A CTL - Lys immune function maintains insect metamorphosis by preventing gut bacterial dysbiosis and limiting opportunistic infections

Abstract

Background: Gut bacteria are beneficial to the host, many of which must be passed on to host offspring. During metamorphosis, the midgut of holometabolous insects undergoes histolysis and remodeling, and thus risks losing gut bacteria. Strategies employed by holometabolous insects to minimize this risk are obscure. How gut bacteria affect host insects after entering the hemocoel and causing opportunistic infections remains largely elusive.

Results: We used holometabolous Helicoverpa armigera as a model and found low Lactobacillus load, high level of a C-type lectin (CTL) gene CD209 antigen-like protein 2 (CD209) and its downstream lysozyme 1 (Lys1) in the midgut of the wandering stage. CD209 or Lys1 depletion increased the load of midgut Lactobacillus, which further translocate to the hemocoel. In particular, CD209 or Lys1 depletion, injection of Lactobacillus plantarum, or translocation of midgut L. plantarum into the hemocoel suppressed 20-hydroxyecdysone (20E) signaling and delayed pupariation. Injection of L. plantarum decreased triacylglycerol and cholesterol storage, which may result in insufficient energy and 20E available for pupariation. Further, Lysine-type peptidoglycan, the major component of gram-positive bacterial cell wall, contributed to delayed pupariation and decreased levels of triacylglycerols, cholesterols, and 20E, in both H. armigera and Drosophila melanogaster.

Conclusions: A mechanism by which (Lactobacillus-induced) opportunistic infections delay insect metamorphosis was found, namely by disturbing the homeostasis of lipid metabolism and reducing 20E production. Moreover, the immune function of CTL - Lys was characterized for insect metamorphosis by maintaining gut homeostasis and limiting the opportunistic infections.

Keywords: C-type lectin; Ecdysone; Lipid metabolism; Lysozyme; Pupariation.

Fig. 1

The abundance of Lactobacillus is significantly decreased in the midgut and significantly increased in hemolymph when larvae transition from the feeding to wandering stage. A Venn diagram of bacterial genus abundance in the larval midgut. Midguts were collected from sixth-instar larvae at 24 h PE (feeding) and 72 h PE (wandering). B Principal coordinates analysis (PCoA) of bacterial communities from the midgut based on genus level. C Relative abundance (%) of bacterial genera between the feeding and wandering stages. D, E Quantification of midgut Lactobacillus (D) and hemolymph Lactobacillus (E) in the feeding and wandering stages. Quantification was by qPCR analysis using primers of Lactobacillus-specific 16S rRNA gene. Statistical differences were analyzed using the Mann–Whitney U test or Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Fig. 2

CD209 is dominantly expressed in the midgut of the wandering stage. A, B Heatmap showing the expression profiles of PRRs (A), AMPs, and Lys (B) in the midgut between the feeding (MgF) and wandering (MgW) stages. CD209 and Lys1 are highlighted in red. C Predicted domain architecture of CD209 protein. Characterization of a predicted signal peptide (SP, 1–19 aa) and a carbohydrate recognition domain (CRD, 23–162 aa) from http://smart.embl.de/ website. D Expression and purification of recombinant CD209 (rCD209), as well as rCD209 recognized by the anti-CD209 antiserum. Lanes 1 and 2, crude protein extracts from bacteria (carrying the CD209-pET32a plasmid) before (lane 1) and after (lane 2) IPTG induction. Lane 3, purified rCD209 protein. Lane 4, rCD209 recognized by the anti-CD209 antiserum. E Spatial and temporal expression profiles of CD209 analyzed by RT-qPCR. Total RNAs were extracted from sixth-instar larvae at 24 h PE (feeding) and 72 h PE (wandering). F Tissue-specific expression of CD209 detected using western blotting. Antiserum against CD209 or β-actin was used for detection. Protein samples were collected from the wandering stage. Pl, plasma. Hc, hemocytes. Fb, fat bodies. Mg, midgut. In, integument

Fig. 3

CD209 depletion increases Lactobacillus load and suppresses the expression of Lys1 and CecD. A, B Quantification of midgut Lactobacillus (A) and hemolymph Lactobacillus (B) in larvae treated with dsCD209 or dsGFP. The midgut and hemolymph were collected from sixth-instar larvae at 72 h PE. C, D Depletion of CD209 in the midgut (Mg) validated by RT-qPCR (C) and western blotting (D). The antiserum against CD209 or β-actin was used for detection. E Heatmap showing that DEGs encoding CD209, Lys1, and CecD were downregulated in CD209-depleted midgut. F, G RT-qPCR analysis confirming inhibition of Lys1 (F) and CecD (G) expression in the CD209-depleted midgut. Statistical differences were analyzed using Student’s t test (*p < 0.05 and **p < 0.01)

Fig. 4

Lys1 contributes to the suppression of Lactobacillus. A, B Quantification of midgut Lactobacillus (A) and hemolymph Lactobacillus (B) in larvae treated with dsLys1 or dsGFP. Midguts and hemolymph were collected from sixth-instar larvae at 72 h PE. C Depletion of Lys1 in the midgut at 48 and 72 h post-dsLys1 injection (hpi) validated by RT-qPCR. D Expression and purification of Lys1 mature peptide (Lys1). Lanes 1 and 2, crude protein extracts from bacteria (carrying the Lys1-pET32a plasmid) before (lane 1) and after (lane 2) IPTG induction. Lane 3, purified rLys1 protein. Lane 4, purified rLys1 cleaved by enterokinase. The two bands represent rTrx and Lys1. Lane 5, purified Lys1. E Representative MRS agar plates showing L. plantarum colonies after each treatment. L. plantarum were incubated with Lys1 or BSA at 25°C with agitation for 1 h, followed by spreading the mixture on MRS agar plates. F The number of L. plantarum colonies for each treatment was determined. Bacterial growth was exhibited as the ratio of viable colonies against BSA controls. Statistical differences were analyzed using Student’s t test (*p < 0.05)

Fig. 5

Delayed pupariation in CD209- or Lys1-depleted larvae, as well as in L. plantarum-injected or L. plantarum-fed larvae. A, B Prolonged duration of sixth-instar in CD209-depleted (A) or Lys1-depleted (B) larvae. The durations of fourth-, fifth-, and sixth-instar were measured individually and the average time was calculated. The dsGFP was injected as control. C Prolonged duration of sixth-instar in L. plantarum-injected larvae. L. plantarum-suspended PBS or PBS (control) was injected into the hemocoel of sixth-instar larvae at 48 h PE. D Prolonged duration of sixth-instar in L. plantarum-fed larvae. Axenic larvae were fed on an artificial diet supplemented with L. plantarum-suspended PBS or PBS (control) at the beginning of the sixth-instar stage. E, F Quantification of Lactobacillus in the midgut (E) and hemolymph (F) from L. plantarum-fed sixth-instar larvae at 24 h PE (6th-24 h) and 72 h PE (6th-72 h). Statistical differences were analyzed using Student’s t test (*p < 0.05 and ***p < 0.001). ns, no significant difference

Fig. 6

Injection of L. plantarum induces a shift from lipid storage to phospholipid synthesis and decreases total cholesterols. A Heatmap showing that most of the metabolites involved in phospholipid synthesis were increased. Hemolymph samples were collected from sixth-instar larvae at 24 h post-L. plantarum (LP) or -PBS injection. Variable importance in projection (VIP) scores for a metabolite represent the contribution of that metabolite to the difference between the two groups. The larger the score, the greater the difference between the two groups. B LDs were detected in the fat bodies of sixth-instar larvae at 24 h post-L. plantarum or -PBS injection. Nile red staining was used for detection. Scale bar = 20 μm. C LD diameter and fluorescence intensity were significantly lower in fat bodies of L. plantarum-injected larvae than that of PBS-injected controls. D The levels of TAGs and total cholesterols were significantly lower in L. plantarum-injected samples than that of controls. E Expression profiles of genes encoding enzymes involved in the synthesis of cholesterol esters, lipids, or phospholipids. Fat bodies were collected from L. plantarum-injected or PBS-injected larvae. ACAT, Acyl-CoA cholesterol acyltransferase (XM_021333888); PAP, PA phosphatase (XM_021327456); DGAT, Acyl-CoA diacylglycerol acyltransferase (XM_021328346); CDS, CDP-DG synthase (XM_049847014); PGSA, PG-P synthase (XM_021335307); PSS, PS synthase (XM_049835809). F Pathways for the synthesis of cholesterol esters, lipids, and phospholipids. LD, lipid droplet; PA, phosphatidic acid; DAG, diacylglycerol; TAG, triacylglycerol; CDP-DG, cytidine diphosphate-diacylglycerol; PG-P, phosphatidyl-glycerophosphate; PS, phosphatidylserine. Statistical differences were analyzed using Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Fig. 7

Challenge by Lysine-type PGN results in delayed pupariation and decreased levels of TAGs, cholesterols, and 20E. A, C Challenge by Lysine-type PGN delays pupariation in H. armigera (A) and D. melanogaster (C). B, D Challenge by Lysine-type PGN reduced the levels of TAGs, cholesterols, and 20E in H. armigera (B) and D. melanogaster (D). Statistical differences were analyzed using Student’s t test (*p < 0.05 and **p < 0.01)

Fig. 8

Model showing CTL − Lys immune function regulates gut homeostasis and controls insect metamorphosis. CD209 and its downstream Lys1 maintain high expression levels in the midgut during the wandering stage due to the high 20E titers. Lys1 suppresses the proliferation of Lactobacillus in the midgut, resulting in a low abundance of Lactobacillus (and the associated PGN) in the hemocoel. In this condition, fat body cells are mainly involved in the storage of lipids and cholesterol esters in LDs, thus providing sufficient energy and 20E titers for timely pupariation. However, low expression levels of CD209 and Lys1 increase the Lactobacillus load in the midgut. The increased loads of Lactobacillus (and PGN) subsequently translocate into the hemocoel and trigger a shift from lipid storage to phospholipid synthesis. Moreover, cholesterol esters stored in LDs decrease. This leads to insufficient energy and 20E titers, thereby delaying pupariation. The low 20E titers, in turn, fail to induce high levels of CD209 and Lys1 expression in the midgut