A link between energy metabolism and plant host adaptation states in the two-spotted spider mite, Tetranychus urticae (Koch)

Energy metabolism is a highly conserved process that balances generation of cellular energy and maintenance of redox homeostasis. It consists of five interconnected pathways: glycolysis, tricarboxylic acid cycle, pentose phosphate, trans-sulfuration, and NAD+ biosynthesis pathways. Environmental stress rewires cellular energy metabolism. Type-2 diabetes is a well-studied energy metabolism rewiring state in human pancreatic β-cells where glucose metabolism is uncoupled from insulin secretion. The two-spotted spider mite, Tetranychus urticae (Koch), exhibits a remarkable ability to adapt to environmental stress. Upon transfer to unfavourable plant hosts, mites experience extreme xenobiotic stress that dramatically affects their survivorship and fecundity. However, within 25 generations, mites adapt to the xenobiotic stress and restore their fitness. Mites’ ability to withstand long-term xenobiotic stress raises a question of their energy metabolism states during host adaptation. Here, we compared the transcriptional responses of five energy metabolism pathways between host-adapted and non-adapted mites while using responses in human pancreatic islet donors to model these pathways under stress. We found that non-adapted mites and human pancreatic β-cells responded in a similar manner to host plant transfer and diabetogenic stress respectively, where redox homeostasis maintenance was favoured over energy generation. Remarkably, we found that upon host-adaptation, mite energy metabolic states were restored to normal. These findings suggest that genes involved in energy metabolism can serve as molecular markers for mite host-adaptation.

www.nature.com/scientificreports/ of energy metabolic responses to xenobiotics.We aimed at elucidating the evolutionary conserved metabolic adaptation to xenobiotic stress that is shared between human β-cells and mites.We performed a comprehensive analysis of transcriptional changes of energy metabolic pathways in xenobiotically adapted and non-adapted TSSM populations and have compared them to transcriptional changes of living human islets from donors with impaired glucose tolerance (IGT) and sincere T2D 28 .Our results indicate a significant overlap in energy metabolism between human T2D islets and host non-adapted mites that are exposed to xenobiotic stress.Thus, an evolutionarily conserved response to stress seems to reflect a continuous attempt in both host non-adapted mites and T2D to limit the production of ROS and increase metabolic defense to counter ROS.However, hostadapted mite populations that effectively counteract plant host xenobiotic compounds are able to revert their energy metabolism to normal despite long-term exposure to severe xenobiotic stress.These findings highlight the potential use of energy metabolism as a measure of stress response in mites.

Results and discussion
Energy metabolism and stress response pathways are highly conserved between humans and the two-spotted spider mite To determine the conservation of energy metabolism under xenobiotic stress, the glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate (PPP), trans-sulfuration (TS), and nicotinamide adenine dinucleotide (NAD) biosynthesis pathways were compared between humans and the two-spotted spider mite (TSSM), Tetranychus urticae.The corresponding hsa00010, hsa00030, hsa00020, hsa00270, and hsa00380 KEGG pathways were selected and used to identify a single protein-coding gene for each enzymatic step.In cases where multiple forms of a particular gene existed, the variant encoding the longest human protein sequence was selected.The representative protein sequences were then used as a query against the Tetranychus urticae database on ORCAE (https:// bioin forma tics.psb.ugent.be/ orcae/).Mite genes with the bidirectional best hits (BBHs) to human counterparts were considered to be orthologues.The percent similarity, E value, and alignment bit score of mite orthologous genes are shown in Table 1.Despite being distantly related to humans, conservation of the rate limiting glycolysis enzyme-Phosphofructokinase (PFKL)-and other key glycolytic regulators-Glucokinase (GCK), Pyruvate kinase (PK), and Glycogen synthase 1 (GYS) was established in T. urticae.Furthermore, the conservation of rate-limiting enzymes Glucose-6 phosphate dehydrogenase (G6PD), Isocitrate dehydrogenase (IDH), and Cystathionine beta-synthase (CBS) was established for the PPP, TCA cycle, and TS pathways, respectively.In addition, the conservation of the rate-limiting enzymes for the Preiss-Handler, de novo, and salvage pathways-Nicotinamide nucleotide adenylyltransferase (NMNAT), Tryptophan 2,3-dioxygenase (TDO2) and Quinolinate phosphoribosyltransferase (QPRT), and Nicotinamide phosphoribosyltransferase (NAMPT), respectively -were also confirmed.The high degree of conservation for rate-limiting enzymes between human and T. urticae orthologues indicate that these pathways are under considerable evolutionary constraint.

Comparative analyses reveal species-specific genes involved in energy metabolism and stress response
Despite a high degree of conservation between humans and mite pathways, there are few notable differences in gene complements associated with energy metabolism.Firstly, we were unable to identify the Phosphoglycerate mutase (PGAM) orthologue in mites.In humans, this enzyme is responsible for the conversion of 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG) in glycolysis.However, this reaction can be catalyzed by two evolutionarily unrelated and distinct proteins with dissimilar amino acid compositions, catalytic residues, protein structure, and size 29 .The enzyme found in humans is cofactor-dependent PGAM (referred to as PGAM/dPGAM), while the other is cofactor independent PGAM (iPGAM).As the conversion of 3PG to 2PG is an essential reaction in the glycolysis pathway, we looked if mites may be using iPGAM for the catalysis of this reaction.Protein sequences of iPGAM from iPGAM-using organisms (including Trichoplax adhaerens, Nematostella vectensis, Caenorhabditis elegans, Ixodes scapularis and Arabidopsis thaliana) were retrieved from NCBI and used as a query against the T. urticae database to identify orthologues.While an organism may possess a single form or both forms of PGAM, sequence alignments using BLAST confirmed that mites exclusively use the iPGAM form for the conversion of 3PG to 2PG.Secondly, we were unable to identify the orthologue of Lactate dehydrogenase A (LDHA) suggesting that mites cannot ferment pyruvate to form lactate.This confirms the previously reported lack of LDHA activity in mites 30 .These collective findings indicate that the aerobic glycolytic pathway is highly conserved between humans and mites with the exception of generation of lactate from pyruvate.Thirdly, we were unable to identify a mite orthologue of 3-mercaptopyruvate sulfurtransferase (MPST) from the trans-sulfuration pathway.MPST has been lost within several eukaryotic taxa including tunicates and insects 31 .It is not an essential enzyme within the trans-sulfuration pathway as there is a redundancy in the number of enzymes that perform the same function 32 .Fourthly, mites do not have an orthologue of NAMPT that in humans catalyzes the conversion of nicotinamide (NA) to nicotinamide mononucleotide (NAM).Pyrazinamidase/Nicotinamidase 1 (PNC-1) is a functionally equivalent enzyme found in the invertebrate NAD salvage pathway 33 .We identified tetur19g00460 as an orthologue of the PNC-1 gene from Caenorhabditis elegans.Lastly, Arylformamidase (AFMID), also involved in the NAD biosynthetic pathway, remained undetected in T. urticae like in some spiders (Theridion grallator and T. californicum) 33 .

The same metabolic endpoints are achieved with different xenobiotic-responsive pathways in humans and the two-spotted spider mite
Metabolic flow through energy-generating pathways is regulated at multiple levels and is modified upon stress.While allosteric inhibition and post-transcriptional modifications regulate important checkpoints in the pathway, www.nature.com/scientificreports/here, we compared transcriptional changes within the energy-generating and stress-response pathways between humans and mites when exposed to xenobiotic stress.To reconstruct transcriptional changes in humans, we used expression data from individuals with impaired glucose tolerance (IGT, i.e. prediabetes) and T2D.Transcriptional changes were presented against samples collected from non-diabetic control individuals.On the mite side, we used expression data from host-adapted and host non-adapted mite populations while feeding on one of two challenging hosts, Arabidopsis thaliana (thale cress) or Solanum lycopersicum (tomato).These data were contrasted against a gene expression set obtained from mites feeding on Phaseolus vulgaris (bean), a nonchallenging host across mite populations.

Aerobic glycolysis
We found no detectable differential stress response between IGT (prediabetic) and non-diabetic samples when we compared the expression of genes encoding enzymes that drive the glycolysis pathway (Fig. 2).Similarly, there were no significant changes in glycolytic gene expression in the Arabidopsis and tomato host-adapted mite samples with the exception of Triose-phosphate isomerase (TPI1), downregulated in Arabidopsis-adapted mites (Fig. 2).Lack of differences between IGT and non-diabetics (ND) conditions 28 , highlights the adaptive nature of IGT.By inference, these data indicate that host-adapted mite populations successfully eliminated the effects of host-induced xenobiotic stresses on glucose metabolism.In contrast, T2D and host non-adapted mites show a plethora of transcriptional changes.In human T2D samples there is an upregulation of genes encoding enzymes that drive the preparatory phase of glycolysis.One of them is Glucokinase (GCK), whose upregulation may affect glucose uptake 34 .In addition, the increased expression of 6-Phosphofructo-2-kinase/Fructose-2,6-biphosphatase 3 (PFKFB3) may promote the synthesis of fructose 2,6-bisphosphate (F2,6BP) that is a potent allosteric activator of PFKL, the rate-limiting enzyme of glycolysis 35 .Furthermore, in the T2D sample, there is an upregulation of Fructose-bisphosphate aldolase (ALDOB) that converts glucose and fructose intermediates to trioses 36 .Based on these expression patterns, one would expect enhanced metabolic flow through the preparatory phase of glycolysis in the T2D sample.This is contrasted by downregulation of genes encoding glycolytic enzymes of the payoff phase such as Triose-phosphate isomerase, Enolase 1, and Pyruvate kinase M (TPI, ENO1, and PKM) indicating suppression of pyruvate metabolism 28 (Fig. 2).In addition, in diabetic mice and human T2D samples, there is an upregulation of LDHA, indicating additional redirection of pyruvate from the mitochondria into lactate generation 19,20 .In xenobiotically-challenged mites, there were no changes in the expression of genes driving the preparatory phase of glycolysis (Fig. 2).However, similar to human T2D, the expression of genes encoding enzymes driving the pay-off phase of the glycolytic pathway was decreased in non-adapted mites upon transfer to either Arabidopsis or tomato (Fig. 2).It appears that mites may have a tighter transcriptional control of pyruvate generation, as genes encoding enzymes of most of the pay-off phase are downregulated.Mites presumably require such a stringency in preventing pyruvate generation under stress as they lack LDHA 30 that enables a depletion of pyruvate by channeling it toward the synthesis of lactate.Thus, under stress, the generation of pyruvate in both www.nature.com/scientificreports/human T2D and non-adapted mite samples seems to be suppressed through downregulation of genes involved in pyruvate generation.A build-up of 6-carbon (C6) glycolytic intermediates in the preparatory phase is expected to be shunted in the PPP 19 in both humans and mites.Thus, under stress, both human β-cells and mites likely shunt intermediary metabolites by funneling glycolytic precursors such as glucose-6 phosphate (G6P) and fructose-6 phosphate (F6P) towards the PPP for NADPH and GSH production or by build-up of fatty acids, while suppressing pyruvate entry into the mitochondrial TCA cycle.

Tricarboxylic acid (TCA) cycle
Mitochondria can function as a primary source of ROS "leakage" in most tissues and cells 37 .It has been estimated that about 0.2-2% of oxygen consumed is converted into O 2-by the electron transport chain 38 that is interconnected with the TCA cycle.This is particularly augmented under oxidative stress that arises as a consequence of xenobiotic exposure 11 .Therefore, there is a general tendency to reduce utilization of mitochondria in order to limit the impact of oxidative and xenobiotic stresses 20 .Similar to the glycolysis pathway, IGT and host-adapted mite samples did not change the expression of genes encoding enzymes in TCA cycle (Fig. 3).However, T2D and host-non-adapted mite samples showed a similar pattern of gene downregulation that is characteristic of diabetic mouse and human islets where there is transcriptional and protein downregulation of the TCA cycle 19,20 .
Remarkably, non-adapted mites on Arabidopsis displayed an elevated Pyruvate dehydrogenase kinase 1 (PDK1) expression similar to diabetic mouse islets 20 (Fig. 3).Interestingly, PDK4 in humans similar to PDK1 in mice and mites, was strongly upregulated in the islets from the donors with T2D 39 .PDK1 and 4 directly block the utilization of pyruvate by the TCA cycle and thus effectively suppress the utilization of this pathway 40 .Decreased expression of genes encoding enzymes of the TCA cycle implied a decline in OxPHOS activity generating ATP, NADH and FADH2, that feed into complexes I, III, and IV of the electron transport chain.The downregulation of the TCA cycle was contrasted by a notable difference in Oxyglutarate dehydrogenase (OGDH) expression between non-adapted mites and T2D.The OGDH complex catalyzes the decarboxylation of 2-HG to succinyl-CoA and reduces NAD+ to NADH 41 .Under homeostatic regulation, the reducing equivalent NADH feeds the respiratory complex I to generate the mitochondrial membrane potential required for ATP production.Unlike in T2D, in www.nature.com/scientificreports/non-adapted mites, OGDH was downregulated, indicating suppression of the mitochondrial metabolism at a larger scale compared to human T2D.Similarly, Drosophila exposed to oxidative stress (that best recapitulates xenobiotic exposure) experienced transcriptional downregulation of pathways enriched for genes responsible for ATP metabolic processes, mitochondrial translation, and generation of precursor metabolites and energy 42 , clearly recapitulating the patterns in mites and T2D 43,44 .The expression pattern of TCA cycle genes suggests that in mites, similar to humans, there is mitochondrial attenuation that slows ATP production in the face of oxidative stress during xenobiotic exposure.The global pattern of mitochondrial attenuation reduces the risk of apoptosis by reducing the risk of ROS accumulation and was shared across the species.

Pentose phosphate pathway (PPP)
The attenuation of the pay-off phase of glycolysis and the TCA cycle in T2D and non-host-adapted mite samples is predicted to lead to rewiring of C6 and C3 sugar metabolism.Glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (G3P) can be redirected from glycolysis into the oxidative and nonoxidative branches of the pentose phosphate pathway (PPP).In both branches, these metabolites ultimately yield ribose-5-phosphate (R5P) 19,20,45 (Fig. 4).However, downregulation of Transketolase (TKT) and Transaldolase (TALDO1) in T2D and host non-adapted mite samples indicates that the PPP redirects glycolytic flux towards its oxidative branch (Fig. 4).R5P biosynthesis by the oxidative branch also yields NADPH that functions as a crucial antioxidant to quench ROS.NADPH forms reduced glutathione, which converts reactive H 2 O 2 into H 2 O by glutathione peroxidase 46 .Thus, the synthesis of NADPH is important for the maintenance of cellular redox homeostasis.In human IGT samples and host-adapted mite samples, there were no changes in the expression of genes encoding PPP enzymes (Fig. 4).However, in the T2D sample, increased funneling of glycolytic metabolites into the PPP was supported by increased expression of the gene encoding 6-phosphogluconolactonase (PGLS), the second enzyme in the oxidative branch of the PPP, coinciding with a downregulation of TALDO1 and Ribulosephosphate 3-epimerase (RPE).In non-adapted mites on tomato, the expression of a gene encoding the enzyme Ribose-5 phosphate isomerase A (RPIA) that catalyzes the final step in R5P biosynthesis was increased similar to diabetic mouse islets 20 .This coincides with a downregulation of TALDO1 and TKT, suggesting that mites under stress maximize the synthesis of R5P (Fig. 4).R5P is the main building block for nucleotide synthesis.
Given that mites use guanine as their main nitrogen excretion product 47 , coupling ROS detoxification (through the oxidative PPP) with nitrogen excretion may increase the requirement for the PPP in mites under xenobiotic stress.These results suggest that in both human T2D and non-host-adapted mite samples, xenobiotic stress stimulates R5P and NADPH generation that is necessary for nucleotide biosynthesis and antioxidant responses, while restricting the re-entry of PPP intermediates into glycolysis/gluconeogenesis.

Trans-sulfuration pathway (TS)
Cysteine availability controls the synthesis of glutathione (GSH), which is the main regulator of cellular redox homeostasis and an important agent in xenobiotic detoxification 48 .The TS pathway does not undergo transcriptional changes in the human IGT sample (Fig. 5).In T2D and host-non-adapted mite samples there is downregulation of the gene encoding Cystathionine gamma-lyase (CTH) that catalyzes the last step in the TS pathway leading to cysteine biosynthesis.Potentially, to preserve the cysteine pool toward the synthesis of GSH, there is coordinated downregulation of genes encoding enzymes that use cysteine toward the synthesis of pyruvate and H 2 S such as Glutamic-oxaloacetic transaminase (GOT1) and Cysteine dioxygenase type 1 (CDO1).A host-specific increase in Glutamate-cysteine ligase catalytic subunit (GCLC) and Glutathione synthase (GSS) expression in Arabidopsis-adapted mites leading to the synthesis of GSH may reflect a need to detoxify Arabidopsis secondary metabolites such as glucosinolates 49 .Even though there are sample/species specific changes in the TS pathway, it appears that cysteine preservation is a priority under diabetogenic and xenobiotic stress.

NAD biosynthesis
Reduced or oxidized NAD is a coenzyme for redox reactions in metabolic pathways 50 .It can be synthesized through three different pathways: (a) the Preiss-Handler pathway (PHP), generating NAD from nicotinic acid, (b) the de novo synthesis pathway (DNP), generating NAD from tryptophan, and (c) the salvage pathway (SP), generating NAD from nicotinamide (NAM) 8 .In human IGT and host-adapted mite samples, genes encoding enzymes in NAD pathways did not change their expression (Fig. 6).The notable exception is the downregulation of a gene encoding 3-Hydroxyanthranilate 3,4-dioxygenase (HAAO) that is part of the de novo synthesis pathway in Arabidopsis-adapted mites.In non-adapted mites there is a downregulation of genes supporting de novo synthesis and salvage pathways (Fig. 6).In addition, the mite orthologue of the human gene encoding Arylformamidase (Kynurenine formamidase, AFMID), a key step in tryptophan metabolism, is missing (Table 1).
In host non-adapted mites and T2D samples, the synthesis of NAD from nicotinic acid is enhanced through the upregulation of NAD synthetase 1 (NADSYN1) and Nicotinate phosphoribosyltransferase (NAPRT), respectively, indicating streamlining of NAD biosynthesis from nicotinic acid.In summary, the comparison of the expression of genes driving cellular energy metabolism between hostadapted and non-adapted mites when transferred to either Arabidopsis or tomato showed that: (a) at the transcriptional level, host non-adapted mites respond to xenobiotic stress similarly to human genes in T2D samples where energy metabolism shifts towards ROS detoxification rather than energy generation, and (b) upon host adaptation, mites revert the expression of genes supporting cellular energy metabolism to a basal level despite the continued presence of xenobiotic compounds in their diet.Therefore, the expression of genes driving cellular energy metabolism can be used as markers of mite host-adaptation status.

Transcriptional changes in genes involved in energy metabolism early in mite host adaptation process
Mite adaptation to a new plant host occurs over 5-25 generations 23,25,26 .Samples used in our analysis so far were collected from host-adapted mites that were maintained on "new" plant hosts for >25 generations.To determine trends in energy metabolism early in the adaptation process we used gene expression data collected from mites that were exposed to a new host for ~5 generations.At that time, there is already a significant improvement in mite fitness relative to non-adapted mites 25,26 and the general gene expression pattern shows similarity to mite populations that are resistant to xenobiotic stress 51 .Figure 7 shows gene expression across five energy and stress metabolism pathways in mites that were maintained on tomato, cotton, maize and soybean leaves for ~5 generations (5G).The expression of selected genes in 5G mites differs from patterns seen in the host-adapted mite samples and have greater similarity to responses observed in host non-adapted mites and the T2D sample.Specifically, upregulation/basal levels of GCK (the first step in the glycolysis pathway) suggests the commitment of glucose for retention in cells and entry into the glycolysis pathway.Even though the expression of genes driving the glycolysis pathway are not downregulated to the same level as seen in T2D and host non-adapted mite samples, the upregulation of genes in the PPP that sequester glucose-6-phosphate away from glycolysis suggests www.nature.com/scientificreports/ that like in human T2D and mite xenobiotically stressed samples, 5G mites still favour detoxification of ROS and the production of R5P at the cost of production of pyruvate.This is consistent with the downregulation of genes that enable pyruvate entry from glycolysis into the TCA cycle (Fig. 7A-C).Consistent with the increased metabolite flow through the PPP and similarly to T2D and mite host non-adapted samples, the expression of genes supporting the trans-sulfuration pathway suggests channeling of cysteine toward the generation of glutathione (Fig. 7D).The expression of genes from NAD biosynthetic pathways suggests that the Preiss-Handler pathway remains equally important for 5G mites as it is for humans under xenobiotic stress (Fig. 7E).While there is a good parallelism in gene expression patterns across the five pathways between T2D, 5G, and mite host non-adapted samples, 5G mite samples show additional interesting gene expression profiles within the TCA and NAD pathways.In the TCA cycle, 5G mites have an increased expression of genes encoding either Dihydrolipoamide acetyltransferase (DLAT), a subunit of the multienzyme complex Pyruvate dehydrogenase (PDH) 52 , Isocitrate dehydrogenases (IDH3), Succinate dehydrogenase (SDH, iron sulfur subunits), or mitochondrial Malate dehydrogenase (MDH1) (Fig. 7B).In humans, these enzymes have broader functions beyond the TCA cycle.For example, MDH2 and IDH1 are promiscuous and able to catalyze the alpha-ketoglutarate (α-KG) conversion to L-2-hydroxyglutarate (L-2-HG) 53 that in turn inhibit the activity of Ten-Eleven Translocation (TET) Methylcytosine dioxygenases, enzymes associated with DNA demethylation 54 .In addition to this route of L-2-HG synthesis, in host non-adapted mites and some 5G samples, there is a downregulation of OGDH that also leads to the accumulation of L-2-HG, indicating potential enhancement of L-2-HG synthesis in 5G mites.Besides, upregulation of either IDH3 or SUCL in 5G mites may reflect their enhanced capacity to deal with ROS, as IDH1 in humans is involved in maintenance of reduced glutathione 55 while SUCLG1 and SUCLA2 are implicated in protein expression of redox-scavenging enzymes 56 .Furthermore, within the de novo NAD synthesis sub pathway, there is an upregulation of a heme-dependent dioxygenase (TDO2) that catalyzes the oxidative cleavage of L-tryptophan (L-Trp) and downregulation of downstream enzyme Kynurenine 3-monooxygenase (KMO) (Fig. 7E).This expression pattern is predicted to yield greater amounts of kynurenine in 5G mites.In humans, kynurenine activates Aryl hydrocarbon receptor (AhR) and causes oxidative stress and pro-inflammatory responses 57 .Interestingly, Kynureninase (KYNU), an enzyme that catalyzes the cleavage of L-kynurenine and is involved in biosynthesis of NAD, is strongly upregulated in all 5G mites, indicating that kynurenine pathway in 5G mites is diverted into de-novo NAD synthesis away from AhR signaling.Collectively, diversion of kynurenine pathway into NAD biosynthesis and the upregulation of selected TCA enzymes may indicate enhancement of antioxidant defenses and epigenetic changes in gene regulation in 5G mites.

Conclusion
We used gene expression data to compare transcriptional responses of five pathways-glycolysis, pentose phosphate (PPP), tricarboxylic acid (TCA) cycle, trans-sulfuration (TS), and NAD biosynthesis-between hostadapted and host non-adapted spider mite populations using responses in pancreatic β-cells of type-2 diabetes patients to model energy metabolism under stress.All five pathways were highly conserved between spider mites and humans, with the exception of some species-specific differences in lactate production, the TS pathway, www.nature.com/scientificreports/and NAD biosynthesis.Gene expression data revealed similar responses to diabetogenic stress and host-plant transfer between humans and spider mites, respectively.The payoff phase of glycolysis and the TCA cycle were downregulated in both samples, likely to prevent excess reactive oxygen species (ROS) production during aerobic respiration.Similarly, genes required to shunt carbon back to glycolysis are downregulated in the PPP, while genes supporting antioxidant production in the PPP and TS pathway were upregulated.Gene expression data would also suggest a preference for NAD biosynthesis from nicotinic acid in both T2D and non-adapted mite samples.Remarkably, our data show that in both host-adapted mite strains, gene expression returns to a basal level in each pathway.Only one notable host-specific difference was observed, where the glutathione synthetase gene of the TS pathway is upregulated only in Arabidopsis-adapted mites, likely reflective of their requirement to detoxify Brassicaceous defense compounds, glucosinolates.Therefore, upon host adaptation, mites revert the expression of genes supporting energy metabolism to normal despite the continued presence of xenobiotics in their diet.We also examined mite gene expression changes after short-term adaptation to a new host.After five generations on a new host, mites still experienced dysregulation at the transcriptional level in each pathway to favour ROS detoxification over energy generation, which parallels the responses seen in T2D and host nonadapted samples.These findings were in line with a previous study that demonstrated a notable decrease in the transcription of genes linked to gluconeogenesis and the generation of ATP in mites subjected to spatiotemporal stress and selection 58 .Collectively, our data show that upon host-adaptation, mites can overcome the metabolic stress associated with host-plant transfer, suggesting that genes involved in energy metabolism can serve as molecular markers for mite host-adaptation.

Identification of human metabolic pathways and protein sequences
The Homo sapiens KEGG database was used to identify all protein-coding genes involved in metabolism associated with glucose homeostasis.Enzymes in the glycolysis, pentose phosphate, citric acid, trans-sulfuration and NAD pathways were retrieved using KEGG IDs hsa00010, hsa00030, hsa00020, hsa00270 and hsa00380, respectively.A representative protein sequence for each enzyme was retrieved from the NCBI database.

BLAST based identification of mite orthologues
Human sequences were used as a query against the Tetranychus urticae protein database using the blastp program 59 with BLOSUM62 similarity matrix.The top scoring matches with bit score of at least 50, E-value below 1E-4, and percent similarity higher than 50 were used in reciprocal blastp searches against human protein database to establish bidirectional best hits (BBH) orthology 60 .

Mites and plant host maintenance and sample preparation
Tomato, Solanum lycopersicum cv Moneymaker, and bean, Phaseolus vulgaris cv California Red Kidney, plants were grown in growth chambers at 25 °C, 60 % relative humidity and with a 16:8 h light:dark photoperiod.Reference mite population Tetranychus urticae "London" was reared on bean plants for >10 years.The T. urticae tomato and Arabidopsis adapted strains were derived from "London" via experimental selection and maintained on tomato cv Moneymaker and Arabidopsis Col-0 respectively 23,61 .For all experiments, adult female mites were used.Both non-adapted and adapted mites were reared in the common garden (bean) for two generations prior to host transfer experiment.Subterminal leaflets of 4-5 weeks old tomato plants were infested with 100 adult female mites.After 24 hours, mites were collected and samples from two plants were pooled to produce a single biological replicate.Experimental plants used in the study were either commercial varieties (tomato, soy, maize, cotton) or laboratory strain (Arabidopsis, seed obtained from The Arabidopsis Information Resource).None of them are endangered species.We confirm that all methods were carried out in accordance with relevant guidelines in the method section.

RNA extraction and sequencing following tomato host plant transfer
RNA extraction was performed using the RNeasy Mini Kit followed by an on-column DNase treatment to ensure genomic DNA removal.The quantity and purity of extracted RNA was assessed using a Thermo Scientific Nan-oDrop 2000.Strand specific paired-end (2 × 75 bp) sequencing was conducted according to Illumina TruSeq protocol (Illumina, San Diego, CA) at the Centro Nacional de Análisis Genómico (CNAG) (Barcelona, Spain).Reads were mapped to the reference T. urticae genome 27 using STAR aligner v.2.7.1a 62 in a two-pass mode with annotation allowing only unique mapping, up to five mismatches per read mapped, a minimum intron size of 20 bp, a maximum intron size of 15,000 bp.Read counts were generated at the level of gene locus using HTSeq v.0.6.0 in "union" mode 63 against T. urticae genome yielding 9-13.5 million mapped fragments per library.Genes expressed at the level at or above 1 fragment count per million reads in at least three samples were considered for the subsequent analysis.Log2 fold changes and associated FDR-adjusted p-values were generated using voom/ limma workflow 64 .

Expression data retrieval and processing
RNA-Seq expression data for human control, impaired glucose tolerance (IGT), and type 2 diabetes (T2D) pancreatic islets were retrieved from the GEO accession GSE164416.Data pre-processing and normalization was conducted as per original publication 28 .Briefly, read counts were retrieved from the GEO public database, genes were filtered by mean raw read count over 5, and samples with INS (ENSG00000254647) as the highest expressed gene were retained for subsequent analysis using voom/limma workflow 64 to generate Log2 fold changes and associated adjusted p-values.

Figure 1 .
Figure 1.Energy metabolism and stress response pathways in a healthy cell.Pyruvate generated from glucose through glycolysis is used as a substrate for the mitochondrial Tricarboxylic acid (TCA) cycle that is coupled with Oxidative phosphorylation (OxPHOS) to generate ATP, ROS and NADH.NAD is mainly generated by the Salvage pathway.Glycolysis intermediates participate in the non-oxidative pentose phosphate pathway (PPP) to reduce oxidized glutathione (GSS) synthesized by the trans-sulfuration pathway.The oxidative branch of the PPP supports biosynthesis of nucleotide precursors of nucleic acids.

FructoseFigure 2 .
Figure2.Glycolysis.Gene expression patterns observed in the glycolysis pathway in impaired glucose tolerance (IGT) and type 2 diabetes (T2D) pancreatic β-cells, and adapted (A) and non-adapted (N) spider mites when reared on non-favourable host plants, Arabidopsis and Tomato, relative to a favourable host plant (bean).NS not significant at FDR-adjusted p-value cut-off of 0.05, ND not detected.

Figure 3 .
Figure 3. Tricarboxylic acid cycle.Gene expression patterns observed in the tricarboxylic acid cycle pathway in impaired glucose tolerance (IGT) and type 2 diabetes (T2D) pancreatic β-cells, and adapted (A) and nonadapted (N) spider mites when reared on non-favourable host plants, Arabidopsis and Tomato, relative to a favourable host plant (bean).NS not significant at FDR-adjusted p-value cut-off of 0.05, ND not detected.

Fructose 6 Figure 4 .
Figure 4. Pentose phosphate pathway.Gene expression patterns observed in the pentose phosphate pathway in impaired glucose tolerance (IGT) and type 2 diabetes (T2D) pancreatic β-cells, and adapted (A) and nonadapted (N) spider mites when reared on non-favourable host plants, Arabidopsis and Tomato, relative to a favourable host plant (bean).NS not significant at FDR-adjusted p-value cut-off of 0.05, ND not detected.

Figure 5 .
Figure 5.Trans-sulfuration pathway.Gene expression patterns observed in the trans-sulfuration cycle pathway in impaired glucose tolerance (IGT) and type 2 diabetes (T2D) pancreatic β-cells, and adapted (A) and nonadapted (N) spider mites when reared on non-favourable host plants, Arabidopsis and Tomato, relative to a favourable host plant (bean).NS not significant at FDR-adjusted p-value cut-off of 0.05, ND not detected.

Figure 6 .Figure 7 .
Figure 6.NAD biosynthesis.Gene expression patterns observed in the NAD biosynthesis pathway in impaired glucose tolerance (IGT) and type 2 diabetes (T2D) pancreatic β-cells, and adapted (A) and non-adapted (N) spider mites when reared on non-favourable host plants, Arabidopsis and Tomato, relative to a favourable host plant (bean).NS not significant at FDR-adjusted p-value cut-off of 0.05, ND not detected.

Table 1 .
Orthologous genes in energy-generating and stress response pathways between human and Tetranychus urticae.Identification of orthologous genes involved in glycolysis, tricarboxylic acid cycle, pentose phosphate pathway, trans-sulfuration pathway, and nicotinamide adenine dinucleotide biosynthesis pathway was conducted via bidirectional best hit analyses utilizing the Online Resource for Community Annotation of Eukaryotes (ORCAE) database and NCBI BLAST.Bit scores, E values, % identity, and % similarity were recorded for each identifiable orthologue.