of 1,314 clusters comprising 5,150 genes in D. citri and 724 clusters comprising 3,133 genes in B. cockerelli (Table S6).In D. citri, 26 percent of lineage-specific genes (1,331 genes) were significantly differentially expressed, where a total of 525 and 804 genes were significantly up-and down-regulated, respectively, in bacteriomes compared to the body tissues (Table S7).The up-regulated lineage-specific genes in D. citri are primarily associated with nucleus-related activities, transposition and the regulation of the Notch signaling pathway (Table S7).The downregulated lineage-specific genes in D. citri are primarily associated with DNA integration, RNA-directed DNA polymerase activity and the tachykinin receptor signaling pathway (Table S7).
In B. cockerelli, 24 percent of lineage specific genes (749 genes) were significantly differentially expressed, where a total of 427 and 322 genes were significantly up-and down-regulated, respectively, compared to the body tissues (Table S8).The up-regulated lineage-specific genes in B. cockerelli are primarily associated with protein processing, telomeric D loop disassembly, and transposition (Table S8).The downregulated lineage-specific genes in B. cockerelli are primarily associated with locomotor rhythm, protein dephosphorylation, positive regulation of transcription, and proteolysis (Table S8).
Additionally, we examined the gene expression patterns of significantly expanding gene families previously identified in B. cockerelli. 22ut of 3,375 significantly expanding genes in 157 clusters, we observed that almost one-third of genes (a total of 1,011 genes) were significantly differentially expressed in bacteriomes relative to the body tissues in B. cockerelli (Table S9).Over 60 percent of these genes in 119 clusters were significantly up-regulated in bacteriomes compared to the body tissues, and the remaining genes in 99 clusters was significantly down-regulated in bacteriomes compared to the body tissues (Table S9).The up-regulated genes are primarily associated with transposons and gene regulation, such as nucleic acid and ATP binding proteins (Table S9A).

KEGG pathway analyses
Using GSEA with KEGG pathways, we investigated the enrichment of biological pathways in the bacteriomes compared to body tissues for both species.In the bacteriomes of both psyllid species, we identified 17 and 39 pathways that were significantly positively and negatively enriched in bacteriomes compared to the body tissues, respectively (Figure 4; Table S10).The significantly positively enriched pathways in the bacteriomes include processes such as cell growth and death, transcription, nucleic acid replication and repair, and amino acid metabolisms (Figure 4; Table S10).The positive enrichment of these latter pathways in bacteriomes suggests that bacteriomes are metabolically highly active compared to other body cells, especially within the amino acid metabolism.The significantly negatively enriched pathways in bacteriomes were primarily involved in signaling pathways, lysosomes, and the digestive and endocrine systems (Figure 4; Table S10).These results indicate that genes involved in cell communication, and hormone and enzyme production, such as in the digestive and endocrine systems, are turned off and/or dampened in expression in the bacteriome.Furthermore, each psyllid species also exhibits species-specific pathways that were either positively or negatively enriched in their bacteriomes.For example, 45 and four unique KEGG pathways were significantly positively enriched in D. citri and B. cockerelli bacteriomes compared to the body tissues, respectively, and eight and 60 unique KEGG pathways were significantly negatively enriched in D. citri and B. cockerelli bacteriomes compared to the body tissues, respectively (Table S10).For example, in D. citri the sulfur metabolism and vitamin pathways related to B6 and folate were only significantly positively enriched in D. citri bacteriomes.In B. cockerelli the N-glycan biosynthesis pathways were only significantly positively enriched in B. cockerelli bacteriomes (Table S10).These results suggest that bacteriomes may play species-specific roles in the expression of pathways that are  We quantified genome copy numbers of the Wolbachia endosymbiont from both psyllid species in the bacteriome and body tissues to assess how the Wolbachia chromosomal copies fluctuate depending on the tissue type and whether it may directly interact with the integrative metabolism inside of the bacteriome.For both psyllid species, Wolbachia genome copies relative to psyllid genome copies were significantly higher in bacteriomes compared to body tissues (Figure 5).The copy number of the single copy Wolbachia gene (wsp) in B. cockerelli relative to the single copy psyllid gene (FORKHEAD) was 16X G 3 (mean G standard deviation, n = 3) higher in bacteriomes and 5X G 0.3 (n = 3) higher for body tissues (Figure 5).In D. citri, the copy number of the single copy Wolbachia gene (wsp) gene relative to the single copy psyllid A B gene (ACTB) was 14X G 5 (mean G standard deviation, n = 3) higher for bacteriomes and 0.4X lower G0.2 (n = 3) for body tissues (Figure 5).These findings indicate a potential direct interaction between Wolbachia and both psyllid hosts and Carsonella (and Profftella in D. citri) within the bacteriome, suggesting that Wolbachia could have a substantial impact on the integrated metabolism between Carsonella and the psyllid host.

Conservation and divergence of gene expression patterns between two psyllid species for the integrated metabolism of Psylloidea and Carsonella
In this study, we observed both conserved and variable gene expression profiles for specialized hemipteran cells (bacteriome) that harbor the nutritional endosymbiont Carsonella for two psyllid species that diverged $86 million years ago 22 (Figures 1 and 2; Table 1).Drawing on previous transcriptomic and proteomic research conducted in Hemiptera, 2,[6][7][8]19,[36][37][38] we specifically investigated homologs that are postulated to collaborate with the obligate nutritional endosymbiont, Carsonella. Five outof nine enzymes (55%) that are predicted to collaborate in six of Carsonella's essential amino acid pathways (arginine, isoleucine, valine, leucine, phenylalanine, and methionine) were significantly up-regulated in psyllid bacteriomes compared to body tissues for both B. cockerelli and D. citri (Figure 2).Interestingly, there is a high level of conservation for the differential expression patterns of these collaborative genes in bacteriomes when compared to the more distantly related psyllid species, P. venusta.7 In both P. venusta and D. citri, as well as P. venusta and B. cockerelli, a notable 75% and 62% of these collaborative genes, respectively, exhibited significant up-regulation in bacteriomes compared to body tissues (Figure 2).

Species-specific control of amino acid biosynthesis and its relation to psyllid microbiome differences
Even though the majority of the psyllid's collaborative genes displayed similar expression patterns in bacteriomes, it is noteworthy that differences in expression patterns were observed between species.Some of these species-specific differences may be attributed to microbiome differences between the psyllid species.For example, a key difference between D. citri and B. cockerelli's microbiomes is the presence of a co-endosymbiont, Profftella, in D. citri.Here we found that the sulfur metabolism and the cysteine and methionine metabolism were only significantly positively enriched in D. citri bacteriomes, suggesting that Profftella may play a key role in collaborating with D. citri for the biosynthesis of sulfur-containing metabolites (Table S10).Indeed, in D. citri, the enzymatic machinery required for sulfate reduction is exclusively found in Profftella (cysDHIN). 24In contrast Carsonella has completely lost all genes associated with this pathway, and there is no evidence suggesting that these genes have been substituted by any functionally acquired HTGs in the psyllid host. 7,22In the model aphid-Buchnera system, the sulfur assimilation pathway is generally present in Buchnera, and it is primarily inferred to have a role in synthesizing cysteine from serine. 39The origin of cysteine for D. citri remains uncertain as the absence of cysEK in Carsonella hinders the completion of the pathway, making it unclear whether cysteine is synthesized from serine. 22A previous hypothesis put forth in P. venusta 7 suggests that cysteine could potentially be synthesized from cystathionine through the activity of cystathionine gamma-lyase (CGL).It is plausible that a similar mechanism may also operate in D. citri.Future studies will be needed to determine the exact role of sulfur assimilation in Profftella and its interactions with D. citri.
Another important species-specific expression difference for collaborative psyllid genes was observed for the enzyme BCAT, which serves as the final step in the biosynthesis of the branched-chain amino acids, isoleucine, valine, and leucine.The expression pattern of the collaborative enzyme BCAT varied among the three divergent psyllid species, where it was up-regulated in bacteriomes in D. citri, down-regulated in B. cockerelli, and non-significantly differentially expressed in P. venusta (Figure 2).One possible explanation for these divergent expression patterns is that, unlike Buchnera in aphids 2 and Tremblaya and Moranella in mealybugs, 6 all Carsonella genomes still encode ilvE, which potentially has a similar enzyme function as BCAT in these latter insect bacteriomes.Therefore, BCAT may not be essential in some psyllid bacteriomes for this collaborative, redundant function.In D. citri, both Profftella and Wolbachia lack the ilvE gene in their genomes (Table S11). 24,40Therefore, the observed up-regulation of BCAT in D. citri, as compared to B. cockerelli and P. venusta, could potentially be attributed to the presence of Profftella and Wolbachia in D. citri's bacteriomes.This presence might lead to an increased nutritional demand for essential amino acids, such as leucine, valine, and threonine, consequently relying more on the psyllid host for the regulation of these branched-chain amino acids.
In this study both psyllid species harbor Wolbachia, which is in contrast to the previous analysis of P. venusta. 7Wolbachia is currently categorized in 16-17 distinct monophyletic lineages known as ''supergroups'' from A to S, 41 where supergroups A and B are found only in arthropods. 42The Wolbachia strains discovered in psyllid species to date all belong to supergroup B. 43,44 Due to its limited capacity for amino acid biosynthesis, Wolbachia supergroups A and B have been traditionally known to be highly dependent on the amino acid metabolism of its hosts, 45,46 while recent studies have provided evidence of evolutionary transitions from facultative to obligate mutualisms in certain members of the Wolbachia F supergroups. 47,48Here higher genome copy counts of Wolbachia were measured in D. citri and B. cockerelli bacteriomes compared to body tissues (Figure 5), suggesting that Wolbachia may have direct metabolic influences on the host and obligate symbiont(s) within bacteriomes for amino acids.A study by Ren et al. (2018)  49 corroborates our data here and found that Wolbachia-DC was detected with the highest density in the D. citri bacteriome compared to other body tissues using Fluorescence In Situ Hybridization (FISH) visualization.Hosseinzadeh et al. (2019), 50 however, found that Wolbachia-DC was the most abundant in D. citri's Malpighian tubules among a variety of organs including heads, guts, testes/ovaries and bacteriomes.To our knowledge, it is not known whether Wolbachia has any role in osmoregulation or nitrogen excretion, which are the primary functions of the Malpighian tubules.][53] While Wolbachia exhibits genomic hallmarks of being parasitic and reliant on amino acids from its psyllid host and Carsonella, it may also play a role in essential amino acid biosynthesis and exhibit facultative or even mutualistic interactions.For example, cysteine-S-conjugate beta-lyase (CBL; EC 4.4.1.13),is encoded in both D. citri and B. cockerelli and is hypothesized to collaborate with Carsonella in the methionine biosynthesis pathway. 7Interestingly, the two copies of CBL in D. citri exhibited significant up-regulation in bacteriomes, while the CBL copies in B. cockerelli showed no differential regulation in bacteriomes (Table 1).The bacterial enzyme metC (EC 4.4.1.13),a cystathionine b-lyase enzyme in bacteria, is widely conserved among different Wolbachia strains (Table S11) 47,54 suggesting that Carsonella might not be entirely dependent on host-encoded CBL for methionine biosynthesis.Instead Wolbachia may help complement the methionine biosynthesis pathway in B. cockerelli.Further functional genomics analyses of Wolbachia in B. cockerelli are needed to further understand this potential metabolic interaction.

Role of the glutamate dehydrogenase pathway in the recycling of waste ammonia in bacteriomes
Sap-feeding insects feed on a diet that is limited in nitrogen, especially essential amino acids. 55,56The recycling of waste ammonia in sapfeeding mutualistic relationships serves as a potential solution to fuel the nitrogen-demanding nature of these symbioses.One enzyme that can upgrade nitrogen from compounds such as ammonia is GS, which is significantly up-regulated in bacteriomes of both D. citri and B. cockerelli here.The GOGAT gene, which works in a cycle with GS to produce glutamate however is down-regulated in B. cockerelli and not statistically differentially expressed in D. citri (Figure 1; Table 1).The GS/GOGAT cycle is known to be an important mechanism in symbiotic relationships between sap-feeding insects and their obligate nutritional symbionts, as highlighted by previous studies on the upgrading of nitrogen from waste ammonia to fuel the amino acid metabolism. 2,6,7,19,38,57,58Interestingly, we identified that glutamate dehydrogenase (GDH) was significantly up-regulated in both D. citri and B. cockerelli bacteriomes, which might be an alternative to GOGAT for recycling ammonia into glutamate (Figure 2).The enzyme GDH is known to play a key role in assimilating waste nitrogen for the production of amino acids in human breast cancer cells where ammonia accumulates rapidly. 59Another recent study also revealed that raised levels of ammonia (hyperammonemia) results in GDH to play an essential role in the recycling of ammonia within non-cancerous brain cells. 60As a result, when ammonia levels rise in bacteriomes, the GS/GOGAT cycle may undergo a shift in its role, favoring GDH for the recycling of ammonia in the biosynthesis of amino acids.
Most studies on hemipteran systems have not reported any involvement of the GDH pathway. 2,6,7,38However, a recent study has found a potential role of the GDH pathway in the glassy-winged sharpshooter (GWSS) symbiosis system. 37In a novel dual obligate symbiosis between Baumannia and Sulcia-GWSS, where the GWSS host has two types of bacteriome tissues for the two endosymbionts, the GDH gene of GWSS is more highly expressed than GOGAT of the GWSS in the yellow bacteriome that harbors both Baumannia and Sulcia-GWSS. 37Future studies are warranted to investigate whether the up-regulation of GDH or the GS/GOGAT cycle in insects for nitrogen upgrading in bacteriomes for amino acid biosynthesis is influenced by the symbiosis, microbiome composition, and/or environmental conditions.

Functional significance of horizontally transferred genes in the amino acid metabolism
In sternorrhynchan insects, a consistent pattern arises with the loss of endosymbiont enzymes involved in the terminal steps of essential amino acid biosynthesis pathways.In response, host genes are hypothesized to step in and bridge these gaps. 39Notably, this pattern extends to the two HTGs in the psyllid's genome, ASL providing the terminal step for the arginine pathway and CM providing the terminal step for the phenylalanine pathway in psyllids.Here we found the significant up-regulation of these HTGs in bacteriomes for both psyllid species (Figures 2  and 3).This finding aligns with previous research conducted in P. venusta, suggesting a consistent pattern of HTG up-regulation in psyllid bacteriomes when compared to body tissues. 7Despite the potential for subfunctionalization and/or neofunctionalization of the paralogous copies of ASL and CM, all of the copies were significantly up-regulated in the bacteriomes compared to the body tissues in both psyllid species B. cockerelli and D. citri (Table S4.2).This suggests the potential importance of gene dosage for these collaborative enzymes within bacteriomes.Further investigation is needed to determine whether these gene copies exhibit differential expression in response to varying environmental conditions.
This study also identified the HTG RSMJ, a bacterial 16S rRNA methylation gene to have one of the highest relative expression levels of any gene in the bacteriome compared to the body for both psyllid species, as determined by comparative transcriptomics analysis using PCA (Tables 2 and S5).Phylogenetic analysis revealed that the RSMJ gene from the three psyllid species cluster within the 'class I SAM-dependent methyltransferase' group within Gammaproteobacteria (e.g., Escherichia coli, Photobacterium spp.) 61 (Figure S3).The InterPro GO term (GO:0008990) and Superfamily annotations (IPR029063) provide further evidence for classifying psyllid RSMJ as a SAM-dependent 16S rRNA methyltransferase, however the precise role of this gene in psyllid bacteriomes and the factors contributing to its conserved high expression levels in psyllids remain uncertain without additional functional assays.In E. coli, ten 16S rRNA methylation genes are involved in the modification of the small ribosomal subunit.The gene rsmJ in E. coli is characterized as a methyltransferase specific for the methylation of guanosine in the 1516 position of the 16S rRNA that has a cold sensitive mutant phenotype. 62In bacteria, rRNA methylation serves various purposes, including facilitating rRNA development, enhancing the stability of rRNA configurations, and modifying translation speed, however, alternative roles have been described, such as conferring resistance against aminoglycoside antibiotics derived from actinomycetes. 63orizontal gene transfer events in psyllids are also hypothesized to play a critical role in B vitamin biosynthesis.Insects require eight B vitamins as coenzymes for essential reactions, but they are unable to produce these vitamins and must obtain them through their diet. 64ome insects can supplement B vitamins using their microbial symbionts, 39,65 while some genes in vitamin biosynthesis have been acquired from bacteria by the insect host. 6,7In psyllids, the RIBC gene, also known as riboflavin synthase, has been found to be horizontally transferred to the host genome. 7,22This gene in bacteria, ribC, catalyzes the final step of the pathway, converting 6,7-dimethyl-8-ribityllumazine (DMRL) into riboflavin. 66Our data shows that RIBC was significantly up-regulated in bacteriomes at very high levels in both B. cockerelli and D. citri (LogFC = 7.88 and 6.15, respectively), similar to RIBC in P. venusta. 7However, only D. citri has been reported to possess a complete riboflavin biosynthetic pathway, where the co-obligate endosymbiont Profftella in D. citri retains all the genes in the riboflavin biosynthesis pathway except for the ribC (Figure 2). 24In contrast, the genomes of both the host and Carsonella in B. cockerelli and P. venusta appear to lack the rest of the genes in the riboflavin biosynthesis pathway.Therefore B. cockerelli and P. venusta may rely on their diet for the acquisition of the intermediate DMRL to produce riboflavin.

Regulation of lineage-specific gene clusters and its implications in evolutionary divergence
Our analysis of lineage-specific gene clusters that are unique to either D. citri or B. cockerelli highlights gene clusters that are differentially expressed in bacteriomes compared to body tissues for each species.Notably, the up-regulation of the Notch signaling pathway was prominent among D. citri-specific gene clusters, while no B. cockerelli-specific gene clusters were related to this pathway (Tables S6 and  S7).The Notch signaling pathway, particularly in insects, plays crucial roles in body segmentation, proliferation, embryogenesis and cell fate determination. 67In the aphid-Buchnera system, it has been observed that the signaling pathways, such as TGF-beta, Wnt, Hippo, Hedgehog and Notch, are significantly enriched in the 3 rd instar bacteriocytes 58 and the 4 th instar bacteriocytes of low-Buchnera titer genotypes. 68mith and Moran (2020) 68 hypothesize that the up-regulation of signaling pathways such as Notch in bacteriomes may result in an increase in both the number and size of aphid bacteriocytes in response to essential amino acid limitation. 69Hence, the observed up-regulation of lineage specific genes associated with the Notch signaling pathway in D. citri suggests that D. citri may possess distinct developmental processes that necessitate precise regulation to maintain a balance between metabolic production and cell growth.This phenomenon could be attributed to the heightened nutritional requirements imposed by Profftella, which is absent in B. cockerelli.
Our analysis showed that telomeric D loop disassembly was one of the most represented up-regulated GO-terms for B. cockerelli lineage specific clusters, and this GO-term was not found in D. citri specific gene clusters.(Tables S7 and S8).Telomeric D loop disassembly is a critical process involved in chromosome stability, integrity, and maintenance. 70The telomere motif (TTAGG)n is considered canonical for insects, 71,72 however, the structure of telomeric repeats can vary depending on the insect group as observed in some coleopteran species. 73,74Interestingly, in a distantly related hemipteran species Myzus persicae, the (TTAGG)n motif has been found to be interspersed with inserted non-LTR retrotransposable elements, 75 which could potentially be an intermediate state between the canonical insect telomere and retrotransposonbased ones. 76It is not clear at this point whether B. cockerelli has a canonical or retrotransposon-based (or intermediate) telomeric structure.To date the telomere structure in psyllids has been investigated for only five species within four genera from the families Psyllidae and Aphalaridae, 77 and all five species in the study appear to have the (TTAGG)n motif according to FISH visualization.Nevertheless, given that transposition is another highly represented GO-term for B. cockerelli specific gene clusters in bacteriomes, and that B. cockerelli shows a significant increase of transposable elements in its genome, 22 the up-regulation of lineage specific genes related to the telomeric D loop assembly in B. cockerelli bacteriomes suggests that there might be a potential association between telomere maintenance and transposable elements that differs from D. citri bacteriomes.

Conclusion
This study found both conserved and variable gene expression profiles for bacteriomes of two divergent psyllid species.Both psyllid species rely on Carsonella for the production of essential amino acids however their regulation of this symbiosis may vary due to host and symbiont genetic factors, differences within their microbiomes, and/or the environment including diet.Future studies aiming to investigate additional psyllid species, as well as considering various factors within species, will provide valuable insights into the evolutionary mechanisms underlying the integrated metabolisms of psyllids.This comprehensive approach will contribute to a deeper understanding of the complex dynamics involved in the evolution of metabolic interactions in psyllids.

Limitations of the study
Functional genetics techniques are still not available in these two psyllid species therefore metabolomic analyses and interpretations here are based on transcriptome data and comparative evolutionary genomics analyses.Due to available insectary and quarantine space, available funding, and timing of life stage dissections of this project, which were conducted around the same time frame to prevent batch effects, only one psyllid genetic line was used per species for this study.

Lead contact
Further information and requests for resources can be directed to and will be fulfilled by the lead contact, Allison K. Hansen (allison.hansen@ucr.edu).High-quality total RNA (>1 mg) from each pooled bacteriome sample and corresponding body tissue sample was submitted to the DNA Technology Core at the University of California, Davis for library preparation and sequencing.Strand-specific and barcode indexed RNA-seq libraries were generated from 300ng total RNA for each sample.Poly-A enrichment and library prep was done using the Kapa mRNA Stranded library preparation kit (KK8421, Kapa Biosystems, Cape Town, South Africa), following the instructions of the manufacturer.Libraries were amplified with 12 cycles of PCR.The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a Bioanalyzer 2100 (Agilent, Santa Clara, CA).The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA) and pooled in equimolar ratios.The pool was quantified by qPCR with a Kapa Library Quant kit (Kapa Biosystems) and sequenced on 1 lane of the Illumina NovaSeq S4 platform (Illumina, San Diego, CA) with paired-end 150bp reads.Reads for all RNA-Seq samples were submitted to the Sequence Read Archive of the National Center for Biotechnology Information (NCBI) under BioProject ID PRJNA939696.

Ortholog analysis and interspecies comparative transcriptomic analysis
Orthologous clusters, lineage-specific clusters, and one-to-one orthologs of D. citri and B. cockerelli were determined using OrthoVenn2 92 using default settings.To visualize the differentially expressed one-to-one orthologs, a Venn diagram was generated using SeqCode Ven-nPlotter. 93Similar to Georgiadou et al. (2022)  33 and Argandona et al. ( 2023), 32 one-to-one orthologs were further examined for inter-species gene expression comparisons.Specifically, the relative magnitude of changes of orthologs in bacteriomes were compared to body tissues for each species using Principal Components Analyses (PCA).To do this, we selected one-to-one orthologs that displayed significant differential gene expression (see above for the thresholds) in both D. citri and B. cockerelli.These orthologs were ranked in descending order of absolute logFC, and each ortholog was then assigned a value of 100 divided by rank, which was then multiplied by the sign of the original logFC.These values were then used as the input for PCA to identify one-to-one orthologs that display the most similar and different gene expression profiles in the bacteriomes compared to the body tissues.The PCA was performed using PC-ORD (version 4.25) 94 .For each principal component (axis 1 and 2), we selected the top 50 genes with the highest correlations (the top 25 genes for positive or negative correlations) from the principal components output loading matrix similar to Korb et al. (2021). 34We examined the GO annotations for these top 100 orthologs using OrthoVenn2. 92We generated a heatmap of logFC for one-to-one orthologs using SeqCode Heatmapper. 93antitative real-time PCR Fifth instar nymphs of both species were collected for bacteriome dissections for Wolbachia endosymbiont quantification similar to the RNAseq analysis (above).Bacteriomes and the rest of the body tissues without the bacteriomes were pooled from 15 individuals of both sexes for

Figure 1 .
Figure 1.A Venn diagram showing the number of significantly differentially expressed one-to-one orthologs in D. citri and B. cockerelli for bacteriomes compared to body tissues (FDR adjusted p value %0.05 and fold change (FC) R 1.5) Four sets of significantly differentially expressed one-to-one orthologs are represented (from left to right; up-regulated one-to-one orthologs in B. cockerelli (BC_up; outlined in yellow), up-regulated one-to-one orthologs in D. citri (DC_up; outlined in green), down-regulated one-to-one orthologs in D. citri (DC_down; outlined in pink), and down-regulated one-to-one orthologs in B. cockerelli (BC_down; outlined in blue).

Figure 2 .
Figure 2. Differential gene expression of psyllid genes involved in the integrative metabolism with Carsonella and Profftella Gene expression data are from NCBI SRA: SRA099681 for Pachypsylla venusta, and from this study for B. cockerelli and D. citri.Asterisks indicate more than one homolog is present in B. cockerelli or D. citri.HTGs are shown in red.(>1.5X, significantly up-regulated; <-1.5X, significantly down-regulated; n.s., not significant; n.a.data not available from P. venusta).
involved in the biosynthesis of sulfur, vitamins, and the modulation of protein folding, stability, and trafficking in D. citri compared to B. cockerelli.Tissue-specific quantification of Wolbachia in D. citri and B. cockerelli

Figure 3 .
Figure 3. Phylogenetic analyses of gene duplications for HTGs in the psyllid genomes of P. venusta (GenBank: GCA_012654025.1),D. citri (GenBank: GCA_024506315.2), and B. cockerelli (GenBank: GCA_024516035.1) for (A) argininosuccinate lyase (ASL) genes and (B) chorismate mutase (CM) genes Branches are colored according to their bacterial classes within the Proteobacteria, and ranges of bootstrap values are indicated by filled or open circles as indicated in the key.

Figure 4 .
Figure 4. Gene Set Enrichment Analyses of D. citri and B. cockerelli displaying all KEGG pathways that were significantly enriched in bacteriomes compared to the body tissues based on transcriptomics data Significance = the normalized p % 0.05 and FDR q % 0.25.

Figure 5 .
Figure 5. Boxplots showing the Wolbachia wsp gene copy numbers in bacteriomes and body tissues for B. cockerelli and D. citri The wsp gene copy numbers were normalized to the psyllid single copy house-keeping genes encoding FORKHEAD and ACTB in B. cockerelli and D. citri, respectively.All data points of three biological replicates of each tissue type for both psyllid species are presented.Tissues for each biological replicate are pooled tissues from 15 fifth instar nymphs.Red dots and asterisks represent means and significance (p < 0.01 **, p < 0.001 ***) of normalized abundance values using a t-test.
cockerelli and D. citri, 60 bacteriomes and the rest of the body, without the bacteriomes, were dissected from 5 th instar nymphs and pooled into two separate tissue samples similar to Hansen & Moran (2011), 2 resulting in three biological replicates per species (N = 12 samples).The D. citri and B. cockerelli nymphs were aged for bacteriome dissections according to their morphological characters and developmental periods as described in Hall et al. (2013)79 and Knowlton & Janes (1931),80 respectively.Samples were stored at À80 C in the RNAprotect bacterial reagent (QIAGEN, Germantown, MD, USA) until RNA extraction.METHOD DETAILS Total RNA sample preparation and RNA sequencingTotal RNA was purified, DNase 1 treated, and cleaned using Quick-RNA Microprep kit and RNA Clean & Concentrator kit-5 (Zymo research, Irvine, CA, USA) following the manufacturer's instructions.Purified RNA sample quality and quantity were measured using the Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) at the Institute of Integrative Genome Biology Instrumentation Facilities Services at the University of California, Riverside.

Table 1 .
Expression of symbiosis-related homologs in bacteriomes compared to the body tissues for D. citri and B. cockerelli Homologs in the same row are one-to-one or one to many orthologs.''N/A'' indicates no ortholog was identified.Bolded gene IDs indicate that the psyllid gene is significantly differentially expressed in bacteriomes compared to the body tissues.

Table 2 .
Differential gene expression for HTGs in bacteriomes compared to the body tissues in B. cockerelli and D. citri Homologs in the same row are one-to-one or one to many orthologs.''N/A'' indicates no ortholog was identified.a All genes in the table are significantly differentially expressed except for ANN12599.
22e B. cockerelli line was derived from a wild population in Temecula, California, USA, in August 2019, and is from the same culture that was sequenced inKwak et al. (2023).22Theestablishedline was maintained on 8-12-week-old Capsicum annuum plants (California Wonder pepper) at 25 C under a 16L:8D light/dark cycle.The Diaphorina citri nymph culture was obtained from the Stouthamer lab at the University of California (UC), Riverside, and was maintained on 6-month-old Murraya koenigii leaves (Curry tree) at 27 C under a 16L:8D light/dark cycle.For both B.