Insect rearing, antibiotic treatment, and PGN challenge

H. armigera larvae were maintained in an insectarium under the photoperiod of 14 h light/10 h dark at 27 ± 1°C. The larvae were fed on an artificial diet comprising soybean powder, wheat germ, compound vitamins, and mineral salt. Axenic H. armigera larvae were obtained by constantly feeding on a diet supplemented with gentamicin (15 μg/mL), penicillin (10 units/mL), and streptomycin (10 μg/mL) from the third instar. The midguts were dissected from sixth-instar larvae at 0 h PE, reared on a diet with or without antibiotics. The efficacy of cultivable bacterial elimination was confirmed by plating gut homogenates onto LB agar plates. PCR analysis against gut homogenates was conducted using universal 16S rRNA gene primers 16S-F and 16S-R (Additional file 1: Table S8). For PGN challenge, sixth-instar larvae at 48 h PE were injected with 5 μL of Lysine-type PGN (500 ng/μL, KALANG, Shanghai, China).

D. melanogaster were raised on a standard cornmeal/yeast diet under the photoperiod of 8 h light/16 h dark at 25 ± 1°C. Drosophila late-third instar larvae (~ 108 h after egg lay) were collected and rinsed in sterile PBS. The larvae were punctured laterally with a needle dipped in Lysine-type PGN or PBS. The punctures were performed in mineral oil, where the punctured larvae were kept for another 10 min before transfer to fresh food, as described [54].

Sequencing and analysis of bacterial 16S rRNA gene from the midgut

Midguts were collected from sixth-instar larvae at 24 and 72 h PE. Before the midguts were dissected, larvae were surface-sterilized with 75% ethanol. Five independent biological replicates were included. A total of 20 midguts from each stage were pooled as a replicate and used for metagenomic DNA extraction. The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified with primers 338F and 806R (Additional file 1: Table S8). The PCR amplicon pools were prepared for sequencing on an Illumina MiSeq platform (San Diego, USA) by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw sequencing reads were demultiplexed, quality-filtered by Trimmomatic, and merged using FLASH. OTUs with 97% similarity cut-off were clustered using UPARSE (http://drive5.com/uparse/). The taxonomy of each OTU representative sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the SILVA database (http://www.arb-silva.de) using a confidence threshold of 0.7.

Isolation, characterization, inoculation, and colonization of L. plantarum

To isolate bacteria from the midgut, sixth-instar larvae at 24 h PE were surface-sterilized in 75% ethanol and rinsed in sterile PBS. The midguts were dissected, homogenized, and diluted (1:1000) in sterile PBS. The diluted homogenates were spread on MRS (Solarbio, Beijing, China) agar plates. The plates were incubated at 37°C for 24 − 48 h. Morphologically distinct colonies were selected for genomic DNA extraction using a bacterial DNA kit (Omega, GA, USA). DNA fragments were amplified using universal 16S rRNA gene primers 27F and 1492R (Additional file 1: Table S8), followed by sequencing. The closest L. plantarum matching the 16S rRNA gene sequence (99% identity) was obtained after BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against the 16S rRNA database.

L. plantarum in mid-logarithmic phase was harvested, washed twice, and resuspended in PBS at 2 × 108 cells/mL. Each sixth-instar larva at 48 h PE was injected with 5 μL of bacterial suspension. L. plantarum was introduced into sixth-instar larvae at 0 h PE by feeding on artificial diet, supplemented with the bacterial suspension. An equal amount of sterile PBS was used as a control. Midguts, hemolymph, and fat bodies from the bacteria-injected or -fed larvae were collected for further assays.

RNAi

To produce dsCD209 and dsLys1, the fragments of these two genes were amplified using the primers listed in Table S8 (Additional file 1). The dsCD209 and dsLys1 were synthesized with their respective PCR products as templates using a MEGAscript kit (Ambion, Austin, TX, USA). The dsGFP was synthesized as a control. The dsRNAs were eluted from the purification column with nuclease-free water and concentrated to 2.0 μg/μL. Each fourth-instar larva at 6 − 12 h PE was injected with 5 μL of dsCD209, dsLys1, or dsGFP. The midguts, hemolymph, and fat bodies were collected from the larvae after dsRNA injection for analysis.

RNA-seq analysis

Total RNA was extracted from the midguts of sixth-instar larvae at 24 h PE (feeding; MgF) and 72 h PE (wandering; MgW), and those at 72 h PE pretreated with dsCD209 or dsGFP using TRIzol (Invitrogen, Carlsbad, CA, USA). The cDNA libraries (MgF-1, -2, -3, -4; MgW-1, -2, -3, -4; dsCD209-1, -2, -3; dsGFP-1, -2, and -3) were constructed as described [59]. Briefly, mRNAs were isolated using oligo(dT) beads and then fragmented. Double-stranded cDNAs were synthesized using EasyScript cDNA synthesis SuperMix (TransGen Bio-tech, Beijing, China), followed by sequencing on the Illumina Novaseq 6000 platform (San Diego, USA) at Majorbio Bio-Pharm Technology Co., Ltd. Clean reads were obtained after filtration of raw reads and aligned to the H. armigera genome (https://www.ncbi.nlm.nih.gov/genome/13316?genome_assembly_id=319039) using TopHat software (http://tophat.cbcb.umd.edu/). Mapped reads were assembled and all unigenes were run in BLASTx against the non-redundant database. DEGs in the libraries (MgW vs. MgF; dsCD209 vs. dsGFP) were determined using a fold cut-off change value ≥ 2 and adjusted p-value < 0.05.

Measurement and injection of 20E

We determined 20E titers using the double antibody sandwich method with an insect 20E ELISA kit (Kmaels Biotechnology, Shanghai, China), as described [60]. Hemolymph was collected from sixth-instar larvae at 72 and 96 h PE treated with dsRNA or L. plantarum. Hemolymph from five larvae was pooled as a replicate, and three replicates in each treatment were obtained for 20E measurement. For Drosophila, larvae were collected at 3 h post-PGN challenge, homogenized, and centrifuged. The supernatants were used for assays. The samples were added to the microwells precoated with 20E antibodies. Subsequently, horseradish peroxidase-labeled detection antibodies were added to the wells. After thorough washing was complete, 3,3′,5,5′-tetramethylbenzidine was used for color development. Absorbance was measured at 450 nm, and 20E titers were calculated from the standard curve.

To determine gene expression, each sixth-instar larva at 48 h PE was injected with 20E (ABMole, Houston, TX, USA) at 200 ng/5 μL, which was close to the amount of endogenous 20E during the wandering stage [26]. An equal amount of dimethyl sulfoxide (DMSO), used for dissolving 20E, was injected as a solvent control. Three biological replicates were included, with each replicate comprising four larvae. Midguts were collected for total RNA or protein extraction at 12 and 24 h post-injection.

Quantification of total bacteria and Lactobacillus in the midgut and hemolymph

Genomic DNA from the midguts and hemolymph of sixth-instar larvae at 24 or 72 h PE was extracted using a genomic DNA isolation kit (Omega). Genomic DNA was extracted from the midguts and hemolymph of sixth-instar larvae at 72 h PE after treatment with dsCD209, dsLys1, or dsGFP. Moreover, genomic DNA was isolated from the midguts and hemolymph of sixth-instar larvae at 24 and 72 h PE fed with a diet containing L. plantarum or PBS. The qPCR analysis was performed on genomic DNA using universal 16S rRNA gene primers 16S-F and 16S-R or Lactobacillus-specific 16S rRNA primers (Additional file 1: Table S8). The qPCR was performed using TransStart Tip Green qPCR SuperMix (TransGen Bio-tech). H. armigera β-actin gene was used as an endogenous control.

Recombinant protein generation, bacterial-binding assay, and antiserum preparation

The DNA fragment encoding the mature peptide of CD209 or Lys1 was cloned into pET32a plasmids, which were transformed into Escherichia coli BL21 (DE3) competent cells. After addition of 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C overnight, rCD209 was induced in a soluble form, whereas rLys1 was present in inclusion bodies. The inclusion bodies were denatured and renatured, as described [61]. The recombinant proteins were purified using High-Affinity Ni–NTA Resin (Novagen, WI, USA), dialyzed against PBS, and concentrated to desired concentrations. The rTrx was prepared simultaneously and used as control.

L. plantarum (2 × 108 cells/mL) was incubated with rCD209 (1.0 mg/mL) in the presence or absence of 10 mM CaCl2 for 30 min. The bacterial cells were pelleted, washed, and eluted with 7% SDS. The rTrx was used as negative control. The samples were analyzed by western blotting.

After homogenization of rCD209 (200 μg) with complete Freund’s adjuvant, the mixture was hypodermically injected into the back of a rabbit. After 3 weeks, the same amount of recombinant protein, homogenized with incomplete Freund’s adjuvant, was subcutaneously injected. The antiserum against CD209 was prepared after a booster injection of 500 μg antigen 2 weeks later.

Western blotting

Total proteins isolated from various tissues, including the midgut treated with 20E or RNAi, were subjected to western blot analyses. Purified rCD209 or rTrx were loaded as controls. Equal aliquots of protein samples (100 μg/lane) were resolved using 12.5% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% skim milk and incubated with the antiserum against rCD209 at a 1:500 dilution. An antibody against β-actin (ABclonal, Cat. AC026) was used as a loading control. Protein samples from bacterial washing and elution were subjected to western blot analyses using anti-His antibody (ABclonal, Cat. AE003). Dosage analyses were performed using ImageJ software (Bethesda, MD, USA), based on three biological replicates.

Analysis of the antibacterial activity of Lys1

To analyze the antibacterial activity of Lys1, rLys1 was incubated with enterokinase (Beyotime Bio-tech, Shanghai, China) to remove Trx (His-tagged). A total of 90 μL of Lys1 (25 μM) was mixed with 10 μL of bacterial suspension at 25°C for 1 h. The mixture was plated on MRS agar plates and cultured at 37°C for 24 h. The number of colonies on each plate was recorded. The same concentration of bovine serum albumin (BSA) was used as a control. Three replicates were used.

RT-qPCR

Total RNA was extracted from various tissues, including the midgut treated with 20E or RNAi, using TRIzol Reagent (Invitrogen). Total RNA was isolated from fat bodies of sixth-instar larvae at 72 and 96 h PE after either dsRNA injection or L. plantarum treatment. First-strand cDNA was synthesized using EasyScript cDNA synthesis SuperMix (TransGen Bio-tech). RT-qPCR analysis was performed using TransStart Tip Green qPCR SuperMix (TransGen Bio-tech). The housekeeping gene β-actin was used as an endogenous control. Primers used for RT-qPCR analysis are listed in Table S8 (Additional file 1).

Untargeted metabolomics analysis

Hemolymph samples (100 μL) from sixth-instar larvae at 24 h post-L. plantarum or -PBS injection were mixed with 400 μL extraction solution (acetonitrile:methanol = 1:1) containing 0.02 mg/mL internal standard (L-2-chlorophenylalanine). After vortexing for 30 s and low-temperature ultrasonic extraction for 30 min was complete, the samples were kept at − 20°C for 30 min to precipitate the proteins and centrifuged for 15 min at 13,000 × g. The supernatants were collected and blow-dried under nitrogen. The samples were resolubilized with 100 μL solution (acetonitrile:water = 1:1) and centrifuged at 13,000 × g. The supernatants were obtained for LC–MS/MS analysis.

The metabolite extracts from hemolymph samples were analyzed using a UPLC-Q-Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA). Chromatographic separation was conducted using an ACQUITY HSS T3 column (Waters, Milford, USA). The mobile phases consisted of 0.1% formic acid in water:acetonitrile (95:5; solvent A) and 0.1% formic acid in water:acetonitrile:isopropanol (5:47.5:47.5; solvent B). Raw data were processed with Progenesis QI software (Waters). Internal standard peaks and known false positive peaks were removed. The metabolites were searched against the HMDB database (http://www.hmdb.ca/). The R package “ropls” (version 1.6.2) was used to perform PLS-DA. Metabolites with VIP > 1 and p < 0.05 were considered significantly different metabolites. Differential metabolites between two groups were mapped into their biochemical pathways through metabolic enrichment based on the KEGG database (http://www.genome.jp/kegg/).

LD staining

Fat bodies were collected at 24 h post-injection of L. plantarum or PBS, incubated in Nile red solution (20% glycerol in PBS, with 1:500 dilution of 10% Nile red in DMSO) for 20 min, and imaged under a laser-scanning confocal microscope (Leica, Germany). LD diameter and fluorescence intensity were determined using ImageJ software. Each group consisted of 15 average LD diameters and fluorescence intensities, each of which was based on one image.

TAG and cholesterol assays

Fat bodies were collected at 24 h post-L. plantarum, -PGN (Lysine-type), or -PBS injection to determine TAG and cholesterol levels, as described [62]. Fat bodies were homogenized in isopropanol and centrifuged at 10,000 × g for 10 min. The supernatants were used for TAG and cholesterol quantification using a triglyceride colorimetric assay kit (Elabscience, Wuhan, China) and total cholesterol colorimetric assay kit (Elabscience), respectively. For Drosophila, larvae were collected at 3 h post-PGN challenge, homogenized, and centrifuged, following which the supernatants were used for assays.

Measurement of developmental timing

To determine the mean duration of the fourth-, fifth-, and sixth-instars, we recorded each individual every 1 h from the beginning of each treatment until pupariation. The duration measurement was based on 23 dsCD209- and 24 dsGFP-injected larvae, as well as 28 dsLys1- and 34 dsGFP-injected larvae. A total of 28 L. plantarum- or PBS-injected larvae, 36 L. plantarum-fed larvae, and 41 PBS-fed larvae were used for the assays. A total of 45 PGN (Lysine-type)- and 44 PBS-injected larvae were assayed. For Drosophila, 22 PGN (Lysine-type)- and 30 PBS-challenged larvae were analyzed. The mean duration of larval stages was determined.

To determine whether administration of 20E rescued delayed pupariation, sixth-instar larvae pre-treated with dsCD209 or dsLys1 were divided into two groups. Sixth-instar larvae injected or fed with L. plantarum were divided into two groups. One group was injected with 5 μL of 20E (500 ng/μL) per larva, whereas the other group was injected with an equivalent amount of DMSO as solvent control. Both injections were committed at 72 h PE of sixth-instar. In the dsCD209-injected group, 34 20E- and 29 DMSO-injected larvae were analyzed. In the dsLys1-injected group, 33 20E- and 30 DMSO-injected larvae were analyzed. In the L. plantarum-injected group, 32 20E- and 30 DMSO-injected larvae were used for calculating the mean of the sixth-instar. In the L. plantarum-fed group, 35 20E- and 34 DMSO-injected larvae were analyzed. Each analysis was conducted twice.

Statistical analyses

Statistical analysis was performed using GraphPad Prism 8 (GraphPad Inc., La Jolla, CA, USA). The Mann–Whitney U test was used to evaluate differences in bacterial composition between the two groups. Unless otherwise indicated, Student’s t test was used to determine differences between two groups. One-way analysis of variance (ANOVA) coupled with Tukey’s multiple comparison test was used to evaluate differences among multiple groups. A value of p < 0.05 was considered statistically significant.