Variation of TNF modulates cellular immunity of gregarious and solitary locusts against fungal pathogen Metarhizium anisopliae

Abstract

Changes in population density lead to phenotypic differentiation of solitary and gregarious locusts, which display different resistance to fungal pathogens; however, how to regulate their cellular immune strategies remains unknown. Here, our stochastic simulation of pathogen proliferation suggested that humoral defense always enhanced resistance to fungal pathogens, while phagocytosis sometimes reduced defense against pathogens. Further experimental data proved that gregarious locusts had significantly decreased phagocytosis of hemocytes compared to solitary locusts. Additionally, transcriptional analysis showed that gregarious locusts promoted immune effector expression (gnbp1 and dfp) and reduced phagocytic gene expression (eater) and the cytokine tumor necrosis factor (TNF). Interestingly, higher expression of the cytokine TNF in solitary locusts simultaneously promoted eater expression and inhibited gnbp1 and dfp expression. Moreover, inhibition of TNF increased the survival of solitary locusts, and injection of TNF decreased the survival of gregarious locusts after fungal infection. Therefore, our results indicate that the alerted expression of TNF regulated the immune strategy of locusts to adapt to environmental changes.

Keywords: cellular defenses; density-dependent prophylaxis; ecological immunology; tumor necrosis factor.

Fig. 1.

Stochastic simulation of hemocytes and pathogen growth in altered immune defenses. (A) The schematic of humoral and cellular defense in host resist pathogen infection. α indicates the increase rate of hemocytes for the total events of the proliferation, migration, and death, and β indicates the increase rate of microbes including the total events of the proliferation and death by humoral attack. K1, K2, and K3 indicates the rate of phagocytosis efficient, phagocytes recovery, and phagocytes death (microbe escape) respectively. (B) To observe the competence of hemocytes and microbe growth, various status of cellular defense, microbe virulence and humoral defenses were examined in simulation. All status (8 combinations) of cellular defense, humoral defense, and microbe virulence (high or low) were observed in the simulation. (C) Kinetic curves of hemocytes and microbes by immune defense of high humoral and high cellular defenses. Left indicates status 3 and Right indicates status 4. (D) Kinetic curves of hemocytes and microbes by immune defense of high humoral and low cellular defenses. Left indicates status 6 and Right indicates status 8.

Fig. 2.

Crowded locusts reduced phagocytosis to conidia of M. anisopliae. To investigate the effects of cellular defense in host resist high-virulence infection, we used the altered immunity of locust to observe the differential cellular defense between locust two phases. (A) Locust displays a reversible phase change between solitary and gregarious phases in response to low and high population density, respectively. Solitary and gregarious locusts. (B) For phagocytosis ability, phagocytic hemocytes of engulfing FITC-labeled conidia were successfully identified by flow cytometry. DIC: differential interference contrast. (C) The higher level of phagocytic cells was displayed in solitary locust than gregarious locusts after counting the positive hemocytes in the 10,000 cell counts. G: hemocytes sample of gregarious locusts; S: hemocytes sample of solitary locusts. (D) Proportion of phagocytic cells in gregarious locust was less than that of solitary locusts (Right, n = 12, P < 0.001), and showed lower capability of engulfing conidia (Left, n = 12, P < 0.001). Asterisk indicates the significance (P < 0.01).

Fig. 3.

Transcriptional analyzed the differential expression of hemocytes between solitary and gregarious locusts. (A) Venn diagram showed the distinct and shared transcripts for hemocytes of pre- and post- conidia infection between solitary and gregarious locusts. (B) Hierarchical analysis of differentially expressed genes (1,990 genes, adjust P < 0.005) of hemocytes before and after conidia infection showed the different defense strategies in the two locust phases. Upper box and Lower box indicates the distinct prophylactically expressed genes for resisting infection in hemocytes of gregarious and solitary locusts respectively. GC: control sample of gregarious locusts; SC: control sample of solitary locusts; GI: conidia-infected sample of gregarious locust; SI: conidia-infected sample of solitary locusts; gnbp1: gram-negative binding protein 1; pirk: poor Imd response upon knockin; tnf: tumor necrosis factor; (C) qPCR results indicated that gregarious locusts prophylactically expressed the effectors transcripts of dfp and gnbp1 but not attacin. (D) qPCR results indicated that solitary locusts prophylactically expressed cytokine tnf and phagocytosis related receptor eater. Asterisk indicates the significance (n = 12, P < 0.01). (E) Immunoblot analysis of the differential activation of JNK phosphorylation in hemocytes of solitary and gregarious locusts. G: sample of gregarious locusts; S: sample of solitary locusts. Data presented as mean ± SEM.

Fig. 4.

Locust TNF simultaneously regulated the phagocytosis and effectors production. In hemocytes of locust, (A) mRNA expression level of tnf and eater can be significantly reduced by RNAi knockdown. (B) After reduced the locust TNF (Lmtnf) expression in solitary locusts, phagocytosis receptor eater was significantly decreased, while effector gnbp1 expression was increased by qPCR analysis. (C) Flowcytometry results showed that knockdown Lmtnf expression by dsRNAi decreased the phagocytic hemocytes and did not affect the capability of phagocytosis in solitary locusts. (D) Flowcytometry results showed that knockdown eater expression by dsRNAi decreased the phagocytic hemocytes and did not affect the capability of phagocytosis in solitary locusts. (E) After injection of recombinant LmTNF, phagocytosis receptor eater was significantly increased while effector gnbp1 expression was decreased by qPCR analysis in gregarious locusts. (F) Flowcytometry results showed that injection of LmTNF increased both the proportion of phagocytic hemocytes and the capability of phagocytosis in gregarious locusts. LmTu: locust Tubulin; LmTNF; locust TNF; inf: infection. Asterisk indicates the significance (P < 0.01). Data presented as mean ± SEM.

Fig. 5.

TNF impaired locust defense against the infection of M. anisopliae. After changing the TNF expression level in locusts, the survival of locusts infected by fungi (M. anisophilae) was analyzed by Kaplan-Meier methods. (A) Increasing TNF level by injection of recombinant TNF proteins reduced the life span of gregarious locusts. (n_G = 49, P_{LmTublin vs. LmTNF} = 0.001). G.LmTubulin: gregarious locusts injected with recombinant tubulin protein; G.LmTNF: gregarious locusts injected with recombinant TNF protein; G.dsTNF: gregarious locusts reduced TNF expression by dsRNAi; G.ctr: gregarious locusts without fungal infections. (B) Knock down of TNF level by dsRNAi extended the life span of solitary locusts. (n_S = 32, P_{dsGFP vs. dsTNF} = 0.013). S.dsTNF: solitary locusts reduced TNF expression by dsRNAi; S.LmTNF: solitary locusts injected with recombinant TNF protein; S. dsGFP: solitary locusts injected with dsGFP; S.ctr: individuals of solitary locusts without fungal infections. (C) Schematic mechanisms showed that TNF affected host survival after pathogens infection. Low level of TNF ensured the effectors production and quarantined the pathogens to limit harmful effects. High level of TNF in solitary locusts enhanced the phagocytosis for pathogen clearance.