Altered immunity in crowded locust reduced fungal (Metarhizium anisopliae) pathogenesis

Abstract

The stress of living conditions, similar to infections, alters animal immunity. High population density is empirically considered to induce prophylactic immunity to reduce the infection risk, which was challenged by a model of low connectivity between infectious and susceptible individuals in crowded animals. The migratory locust, which exhibits polyphenism through gregarious and solitary phases in response to population density and displays different resistance to fungal biopesticide (Metarhizium anisopliae), was used to observe the prophylactic immunity of crowded animals. We applied an RNA-sequencing assay to investigate differential expression in fat body samples of gregarious and solitary locusts before and after infection. Solitary locusts devoted at least twice the number of genes for combating M. anisopliae infection than gregarious locusts. The transcription of immune molecules such as pattern recognition proteins, protease inhibitors, and anti-oxidation proteins, was increased in prophylactic immunity of gregarious locusts. The differentially expressed transcripts reducing gregarious locust susceptibility to M. anisopliae were confirmed at the transcriptional and translational level. Further investigation revealed that locust GNBP3 was susceptible to proteolysis while GNBP1, induced by M. anisopliae infection, resisted proteolysis. Silencing of gnbp3 by RNAi significantly shortened the life span of gregarious locusts but not solitary locusts. By contrast, gnbp1 silencing did not affect the life span of both gregarious and solitary locusts after M. anisopliae infection. Thus, the GNBP3-dependent immune responses were involved in the phenotypic resistance of gregarious locusts to fungal infection, but were redundant in solitary locusts. Our results indicated that gregarious locusts prophylactically activated upstream modulators of immune cascades rather than downstream effectors, preferring to quarantine rather than eliminate pathogens to conserve energy meanwhile increasing the "distance" of infectious and target individuals. Our study has obvious implications for bio-pesticides management of crowded pests, and for understanding disease epidemics and adaptiveness of pathogens.

Figure 1. Locust phenotypic life span after lethal fungal infection (M. anisopliae).

(A) Adult gregarious and solitary locusts were topically infected with fungi (M. anisopliae) under the protonum to prevent septic injury. The arrow indicates the inoculation site. Scale bar: 1 cm. (B) Gregarious (G) and solitary (S) locusts were randomly selected (female G:20, S:24 and male G:40, S:27) for fungal infection assay. The life span of the locusts was calculated by Kaplan-Meier methods, and Cox proportional hazards model analysis was used for assessing variables affecting locusts survival. The curves of control treatments (blue and black lines) largely overlapped and displayed as a single black line. (Three replicates for each locust phase, χ² = 25.959, P<0.001).

Figure 2. Transcriptome analysis of the fatbody of gregarious and solitary locusts before and after infection.

The abundance and differential expression of transcripts were detected by software packages of Trinity and DEGseq respectively after assembling reference transcripts from raw reads from illumine Truseq experiments. (A) Hierarchical cluster analysis of fatbody transcripts that were significantly regulated (P<0.001, q-value<0.05) in at least two samples of four experimental conditions, and two phase locusts displayed distinct response to fungal infection. Heatmap was calculated by implement package of Trinity software. (B) Differential expressed transcripts (P<0.001, q-value<0.05) were classed by function (blast2go 1.0E-6). GC: fatbody sample of control gregarious locusts; SC: fatbody sample of control solitary locusts; GI: fatbody sample of gregarious locust infected by M. anisopliae; SI: fatbody sample of solitary locusts infected by M. anisopliae. IMM: immune defenses; MET: metabolism functions; OTHER: other biological process including unidentified. (C) Venn diagram representing unique and shared transcriptome regulation of prophylactic immunity and responsiveness of the two phases of locust to M. anisopliae (D) Prophylactically presented immune molecules of gregarious locust were showed as red in schematic immune pathways of resistance to fungal infection. PRPs: pattern recognition proteins; SP: serine protease; proPAP: pro-phenoloxidase activating proteinase; PPAE: proPO activating enzyme; PO: phenoloxidase; ROS: reactive oxygen species; Serpins: serine protease inhibitors; Grass: Gram-positive Specific Serine protease; SPE: Spätzle-processing enzyme; PSH: Persephone; PR1: fungal virulence protease PR1.

Figure 3. Determination of prophylactically expressed immune genes between two phase locust.

(A) Differential expressed transcripts between pre-infected solitary and gregarious locusts in main immune tissues (Fb: fat bodies; Hc: hemocytes; Mg: midgut) were confirmed by quantitative real time PCR. Data was presented as mean (±SE) and analyzed by Mann-Whitney U test (B) Immunoblot assay examination of circulating GNBPs between pre-infected two phase locusts. Upper panel indicated the immunoblot results of polyantibodies against locust GNBP1 and GNBP3 respectively; Lower panel was coomassie brilliant blue stained SDS-PAGE for protein amount calibration; positive sample was fungi infected locust hemolymph after 4 days. S: solitary locust; G: gregarious locust. (C) The circulating GNBP3 in hemolymph of pre-infected two phase locusts was measured by ELISA assay. G: gregarious locust; S: solitary locust. Briefly, polyantibodies against C-terminal of GNBP3 was coated on plate to capture circulating GNBP3, and polyantibodies against N-terminal of GNBP3 were conjugated with HRP to recognize the captured GNBP3 proteins. OD450 values indicated the relative abundance of GNBP3 in hemolymph, total hemolymph proteins were used for calibration (one-way ANOVA, F = 12.61, P = 0.002).

Figure 4. Locust GNBP1 responded to M. anisopliae invasion.

(A) mRNA expression of locust GNBPs in fat body in respond to injected pathogen associated molecular patterns (PAMPs) were analyzed by quantitative real time PCR. LM: laminarin; PG: peptidoglycan; LPS: lipopolysaccharide. Data was presented as mean (±SE) (B) Immunoblot analysis of GNBPs expression in response to injected conidia (M. anisopliae) fat body. CTR: locust saline; fungi: conidia of M. anisopliae.

Figure 5. Locust GNBP3 affected attacin expression and was susceptible to humoral protease.

(A) Immunofluorescence observed the locust GNBPs binding to fungi cell wall by confocal microscope. DIC: differential interference contrast; FITC: fluoresceinisothiocyanate staining; DAPI: 4′,6-diamidino-2-phenylindole staining; Ctr: Drosophila hemolymph injected with M. anisopliae was used as negative control for examining reactivity of anti-GNBP1 and anti-GNBP3 pAbs. White arrow indicated positive signals. (B) Locust attacin expression in fat body after RNAi knockdown of GNBP1 or GNBP3 was examined by quantitative real time PCR. Fold change was normalized to GFP knockdown locusts. (Mann-Whitney U test, n_GNBP1 = 9, P<0.001; n_GNBP3 = 9, P<0.001) (C) After 2-hours incubation of laminarin-stimulated hemolymph at room temperature, the proteolysis samples of GNBP1 and GNBP3 proteins in supernatant and clot were analyzed by immunoblot. Ctr: before incubation; In: 2-hours incubation; Pe: pellets after incubation. The black arrow indicates the degraded fragment of GNBP3 recognized by anti-N-terminal pAbs.

Figure 6. Knockdown GNBP3 affected the locust phenotypic resistance to M. anisopliae.

Adult male locusts of two phase were inoculated by M. anisopliae without septic injury under protonum after knockdown gnbp1 and gnbp3 expression for 48 hours by injection of dsRNA. (A) Log survival curves of gregarious locusts after GNBP1 and GNBP3 knockdown by dsRNAi injection (n_GNBP1 = 36, n_GNBP3 =44, n_GFP =32, n_control = 27; P _{GNBP1 vs. GFP} = 0.796, P _{GNBP3 vs GFP} = 0.006). (B) Log survival curves of solitary locusts after GNBP1 and GNBP3 knockdown (n_GNBP1 = 33, n_GNBP3 = 30, n_GFP =22, n_control = 27; P _{GNBP1 vs. GFP =}0.389, P _{GNBP3 vs. GFP =}0.941). Animals injected with dsGFP for infection were used as negative control; animals without RNAi knockdown and M. anisopliae infection were used as naïve group. Kaplan-Meier method in SPSS 13.0 was used to analysis locust survival data.

Figure 7. A model for prophylactic immunity in suppression of pathogens spread.

A) Pathogens spread through entire population from the infective individuals to susceptible without any obstacles; B) The clustered individuals applied prophylactic immunity to increase the “distance” between the susceptible and infective individuals, as well as the probability of stochastic extinction of the pathogens.