Annotation of glycolysis, gluconeogenesis, and trehaloneogenesis pathways provide insight into carbohydrate metabolism in the Asian citrus psyllid

Citrus greening disease is caused by the pathogen Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid, Diaphorina citri. No curative treatment or significant prevention mechanism exists for this disease, which causes economic losses from reduced citrus production. A high-quality genome of D. citri is being manually annotated to provide accurate gene models to identify novel control targets and increase understanding of this pest. Here, we annotated 25 D. citri genes involved in glycolysis and gluconeogenesis, and seven in trehaloneogenesis. Comparative analysis showed that glycolysis genes in D. citri are highly conserved but copy numbers vary. Analysis of expression levels revealed upregulation of several enzymes in the glycolysis pathway in the thorax, consistent with the primary use of glucose by thoracic flight muscles. Manually annotating these core metabolic pathways provides accurate genomic foundation for developing gene-targeting therapeutics to control D. citri.

HLB causes development of small, bitter fruits, loss of tree vigor, fruit drop, and ultimately tree decline and death [1][2][3][4]. This bacterium is transmitted by the psyllid vector, Diaphorina citri (NCBI:txid121845), when feeding on citrus [5,6]. Pesticide application to eliminate D. citri has been unsuccessful and no cure for HLB exists [7,8]. To develop new psyllid control strategies, the International Psyllid Genome Consortium was established in 2009 [9] to provide the genome, transcriptome resources, and an official gene set of D. citri [10,11].

Context
A community-driven annotation strategy was used to identify and characterize the genes encoding enzymes involved in glycolysis, gluconeogenesis, and trehaloneogenesis ( Figure 1).
Glycolysis is vital metabolic pathway in core energy processing reactions, and provides a source of metabolites for other biochemical processes. Insects utilize much glucose in flight muscles in the thorax [28]. Accordingly, the activities of glycolytic enzymes are increased in insect flight muscle compared with vertebrate muscle tissue [29]. Gluconeogenesis is essential in insects to maintain sugar homeostasis and serves as the initial step towards generating glucose disaccharide, also known as trehalose. Trehalose is the main circulating sugar in the insect hemolymph [30][31][32]. In trehaloneogenesis, glucose-6-phosphate is converted into trehalose by trehalose-6-phosphate synthase (TPS). Trehalase enzymes then degrade trehalose into two glucose molecules [33]. Genes involved in psyllid glycolysis, gluconeogenesis, and trehaloneogenesis have been targeted by several RNAi studies (Table 1) as a promising avenue for psyllid population suppression. In particular, one proof of concept experiment targeting trehalase led to the release of the first RNAi patent to control psyllid populations [49]. RNAi, as a biopesticide, and strategies for delivery and applications to target insect pests and viral pathogens have been thoroughly reviewed [50][51][52][53][54].

METHODS
The D. citri genome was manually annotated through a collaborative community-driven strategy [11] with an undergraduate focus that allows specific students to focus on main gene sets [55]. Orthologous protein sequences for the glycolysis, gluconeogenesis, and trehaloneogenesis pathways were obtained from the National Center for Biotechnology Information (NCBI) protein database [56] and were used to BLAST the D. citri MCOT (Maker (RRID:SCR_005309), Cufflinks (RRID:SCR_014597), Oases (RRID:SCR_011896), and Trinity (RRID:SCR_013048)) protein database to find predicted protein models [25]. MCOT predicted protein models were used to search the D. citri genomes (versions 2.0 and 3.0) [55]. Regions of high sequence identity were manually curated in Apollo v2.1.0 (RRID:SCR_001936) using Overview of the glycolysis, gluconeogenesis, and trehaloneogenesis pathways. The pathway image shows the enzymes that produce and utilize glucose and trehalose in insects [25]. The glycolysis pathway comprises 10 enzymes that convert glucose into pyruvate as the final product. These are divided into the energy investment phase (light green) and the energy production phase (dark green). The gluconeogenesis pathway comprises eight enzymes (blue), with three being unique to the pathway that bypasses the irreversible reactions in glycolysis to convert non-carbohydrate molecules into glucose. The trehaloneogenesis pathway comprises three enzymes: trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), and trehalase (TREH), as well as trehalose transporters (TRET) and glucose transporters (GLUT1). Image adapted from a diagram in [26] and created with BioRender.com [27].
de novo transcriptome, MCOT gene predictions, RNA-seq, Iso-seq, and ortholog data to support and evaluate gene structure ( Table 2). The curated gene models were compared with other orthologous sequences, such as hemipterans, available through NCBI for accuracy. A more detailed description of the annotation workflow is available ( Figure 2) [58].
Neighbor-joining phylogenetic trees of the annotated hexokinase gene models in D. citri and orthologous sequences were created with MEGA v7 (RRID:SCR_000667) using the MUSCLE (RRID:SCR_011812) multiple sequence alignment with p-distance for determining branch length and 1,000 bootstrap replicates [59]. Tribolium castaneum HexA1 role in glucose metabolism is essential during oogenesis and embryogenesis [34] Aldolase UAS-Aldolase-RNAi Drosophila melanogaster Knockdown in Drosophila neurons and glia resulted in reduced lifespan; essential in glia for neuronal maintenance [35] Enolase -enolase Nilaparvata lugens Knockdown reduced egg production, offspring and hatching rate; mortality of adults was unaffected [36] Pyruvate kinase (PYK) NlPYK Nilaparvata lugens RNAi treatment including triazophos and dsNlPYK led to reduced ovarian protein content, ovarian and fat body soluble sugar contents, and fecundity [37] Phosphoenolpyruvate carboxykinase (PEPCK) Drosophila melanogaster Knockdown of two PEPCK mutant isoforms led to reduced circulating glycerol levels and reduced triglyceride levels in pepck1 mutant flies [38] Trehalose-6-phosphate synthase (TPS) Diaphorina citri Knockdown of the Trehalose-6-phosphate synthase gene using RNA interference inhibits synthesis of trehalose and increases lethality rate in Asian citrus psyllid [39] Trehalose phosphate synthase (TPS) NlTPS Nilaparvata lugens Feeding N. lugens larvae with NlTPS dsRNA led to disrupted expression and lethality [40] Trehalose-6-phosphate synthases Nilaparvata lugens Silencing of two TPS genes can lead to increased molting deformities and mortality rates leading to misregulation of chitin metabolism genes [41] chitin List of annotated genes in glycolysis (HK, aldolase, enolase, PYK), gluconeogenesis (PEPCK), and trehaloneogenesis (TPS and TREH), with their corresponding RNAi studies and references. ‡ indicates that additional genes were added, but not annotated in D. citri, such as muscle protein 20 and sucrose hydrolase. *indicates that the chitin synthase gene in the chitin synthesis pathway was also annotated in D. citri [48].

DATA VALIDATION AND QUALITY CONTROL
There are four phases of the carbohydrate metabolism pathways in D. citri: the energy investment phase of glycolysis, the energy production phase of glycolysis, gluconeogenesis, Each manually annotated gene in glycolysis, gluconeogenesis, and trehaloneogenesis associated with a D. citri identifier shows supporting evidence used in the curation of the gene model [57]. Evidence tracks are as follows: RNA-seq, long-read Iso-seq, MCOT, de novo assembled transcripts and orthologous proteins. A gene marked with an "x" within the table indicates that the gene model is supported by the evidence track. A gene followed by "*" indicates that it is involved in both glycolysis and gluconeogenesis.

Energy investment phase of glycolysis
HK catalyzes the first step in glycolysis, utilizing adenosine triphosphate (ATP) to phosphorylate glucose, creating glucose-6-phosphate. Most insects have multiple HK genes and three copies of HK are present in the D. citri genome (Figure 3, Tables 2 and 4). In insect flight muscles, HK activity is inhibited by its product, glucose-6-phosphate, to initiate flight muscle activity [69]. Drosophila melanogaster has four duplicated HK genes, with Hex-A being the most conserved and essential flight muscle HK isozyme among Drosophila species [70,71]. For Diasporina citri, one of the copies of HK type 2-2 (Dcitr03g19430.1.1) showed moderate expression in the male and female thorax. In contrast, another copy HK PFK, which catalyzes the phosphorylation of fructose-6-phosphate using ATP to generate fructose-1,6-bisphosphate and adenosine diphosphate (ADP), is the key regulatory enzyme controlling glycolysis in insects, as it catalyzes a rate-determining reaction [76,77]. One copy of PFK (Dcitr01g16570.1.1) was found and annotated in D. citri (Table 4). Aldolase catalyzes the fourth step, the reversible aldol cleavage of fructose-1,6-bisphosphate to form two trioses, glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP).
Although most insects have a single copy of this gene, two well supported copies were found in D. citri (Table 4). One of the aldolase annotated copies, fructose-bisphosphate aldolase 1, (Dcitr04g02510.1.1) appears to have moderate expression in the male abdomen and terminal abdomen, and highest expression in the adult whole body (Figure 4). TPI catalyzes the fifth step, the reversible interconversion of DHAP and GAP. TPI is also important to sustain DHAP to maintain insect flight muscle activity [78]. D. citri contains a single copy of this gene (Dcitr10g08030.1.1), which is consistent with other insects (Table 4). Hexokinase amino acid sequence of D. citri compared with sequences from other insects. MUSCLE multiple sequence alignments of HK in D. citri and orthologs were performed using MEGA7 and neighbor-joining phylogenetic trees were constructed with p-distance for determining evolutionary distance and 1000 bootstrapping replicates [59]. Accession numbers for the orthologous sequences used in phylogenetic analysis are in Table 3.

Energy production phase of glycolysis
GAPDH catalyzes the reversible conversion of GAP to 1,3-bisphosphoglycerate during glycolysis. Two GAPDH genes were annotated in D. citri and the expression data for the two paralogs show that GAPDH-like 1 (Dcitr10g11030.1.1) has higher expression in the male terminal abdomen and whole body and GAPDH-like 2 (Dcitr01g03200.1.1) has higher expression values overall with a considerable increase in male thorax, female thorax and whole body (NCBI BioProjects PRJNA609978 and PRJNA448935) (Figure 4 and Table 4 in GigaDB [79]).  The number of genes identified in glycolysis, gluconeogenesis and trehaloneogenesis in D. citri and related organisms. †indicates that there are possibly more PYK genes in D. melanogaster and potentially six in A. mellifera. * indicates that there is phosphoglucomutase 2a and 2b in D. melanogaster. Copy numbers for the orthologs were obtained from NCBI [56], OrthoDB [67], and Flybase [68]. (Table 4). PGAM is an enzyme that converts 3-phosphoglycerate to 2-phosphoglycerate.
Members of the PGAM family share a common PGAM domain, and function as either phosphotransferases or phosphohydrolases [80]. Two copies of PGAM were annotated in the D. citri genome (Table 4). PGAM 1 (Dcitr03g11640.1.1) has high expression evident in the midgut and the other paralog, PGAM 2 (Dcitr03g17850.1.1) is highly expressed in the whole body ( Figure 4).
Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the ninth step of the glycolytic pathway and a single copy was annotated in the D. citri genome (Table 4). RNAi knockdown of the -enolase in Nilaparvata lugens reduced egg production, offspring, and hatching rate; however, mortality of adults was unaffected [80]. Pairwise  (Table 4). In D. citri, two PYK genes were characterized and annotated ( Table 2). One of the PYK genes (Dcitr07g06140.1.1) is highly expressed in male and female thorax and the other PYK gene (Dcitr01g11190.1.1) has relatively low overall expression with the highest expression in the male terminal abdomen (Figure 4). Expression analysis of the enzymes from this phase of glycolysis in thoracic tissue shows that the highest expression is observed for GAPDH-like 2 and PYK-like 1 and the lowest occurs for both GAPDH-like 1 and PYK-like 2 ( Figure 5). In addition, PGK (Dcitr00g01740.1.1) and enolase (Dcitr02g07600.1.1) also have high expression in the male and female thorax and PGAM 2 (Dcitr03g17850.1.1) has high expression in whole body besides the male and female thorax (NCBI BioProject PRJNA609978, NCBI BioProject PRJNA448935) (Figure 4 and Table 4 in GigaDB [79]).

Enzymes of gluconeogenesis
Gluconeogenesis is the metabolic process to re-generate glucose from non-carbohydrate substrates. It uses four specific enzymes. PC catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. The curated PC model (Dcitr08g01610.1.1) in D. citri shows highest overall expression in the male and female thorax, male and female head, and male and female antenna (Figures 6, 7 and Table 5 in GigaDB [79]). PEPCK controls cataplerotic flux and converts oxaloacetate from the tricarboxylic acid cycle to form phosphoenolpyruvate (PEP). Two PEPCK genes were annotated and   [25]. Data in the heatmap show transcripts per million scaled by gene. RNA-seq data are available from NCBI Bioprojects PRJNA609978 and PRJNA448935 and published datasets [72]. characterized in the D. citri genome ( Table 2). The first PEPCK copy (Dcitr05g10240.1.1) has higher expression in most tissues than all of the other gluconeogenesis genes as is evident in the male and female antenna, male and female thorax, and the male and female head. copy of this gene was annotated in D. citri, similar to other insects, although two copies are present in the pea aphid, A. pisum, and the honeybee, A. mellifera (Table 2). FBPase (Dcitr11g08070.1.1) shows highest expression in the egg ( Figure 5). Glucose-6-phosphatase (G6Pase or G6P), which is specific to gluconeogenesis, catalyzes the conversion of glucose-6-phosphate to glucose [31]. However, this enzyme is not present in most insect species, including D. citri. Though present in N. lugens, RNAi studies showed that knockdown of G6Pase in N. lugens had no effect on the genes involved in trehalose metabolism [82].

Enzymes of trehaloneogenesis
Trehalose is a non-reducing disaccharide present in many organisms, including yeast, fungi, bacteria, plants and invertebrates. As the main hemolymph sugar in insects, it is found in high concentrations [32,83]. Trehalose is synthesized from glucose by trehalose-6-phosphate (Tre-6-P), where the mobilization of trehalose to glucose is considered critical for metabolic homeostasis in insect physiology [30]. Synthesis of trehalose occurs in the fat body, when stimulated by neuropeptides from the brain [32]. These peptides decrease the concentration of fructose 2,6-bisphosphate, which strongly activates the glycolytic enzyme PFK and inhibits the gluconeogenic enzyme fructose 1,6-bisphosphatase. Fructose 2,6-bisphosphatase is thus a key metabolic signal in regulating trehalose synthesis in insects. After synthesis, trehalose is transported through the hemolymph and enters cells through trehalose transporters, where it is converted into glucose by trehalase.
However, many insects appear to lack this gene, including D. citri as it was not found in the v3 genome. Most insects with multiple TPS genes encode proteins with TPS and TPP domains [85,86]. TPS in Drosophila appears to have the functions of both TPS and TPP [87].
Trehalase (TREH) catalyzes stored trehalose by cleaving it to two glucose molecules. There are two trehalase genes: TREH-1, which encodes a soluble enzyme found in hemolymph, goblet cell cavity and egg homogenates, and TREH-2, which encodes a membrane-bound enzyme found in flight muscle, ovary, spermatophore, midgut, brain and thoracic ganglia [84]. The two curated TREH genes in D. citri show different expression in the psyllid.
TREH is the only enzyme known for the irreversible splitting of trehalose in all insects [84] and D. citri and T. castaneum are the only insects with the second copy, TREH-2 (Table 2).
The two main trehalose transporters are trehalose transporter 1 (TRET1) and trehalose transporter 2 (TRET2), which both transport trehalose to and from cells with TREH. One gene copy for each of these trehalose transporters was annotated in D. citri (Table 2).

CONCLUSION
Manual annotation of the central metabolic pathways of glycolysis, gluconeogenesis, and trehaloneogenesis provides the accurate gene models required for development of molecular therapeutics to target D. citri. RNAi studies targeting genes involved in trehalose metabolism produced significant mortality in D. citri, [39,88] [79]). Annotation of the carbohydrate metabolism genes advances the understanding of the basic biology of D. citri and will aid in the development of RNAi-based applications.

REUSE POTENTIAL
The manually curated gene models were annotated through a collaborative community project [11] to further understand psyllid biology and with a goal to annotate gene families species-specific gene targets to control psyllid populations (potentially through RNAi) and reduce the effects of pathogens such as CLas.

DATA AVAILABILITY
The datasets supporting this article are available in the GigaScience GigaDB repository [79]. The gene models are part of an updated official gene set (OGS) for D. citri submitted to NCBI under Bioproject PRJNA29447. The OGS (v3) is also publicly available for download, BLAST analysis and expression profiling on Citrusgreening.org and the Citrus Greening Expression Network [25]. The D. citri genome assembly (v3), OGS (v3) and transcriptomes are accessible on the Citrusgreening.org portal [12]. Accession numbers for genes used in multiple alignments or phylogenetic trees are provided in Table 1.

EDITOR'S NOTE
This article is one of a series of Data Releases crediting the outputs of a student-focused and community-driven manual annotation project curating gene models and, if required, correcting assembly anomalies, for the Diaphorina citri genome project [95].

ETHICAL APPROVAL
Not applicable.

CONSENT FOR PUBLICATION
Not applicable.