Infection with a wide variety of parasites, including baculovirus, can result in behavior changes in the host. For survival and replication, the parasites have to conquer host defenses barriers of immunity, even manipulate the host’s physiology1–3. Some reports have provided insight into how baculovirus producing influences on host, depending on some core genes or even just one gene9, 13–16. Within the last decade, the primary host behavior manipulation gene of NPV had been brought to light5, 9, 13–18, 26–29. The details on host reacting and its mechanism to virus-induced activity rarely had been addressed. In this study, through the integrative analysis of metabolome and transcriptome between the key time points, which were chosen by critical moment of replication characteration and infected host larval behavior changes, and we sum up the alteration of MI, PI and relevant genes involved in larval extended instar duration, and the changes of caffeine, glutamate and GABA involved in LdMNPV inducing behavior change.
Parasites are capable of inducing host behavior alterations in many aspects to increase transmission rates1–3, 5. The NPV infection triggered abnormal physiological status, like increasing food intake, ELA, extended instar duration and climbing to elevated location before death foraging5, which leading by alteration of host cellular metabolism and manipulation of signaling transduction. The infected L. dispar larvae had prolonged their 3th instar duration, which was 4 to 5 days in healthy larvae, to 7 to 8 days10. The survival curve in LD90 of LdMNPV-infection (Supplemental Figure S1) was shown that the 3 and 6 dpi were two inflection points. Baculoviuses specially have a biphasic replication cycle, which including two individual virion phenotypes, the ODV and the BV5, 7. The ODV and BV were genetically identical but morphologically different, which also were related-time produnction. Combined with the biphasic infections and the early infection mainly happening in midgut which only regional impact, the time point Ld3 and Ld6 were chosen to indicate the proceeding of systemic infection at early and imminent stages of massive replication.
In the four groups (NB3, CK3, Ld3 and Ld6) of L. dispar larvae, twelve transcriptomic samples were sequenced. On the whole, the CK3 and Ld3 were more similary than others groups (Fig. 1A). Based on DEGs annotation, it is showed that the pattern of NB3 versus CK3 and Ld3 versus Ld6 were approximate, and the alteration in cellular mainly concentrates on benefiting virus DNA replication, the energy supply and detoxication relevant metabolism were visibly affected, and immune function relevant pathways, like the JAK/STAT signaling pathway, NF/kB signaling pathway and Toll-like receptors pathway, were not significantly affected (Fig. 1B). After being infenction, the DEGs analysis showed regulatory strategy change in different infection stages, involving affect material supply of virion production and anti-apoptosis to prolong infected cell survival. Furthermore, by KEGG annotations, the DEGs enrichment showed that between CK3 and Ld3, the violently upregulated pathways were metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450 and galactose metabolism; and the drastically downregulated pathways were ribosome, carbon metabolism and phagosome, which reveals alteration provide the condition of viral replication, alteration of energy supply and avoid cell damage, toxin and reactive oxygen species (ROS) stress causing by virus repilication. Between Ld3 and Ld6, the violently upregulated pathways were carbon metabolism, phenylalanine metabolism and biosynthesis of amino acids; and the drastically downregulated pathways were lysosome, mTOR signaling pathway and longevity regulating pathway-multiple species. The upregulated of carbon metabolism, phenylalanine metabolism and biosynthesis of amino acids confirmed infection causing globally alteration of material utilization. The downregulated of lysosome, mTOR signaling pathway and longevity regulating pathway were benefit in keeping host cell survival. The genes, which were enrichment in lysosome pathway involving in metabolism disorder, and the upregulated gene could promote glycerolipid collection30. The drastic changes of longevity regulating pathway may relate to prolonging instar duration of infected larvae. It is also reported that dietary restriction and genetic down-regulation of nutrient-sensing pathways, like Insulin-like signaling pathway and mTOR signaling pathway can substantially increase life span31.
Generally, the GESA result (Fig. 2A-E) is shown that NB3 versus Ld3 and NB3 versus Ld6 the gene sets which were enriched in have more volume, reveals the larger variations in these two comparisons. From NB3 to CK3 (Fig. 2A), the enrichment was involved in material metabolism and surroundings signal receptor, which reflecting the larval physiological change of growing process. Between CK3 and Ld3 (Fig. 2E), only the Citrate cycle (TCA cycle) and tyrosine metabolism showed the upregulation, and lots of gene sets downregulated mainly covered maintenance and development signal and genetic information processing, which was accords with the viral manipulation in early replication. The transcriptomic analysis shown that pathways of metabolism, signal transductions and detoxication relevant metabolism were visibly affected in infection L. dispar larvae. Further more, with the infection proceeding, it is revealed that in Ld3 versus Ld6 (Fig. 2D) the alteration mainly consists of material metabolism and protein export, especially in fatty acid metabolism, and the apoptosis was continuous downregulation.
Twenty-four metabolomics samples obtained 1,930 metabolins in POS mode and 1,831 metabolins in NEG mode. In contrast between groups, a large variety of metabolites show significant reduction in the virion production stages (Fig. 3A-B). Results of the amount of different metabolites between groups, the variability of different metabolins and the Wayne diagrams (Fig. 3C-F) reflected that the metabolite changes in NB3 versus CK3 and Ld3 versus Ld6 were easement, but in CK3 versus Ld3 was fierce. The metabolin enrichment showed the metabolic pathway, ABC-transporters, 2-Oxocarboxylic acid metabolism, biosynthesis of amino acid and protein digestion and absorption merged to produce a violent change in all comparisons. Meanwhile, the alteration of central carbon metabolism in cancer, which was accredited in lots of human source virus infections, only emerged in NB3 versus Ld3, NB3 versus Ld6 and CK3 versus Ld3. In rapidly replication viruses, the hyper-activation of sustained metabolic pathways was sufficed to increasing viral titers32, 33. The results of different metabolites KEGG enrichment revealed that the different metabolites were concentrated in some key pathways. From NB3 to CK3, as normal physiology status, the different metabolites have been enriched evenly distribute in amino acid, lipids, and other small molecular metabolistes. After be infected, from CK3 to Ld3, the enriched pathways covering amino sugar and nucleotide sugar metabolism, biosynthesis of unsaturated fatty acid biotin metabolism, D-glutamine and D-glutamate metabolism, nitrogen metabolism and galactose metabolism were enriched dramatic change. The nutrients uptake and utilization alteration tend to provide enough ATP to meet the energy costs of virus DNA replication. The increasing of lipid material was induced by the needed of additional membrane material for the viral particle envelopment. Though different viruses appear to activate different metabolic altering pattern, the reprogramming of host cell is determinant to viral growth. Since Munger et al reporting the first eukaryotic virus tampering cellular metabolomics, different studies shown variously manipulate multiple major metabolic pathways to reprogram host cellular nutrients uptake and utilization to meet the increasing material and energy costs to guarantee the optimal microenvironment34–38. The research also shown that the viruses inducing aerobic glycolysis as known as the “Warburg effect” 32,33. The alterations of nutrients uptake and utilization could provide ATP in a rapid fashion to meet the high energy costs and avoid cell ROS stress of virus replication, and the increasing of lipids by altering the fatty acid synthesis, is needed to afford the additional membrane material for the viral particle envelopment.
In comprehensive consideration of existing research and the data of transcription and metabolism, more detail about LdMNPV and host interaction was uncovered. The integrative analysis of metabolome and transcriptome totally enriched 52 correlational pathways in the POS mode and 56 correlational pathways in the NEG mode. The results showed that the specific differences in NB3 versus CK3 (physiological status) and Ld3 versus Ld6 were covering more KEGG pathways with less enrichment amount of different express genes and metabolites, between which have more similarity than others compares (Fig. 4A-C). The Global and overview maps, Carbohydrate metabolism and Amino acid metabolism present significant difference in both of POS and NEG mode (Fig. 4C), but the amount of difference expression genes and metabolites in Ld3 versus Ld6 were smaller than others, especially the CK3 versus Ld3 (Fig. 4D-E). Comparing with the normal physiological status, the alteration of infected status refers to signal transduction, nucleotide metabolism, fatty acid metabolism, amino acid metabolism and cofactors metabolism.
The figure.4F and table.2 were showed that Carbohydrate metabolism, Amino acid metabolism and Lipid metabolism were enriched to most pathways, Carbohydrate metabolism have 15 pathways, Amino acid metabolism have 14 pathways, and Lipid metabolism have 11 pathways. In metabolism relevant pathway the dynamic change of the Alanine, aspartate and glutamate metabolism pathway, Lysine degradation pathway and Tyrosine metabolism pathway in Amino acid metabolism, Glycerophospholipid metabolism pathway in Lipid metabolism, ABC transporters pathway in Translation and neuroactive ligand-receptor interaction pathway in Membrane transport were striking. It is showed that the specific enriched KEGG pathways were arachidonic acid metabolism, propanoate metabolism, mTOR signaling pathway, parkinson disease and alcoholism in NB3 versus CK3; oxidative phosphorylation in NB3 versus Ld3; D-Glutamine and D-glutamate metabolism, nicotinate and nicotinamide metabolism, one carbon pool by folate and FoxO signaling pathway in Ld3 versus Ld6; and glycerolipid metabolismin CK3 versus Ld3. For another, the enrichments excepting metabolism relevant only covered 10 pathways, including aminoacyl-tRNA biosynthesis pathway, neuroactive ligand-receptor interaction pathway, ABC transporters pathway, phosphatidylinositol signaling system, FoxO signaling pathway, mTOR signaling pathway, protein digestion and absorption pathway, bile secretion pathway, parkinson disease pathway and alcoholism pathway. Except the aminoacyl-tRNA biosynthesis pathway, neuroactive ligand-receptor interaction pathway, ABC transporters pathway and phosphatidylinositol signaling system, the others just change a few in limited compares. The aminoacyl-tRNA biosynthesis pathway was violent alteration in CK3 versus Ld3, reflecting that the reprogram of host cell transcription preferences which leading by LdMNPV. There have not enrichment any difference in Ld3 versus Ld6, which means alteration of aminoacyl-tRNAs were keeping stable with infection processing. Moreover, it is reported that after infected by BmNPV in Bombyx mori larvae the trehalose, malate, fumarate, citrate, succinate, α-ketoglutarate, phenylalanine and tyrosine were accumulated in the resistant strain YeA, but in susceptible strain YeB the gluconate, glutamate and aspartate showed upregulation25. The integrative analysis showed in L. dispar larvae the phenylalanine, tyrosine, gluconate and glutamate were continued accumulation in CK3 versus Ld3 and Ld3 versus Ld6, which revealed different patterns of host-virus interaction. The gluconate and glutamate were gived further attention, in Ld3 versus Ld6, the accumulation glutamate indicate targeted utilization by glul gene upregulated leading glutamine showed increase and the glutamate decarboxylase gene was downregulated causing GABA, a chief inhibitory neurotransmitter, remains stable. In the insect central nervous system (CNS), GABA and glutamate working as antagonists and agonists in neuronal excitability control39–42. It is showed that GABA and glutamate are involved in the mediation of aggressive behavior in invertebrates43. In honeybee, the connection between scouting behaviors and catecholamine, glutamate and GABA reflect effects of neurotransmitter to behaviors, and glutamate treatments increased the probability of foragers scouting behaviors42. Moreover, in CK3 versus Ld3 the caffeine was increase. It is reasonable to consider that content patterns of caffeine, glutamate and GABA involved in LdMNPV inducing behavior change.
Metabolisms like spermidine, pyruvate, GluNAc and NAD+ were relevanted to life prolonging44–47. Among them, only GluNAc showed increasing, which was enriched in Ld3 versus Ld6. In C.elegans and Drosophila melanogaster cyclohexanehexol was reported to extend lifespan23, 24, 49. The inositol phosphate metabolism pathway and phosphatidylinositol signaling system pathway, which linking by cyclohexanehexol, were both enriched after LdMNPV infection. In CK3 versus Ld3, the MI was increase and PI was decrease, and relevent genes, the inn5a and pi3k were upregulated, and pi3r gene was downregulated. And in Ld3 versus Ld6, the PI was increase and MI was decrease, and the pten/mmac1, minpp1 and ip3r gene were upregulated, the pi3k gene was downregulated. As important second messenger, the phosphatidylinositol-3,4,5-triphosphate (PIP3) mediated lots of intracellular signaling molecule which was activating types of growth factors, cellular stimulus or toxic insults and regulates fundamental functions affected transcription, cell cycle progression, proliferation and survival50, 51, that keeping stable in all groups. The accumulation of PIP3 in cell will trigger stress resistant and apoptosis. Moreover, intracellular phospholipid signal involved in managing endocytic and exocytic processes, regulating iron channaels, pumps and transporters, coordinating vesicular trafficking and modulating lipid distribution and metabolism52. Numbers of pathogens, especially virus, has been recognized reprogram cellular processes by hijack host cell phospholipid signal53. Refer to alteraction of the inositol phosphate metabolism pathway and phosphatidylinositol signaling system pathway in infected larvae, it is hypothesis that MI, PI and relevant genes involving in larval extended instar duration and apoptosis avoiding.
Considering all the results, LdMNPV biphasic infection mode causing host manipulation changes continuously in different time points of infection. Comparing with the normal physiological status, alterations of nutrients uptake and utilization could provide ATP in a rapid fashion to meet the high energy costs and avoid cell stress resistant and apoptosis of virus replication, and the increasing of lipids by altering the fatty acid synthesis, is needed to afford the additional membrane material for the viral particle envelopment, and immune function relevant pathways were not significantly affected. Moreover, maintaining infected cell stability is important to virion production and assembly at terminal of infection and the extended instar duration of host was benefitted to virus production.
In summary, we explored the complicated interactions between LdMNPV and 3th-instar L. dispar larvae at the metabolome and transcriptome levels, conducted the preliminary analysis, uncovered that the content patterns of caffeine, glutamate and GABA involved in LdMNPV inducing behavior change, and the MI, PI and relevant genes involving in larval extended instar duration and apoptosis avoiding. It is anticipated that further research in relevent pathways will reveal in the complex reactions in conflict between L. dispar and LdMNPV.