Molecular and biochemical changes in Locusta migratoria (Orthoptera: Acrididae) infected with Paranosema locustae

Abstract Microsporidia are a group of eukaryotic intracellular parasitic organisms that infect almost all vertebrates and invertebrates. Paranosema locustae are specialized parasites of Orthoptera that are often used as biological controls of locusts, with slow effects of action. In this study, we found that after infection with P. locustae, changes in energy metabolism in male and female Locusta migratoria as were consistent, with no gender differences. During the first 8 days of infection, L. migratoria used sugar as a source of energy. After 8 days, lipids and proteins were consumed to provide energy when the spore load was considerably heavy, and energy supply was insufficient. With increasing infection concentration and time, energy conversion from sugar, fats, and proteins was improved, which may explain why high mortality did not occur until about 15 days after P. locustae infection. The tandem mass tag-based quantitative proteomics analysis revealed that most altered metabolism-related proteins were upregulated (27 of 29 in the metabolic pathway). This result suggests that P. locustae infection accelerated metabolism in L. migratoria, which facilitated the pathogen’s life cycle, inhibiting the growth and development of the locusts and eventually killing them. Our findings will be useful to better understand of the chronic pathogenic mechanisms of P. locustae and inform on applications of P. locustae to control locusts.


Introduction
Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates (Li et al. 2018).Paranosema locustae is an obligate parasite of Orthoptera.Due to its high spore production potential in hosts, the horizontal and vertical transmission of the disease, and its wide host range within acridid grasshoppers, this parasite can be comprehensively applied to control grasshoppers in grasslands (Shi and Tan 2019).The use of P. locustae to control locusts presents environmental advantages over the use of conventional insecticides.This fungal species offers a higher safety profile for humans and other nontarget organisms and allows long-term locust suppression (Henry 1985, Wang 2015).The exact mechanisms by which P. locustae causes disease in locusts are unknown.There is a need to determine whether it affects host metabolism in a manner that is similar to other insects, such as silkworms (Hu et al. 2021).
P. locustae lack the mitochondria and only contain a mitosome, which is a relic of the mitochondria; some genes involved in energy and metabolism have been lost (Katinka et al. 2001, Williams et al. 2002, Goldberg et al. 2008).All microsporidia have no capacity for the tricarboxylic acid (TCA) cycle, fatty acid oxidation, oxidative phosphorylation, and lack the ATP synthase complex.Some microsporidians, such as Enterocytozoon bieneusi also lack the glycolytic pathway (Keeling andCorradi 2011, Timofeev et al. 2020).Therefore, the microsporidia strictly depend on infected host cells for substrates and ATP to complete their proliferation (Cuomo et al. 2012, Dean et al. 2016, Qiang et al. 2019, Timofeev et al. 2020).The most essential energy substrates for insects are soluble proteins, soluble sugars, and fats (Han et al. 2019).We investigated whether P. locustae disrupts the normal metabolism of host cells, resulting in the need for the host to consume more energy substrates and synthesize large amounts of ATP to resist microsporidia infection.
Microsporidia infection upregulates host energy synthesis and they depend on the energy provided by the host to complete their proliferation and life cycle, resulting in host death (Luo et al. 2021).The microsporidia usually cause chronic infections in hosts, inhibiting their growth and development (Cassal et al. 2020).The P. locustae take 10-20 days to induce locust morbidity and mortality (Wang and Guang 1994), during which infection-induced specific metabolic changes in energy substrates and ATP have yet to be established.
The main metabolic pathway of sugar is glycolysis, while lipid synthesis is associated with the tricarboxylic acid cycle.In this study, the key metabolites, that is, glucose-6-phosphate (G6P) (associated with glucose metabolism) and citrate (associated with lipid synthesis), were assessed.
The model organism, Locusta migratoria (Orthoptera: Acrididae) (Misof et al. 2014) was used as the host to investigate the utilization of host energy substance by pathogenic.L. migratoria were infected with different concentrations of P. locustae.After infection, levels of G6P, citrate, total protein, and ATP were measured in locust hemolymph at different time points.The effects of P. locustae infection on locust energy substrates were elucidated using its alterations as indicators.
In our previous studies on total protein levels in locust hemolymph after microsporidia infection, electrophoretic profiles of proteins before and after infection were found to be significantly different.This is the first study to use tandem mass tag (TMT) quantitative proteomics techniques to understand these differences.Mass spectrometry-based quantitative analyses of TMT isotope labeling combined with liquid chromatography-mass spectrometry (LC-MS/MS) analysis have become the mainstream technique for studying proteomics and post-translational modifications (Li et al. 2018).Differentially expressed proteins in locust hemolymph before and after P. locustae infestation were determined to investigate the changes in L. migratoria protein expressions after infection.
Biochemical assays and proteomic analyses will be useful to explore the interactions between pathogenic fungi and hosts, improving our understanding of the pathogenic mechanisms of P. locustae and providing a scientific basis for future applications of P. locustae in locust control.

Insect Culture
Migratory locusts used in this study were obtained from laboratory incubation.The populations of L. migratoria were reared at 30 ± 2 °C, 50 ± 5% relative humidity, and under a 14:10 h light:dark (L:D) cycle.The locusts were fed daily on fresh wheat shoots and water.

Experimental P. locustae
The P. locustae used in this study were acquired from the China Agricultural University.

Microsporidian Infection
The spores were stored at -20 °C.Third instar nymphs were starved for 12 h.Using a pipette, 5 µl of 1 × 10 4 , 1 × 10 6 , or 1 × 10 8 spores of P. locustae were slowly dripped onto each nymph mouthpart for sucking (Panek et al. 2018).Cages of inoculated and untreated nymphs (controls) were kept under the same conditions as those in locust rearing.

Collection of Locust Hemolymph
On days 1, 8, and 15 after microsporidia infection, 27 locusts were obtained from 3 concentration gradients for the male, female, and control groups, respectively.Hemolymph (10 µl) was obtained from each locust and mixed with 360 µl of saline.Hemolymph for 9 locusts from the same group was pooled in 1 tube.The hemolymph was collected from the hind legs of locusts.Briefly, the hind legs and surrounding areas were disinfected with 70% alcohol, dried, and 1 hind leg was cut off using scissors.The thorax and abdomen were squeezed, and the hemolymph was quickly drawn and placed in precooled 1.5-ml centrifuge tubes with preadded saline.The samples were immediately mixed in the tubes, and stored at -80 °C for analyses.

DNA Extraction and Detection by TaqMan Assay
The DNA was extracted from 216 locusts.Experimental locusts were placed in 1.5-ml centrifuge tubes supplemented with 200 μl lysis solution (including 0.2M Tris, 0.3M NaCl, 0.025M EDTANa2, and 0.017M SDS), thoroughly ground and maintained in a water bath at 56 °C for 5 min.Then, the tubes were supplemented with 600 μl of a mixture of phenol, chloroform, as well as isoamyl alcohol and centrifuged at 14,000 rpm for 5 min.The supernatants were discarded.The mixtures were washed with 70% ethanol and centrifuged at 14,000 rpm for 5 min.The ethanol solution was discarded, after which the contents in the tubes were dried and dissolved to extract the DNA by adding 50 μl ddH 2 O.The extracted DNA was measured concentration, and were stored at -20 °C.Finally, the acquired DNA was tested for microsporidia by TaqMan probe assay.
For the TaqMan assay, a reaction mixture consisting of 10 μl Platinum Quantitative PCR SuperMix-UDG (catalog no.11730-017; Invitrogen, America), 0.4 μl (10 μM) of each forward and reverse primer, 0.05 μl (10 μM) of the probe, 0.04 μl (50 nM) of the Rox, and 100 ng of the DNA template was prepared and supplemented with nuclease-free water to 20 μl total volume.A positive control consisting of the DNA of P. locustae and a negative control were included in all PCR runs.The TaqMan assay was run in a LightCycler96 Real-Time PCR machine (Roche, Switzerland) under the following conditions: initial denaturation at 95 °C for 2 min followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 63 °C for 30 s.

Determination of Energy Materials
In experimental and control groups, concentrations of glucose-6phosphate dehydrogenase (G6PDH), citrate, and ATP were determined using the Glucose-6-phosphate dehydrogenase (G6PDH) assay kit (catalog no.BC0265; Solarbio, China), citric acid (CA) colorimetric assay kit (catalog no.EBCK351M; Elabscience, China), and the Na + K + -ATP enzymatic activity assay kit (catalog no.BC0065; Solarbio, China), respectively.Total protein concentrations were determined using the Bradford method (Bradford 1976).Three independent biological replicates were used in this assay, and 3 technical replicates were performed.

Quantitative Proteomics Analysis
Hemolymph was extracted from L. migratoria infected with 10 6 spores using the same procedure described above, diluted tenfold with 1× PBS buffer and stored in a -80°C freezer until transportation to Applied Protein Technology (Shanghai, China) for TMT quantitative proteomics analysis.The quantitative proteomics has 3 biological replicates.
Functional annotations of proteins were performed using the Blast2GO (https://www.blast2go.com/)program against the nonredundant protein database.The Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/pathway.html)(KEGG) was used to classify and group the identified proteins.Gene Ontology (GO) is an international standardization of the gene function classification system.It has 3 ontologies; molecular functions, cellular components, and biological processes.The KEGG pathway is a collection of manually drawn pathway maps representing our knowledge of molecular interactions and reaction networks.

Statistical Analysis
Data are expressed as mean ± standard deviations for n = 3. Comparisons of means among groups were performed by one-way ANOVA followed by Duncan's test.P ≤ 0.05 was set as the threshold for statistical significance.Analyses were performed using the Statistical Package for Social Sciences, version 26.0 (SPSS, Chicago, USA).Graphs were drawn using PRISM (version 5.00, GraphPad Company, USA).

Confirmation of P. locustae Infection in L. migratoria
Quantitative Cq values (Table 1) showed that all samples were positive at 1, 8, and 15 days postinfection.Copy number of spores in locusts increased with increasing number of infection days and concentration, confirming that L. migratoria had been successfully infected with P. locustae.

Energy Metabolism After L. migratoria Infection With P. locustae
Changes in G6P levels of L. migratoria infection with P. locustae Figure 1 shows that male and female L. migratoria exhibited consistent changes in G6P levels.The G6P levels in hemolymph gradually increased with increasing infection time.On day 15, the G6P levels were significantly higher than those of the control group (P < 0.05).

Changes in CA levels of L. migratoria infection with P. locustae
Hemolymph samples were collected from L. migratoria at different time points after P. locustae infection to measure citric acid activities.Figure 2 shows that CA concentrations in hemolymph samples from male and female L. migratoria first increased and then decreased.The levels of CA in hemolymph from L. migratoria infected with P. locustae were significantly higher than those of the control group, particularly on day 15 (P < 0.05).In contrast, CA levels in the hemolymph of L. migratoria infected with high concentrations of P. locustae exhibited a downward trend.

Changes in total protein levels of L. migratoria infection with P. locustae
Total protein levels in the hemolymph of male and female L. migratoria infected with P. locustae exhibited the same trend compared to the control group (Fig. 3).On days 1 and 8, differences in protein expressions between the experimental and control groups were insignificant.On day 15, total protein levels in the hemolymph of L. migratoria infected with medium and low concentrations of P. locustae were significantly higher than those of the control group, while total protein levels in the hemolymph of L. migratoria infected with high concentrations of P. locustae were significantly lower than those of the control group (P < 0.05).

Changes in ATP levels of L. migratoria infection with P. locustae
The overall change in ATP levels between male and female L. migratoria was not significantly different (Fig. 4).On day 15, ATP levels in the hemolymph of L. migratoria infected with high concentrations of P. locustae were significantly higher than those of the control group (P < 0.05).

Protein identification
Interactions between pathogens and hosts are complex as they involve numerous gene regulatory and signaling pathways.To understand the interactions between L. migratoria and P. locustae, we used TMT as a quantitative proteomic approach to investigate the proteins in L. migratoria that were altered following P. locustae infection.
A total of 1,506 unique peptides and 266 proteins were detected.Only the proteins with a fold change > 1.2 and a P-value < 0.05 were considered to be differently expressed.As a result, 128 proteins were significantly differentially expressed (66 upregulated and 62 downregulated) in L. migratoria (Supplementary S. 4).

GO enrichment analysis
Biological process-based enrichment analyses of differentially expressed proteins revealed that some GO terms were enriched.Most of the proteins in L. migratoria were enriched to cellular and metabolic processes (Fig. 5; Supplementary S. 1).Alterations in metabolic process-related proteins imply that P. locustae infection impacts host metabolism.
Molecular function-based enrichment analysis was performed to investigate the potential mechanistic roles that these proteins play in the cell (Fig. 5).Two GO terms, binding and catalytic activity, were highly enriched.The downregulated proteins were enriched in a single category; transcription regulator activity.Cellular component-based enrichment analysis was performed to investigate the localization of the differentially expressed genes (Fig. 5).Cell and cell part were the most enriched GO terms.Some of the upregulated and downregulated proteins were present in the same categories.

KEGG pathway analysis
The KEGG pathway analysis was performed to interpret systemic functions of differentially expressed proteins.In this study, 16 of the proteins were significantly enriched in 5 pathways (P < 0.05; Supplementary S. 2), including peroxisome (P = 0.015), glycolysis (P = 0.035), biosynthesis of nucleotide sugars (P = 0.048), HIF-1 signaling pathway (P = 0.048), and endocytosis (P = 0.048) pathway.Most of the proteins enriched in metabolic pathways were significantly upregulated, while proteins enriched in the protein export pathway were significantly downregulated.

Discussion
Microsporidia lack the core energy metabolism pathways, therefore, they depend on energy supply from the host for their replication (Pan et al. 2013, Boakye et al. 2017).In cells, sugar is converted into energy through glycolysis and other pathways.The pentose phosphate pathway (PPP) is an integral part of glucose metabolism.Insects convert glucose to glucose-6-phosphate (G6P) (Ge et al. 2020).The G6P is the primary metabolite in glycolysis and serves as a substrate for nucleotide synthesis after oxidization in the pentose phosphate pathway (Luo et al. 2021).In this study, G6P levels significantly increased with time after P. locustae infection of both sexes (P < 0.05).This was accompanied by increased expressions of 5 proteins involved in glucose metabolism, particularly hexokinase (P < 0.05).These results agree with those of .Host glycolysis and TCA cycle can be initiated by microsporidia-secreted hexokinase.Hexokinase induces host biosynthesis to benefit the parasite (Senderskiy et al. 2014, Reinke et al. 2017, Timofeev et al. 2017, Huang et al. 2018), and phosphorylates glucose, which is then absorbed by the parasite (Dolgikh et al. 2019, Luo et al. 2021).The RNA-interfering hexokinase of N. bombycis suppresses pathogen proliferation (Huang et al. 2018), suggesting an active glycolytic pathway after P. locustae infection, promoting G6P production (Huang et al. 2018).It is corroborative evidence for that the sugar (P < 0.05) is an important energy source for P. Locustae to proliferate in locusts.
Citrate is involved in the tricarboxylic acid cycle and can activate acetyl-coenzyme A (CoA) carboxylase, the rate-limiting enzyme for the synthesis of fatty acids (Lewis and Majerus 1969).In this study, the lipid metabolism pathway-related proteins,  including glycerol-3-phosphate dehydrogenase (NAD + ), were significantly upregulated.Physiologically, NAD + is mainly involved in glycerophospholipid metabolism.Transcriptome analysis indicated that infection with N. bombycis downregulated most of the genes in the long-chain fatty acid synthesis pathway.In contrast, the fatty acid chain degradation pathway significantly upregulated the expressions of thiolytic enzymes (Hu et al. 2021).This shows that microsporidian infection promotes lipid degradation.In this study, citrate levels first increased and then decreased.The experimental results do not match those of proteomic analysis.In contrast with our findings, Hu et al. (2021) infected silkworms with N. bombycis and found that host fatty acid levels continuously decreased after infection with microsporidia.These differences were attributed to the different hosts and pathogenic fungi, which may have affected the course of infection.On day 15 after infection with medium-low concentrations of P. locustae, citrate levels were significantly elevated (P < 0.05), most likely due to high energy requirements for P. locustae to proliferate in the host.When the spore load reaches a certain value, citrate is produced, indirectly promoting lipid formation to provide energy.A high number of P. locustae progeny were produced on day 15 of the late stage of P. locustae infection, leading to insufficient energy supply to host cells and causing lipids to be consumed for energy supply (Li et al. 2018).Therefore, compared with medium-low concentrations, citrate content showed a decrease.After infection with P. locustae, hemolymph lipase activities of L. migratoria were increased while total fat and hemolymph glycerolipid levels decreased (Chen et al. 2000), which would die after some time and reduce the density of the contemporary locust.This shows the possibility that microsporidia proliferation and microsporidia pathogenicity to locust infection are significantly influenced by lipid metabolism (Franchet et al. 2019).
When the energy provided by fats and sugars is insufficient to meet physiological needs, proteins can be used as energy substrates.
A study investigating the relationship between microsporidia infection and host energy metabolism measured the metabolome, including metabolites such as α-ketoglutarate, which is a precursor for the synthesis of amino acids, e.g., glutamic acid as well as proline and found it to be significantly upregulated in the TCA cycle (Luo et al. 2021).The G6P serves as a substrate for nucleotide synthesis after oxidization in the pentose phosphate pathway (Aiston et al. 2004, Ge et al. 2020).We found that G6P levels were significantly upregulated (P < 0.05) after P. locustae infection.Upregulation of these metabolites promotes the synthesis of nucleotides and amino acids (Williams et al. 2008, Dean et al. 2018).These are accord with the significant increase in total protein levels on day 15 (P < 0.05) in the hemolymph of L. migratoria infected with medium and low concentrations of P. locustae.However, the total protein contents in the hemolymph of L. migratoria infected with high concentrations of P. locustae were significantly suppressed (P < 0.05).This may be due to excess spore loads in the later stages of infection.When the host is low on energy, proteins must be consumed for energy production.Proteomics analyses revealed that protein-associated amino acid metabolism pathways were significantly upregulated.When many spores are present in the locust, and the ATP supply is insufficient, the host consumes proteins to provide energy for the organism to defend against microsporidian infection.
P. locustae infection can affect host metabolism.Microsporidia cannot grow or divide outside their host cells.Infection induces metabolic-dependent changes in the host (Dolgikh et al. 1997, Panek et al. 2017).However, how they interact with their hosts and use their resources has not been established.Genomic sequencing of microsporidia revealed that they lack specific metabolic pathways, such as oxidative phosphorylation, electron transport, and the tricarboxylic acid cycle (Luo et al. 2021).Most of the differentially expressed metabolism-related proteins in our study were upregulated (27 of 29 in metabolic pathways), while only 2 were downregulated, supporting the hypothesis that P. locustae infection accelerates L. migratoria metabolism to benefit the life cycle of the pathogen (Supplementary S. 2).
Proteomics analysis revealed that host energy metabolismassociated proteins were upregulated.P. locustae infection promoted glycolysis, fatty acid degradation, and the TCA cycle in the host, which eventually led to increased ATP production (Hoch et al. 2002).Therefore, we determined the ATP concentrations in infected and noninfected L. migratoria and found that ATP levels in the hemolymph of L. migratoria infected with medium and high concentrations of P. locustae were significantly increased on day 15 (P < 0.05), consistent with findings from proteomics analysis.However, ATP levels in the low P. locustae concentration infected locusts were not significantly increased, presumably because the spore load had not reached the threshold, thus, ATP levels in the host were maintained at the homeostatic equilibrium.This is an important strategy for microsporidia proliferation in host cells over a long period (Luo et al. 2021).
Sugar and lipid levels in the locust hemolymph began to exhibit significant changes on day 1 (P < 0.05) after infection with P. locustae, whereas total protein levels were significantly increased on day 15 (P < 0.05) and changed at different times, suggesting that P. locustae are selective in promoting host metabolism (Hu et al. 2021).Regarding the intensity of the content changes, the overall changes in lipid and soluble protein were not as dramatic as those in sugar.The levels of G6P were significantly increased after 15 days (P < 0.05) of infection with different P. locustae concentrations.It has been reported that microsporidia convert G6P to trehalose during the infection phase, which is the primary carbohydrate source for most of the microsporidian species (Undeen andSolter 1997, Hoch et al. 2002).This implies that during the infection phase, L. migratoria first consumes trehalose to provide energy.Lipids and proteins are used as alternative energy supplies when the spore loads are high in the late stages of infection and sugar supply is low.Therefore, ATP levels in the hemolymph of L. migratoria were significantly increased (P < 0.05) in the later stages of infection because microsporidian invasion increased the metabolic levels of the host.
Female and male L. migratoria were separately used in this study because microsporidia exhibit vertical transmission properties.The trends in levels of sugars, lipids, and soluble proteins in male and female locusts after infection were the same, indicating that P. locustae infection exerted the same effects on energy substrates in male and female L. migratoria.The synthesized energy is initially intended to be used for the growth and development of the host.In a previous study, infection with N. bombycis resulted in significant decreases in silkworm body weight and larvae sizes compared to the control group.This indicates that the hosts produce much energy to defend themselves against pathogenic microorganisms, while microsporidia partially absorb and use this energy for their development, causing the hosts to shrink in size.Thus, microsporidia infection significantly impacts host physiology and development (Ma et al. 2013).
In this study, changes in energy metabolism in male and female L. migratoria after infection with P. locustae were consistent, and there were no gender differences.In the first 8 d of infection, L. migratoria used sugar as the energy source.After 8 d when the spore loads were considerably heavy and energy supply was insufficient, lipids and proteins were consumed to provide energy.Energy conversion from sugar, fats, and proteins improved with increasing infection concentration and time.This may be one of the reasons why the high mortality did not occur until about 15 days after P. locustae infection.The TMT-based quantitative proteomics analysis revealed that most altered metabolism-related proteins, including hexokinase and glycerol-3-phosphate dehydrogenase (NAD + ), were upregulated.This indicates that P. locustae infection accelerated L. migratoria metabolism, which caused the host to produce large amounts of energy to support the proliferation of the pathogen, inhibiting the growth and development of the locusts and eventually causing their death.Our studies on the effect of pathogens on host metabolism may contribute to a better understanding of the chronic pathogenic mechanisms of P. locustae and provide a scientific basis for future improvements in P. locustae control.However, the specific reason for P. locustae long incubation period are unclear and should be further studied.

Fig. 1 .
Fig. 1.Analysis of changes in G6P levels after infection.A) Male; B) female.Error bars show means ± SD.Values marked with the different letters are significantly different, P < 0.05.

Fig. 2 .
Fig. 2. Analysis of changes in citrate levels after infection.A) Male, B) female.Error bars show means ± SD.Values marked with the different letters are significantly different, P < 0.05.

Fig. 3 .
Fig. 3. Analysis of changes in total protein levels after infection.A) Male, B) female.Error bars show means ± SD.Values marked with the different letters are significantly different, P < 0.05.

Fig. 4 .
Fig. 4. Analysis of changes in ATP levels after infection.A) Male, B) female.Error bars show means ± SD.Values marked with the different letters are significantly different, P < 0.05.

Fig. 5 .
Fig. 5. GO categories of upregulated and downregulated proteins.Enrichment of differentially expressed proteins in biological processes, cellular components, and molecular functions was performed by Blast2GO.The number of proteins mapped to GO terms is shown in the left panel.

Table 1 .
Results of TaqMan assay after infection of L. migratoria with P. locustae Standard deviations of means (SD) is shown; "+" represents a positive result; "−" represents a negative result.