Nematode and insect culture
Entomopathogenic nematode H. beicherriana LF were cultured in vivo on larvae of the greater wax moth (Galleria mellonella) at 25 °C and 80% relative humidity (RH). Released infective juveniles (IJs) were collected and stored at 7 ± 1 °C for no more than 15 days before use [40]. The larvae of H. parallela were fed germinated wheat seeds, and actively feeding grubs (approximately 13 days old and at the beginning of second instar) were kept individually in the holes of six-well bioassay trays (Corning Inc., NY, USA) filled with 15 g and 18% soil moisture (w/w) of high temperature sterilized sandy loam soil (76% sand, 16% silt, 6% clay, 0.5% organic matter; pH 6.7) for use.
Nematode infection to Holotrichia parallela
One hundred IJs were added into each hole of the six-well bioassay trays described above. A total of 60 H. parallela larvae were treated with nematodes, and control group was treated with sterile water. The six-well bioassay trays were then transferred into a dark incubator under the following conditions: 25 °C and 80% RH. The midgut samples (n = 3 for each treatment) of H. parallela used for RNA-Seq were collected in RNase-free tubes at 24 h and 72 h after treatment with nematodes or sterile water following the method below.
Disintegration of Holotrichia parallela midgut
H. parallela larvae treated with nematodes or sterile water were quickly transferred on ice for 1 min and then placed on a precooled petri dish (d = 15 cm). After adjusting the focal length of SMZ-168 Stereo Zoom microscope (Sinolab Inc., Beijing, China) until the insect body could be observed clearly, the head and tail of the larvae were cut off with scissors and the whole midgut was carefully pulled out using tweezers. The malpighian tubule and fat around the midgut were removed with tweezers, and the peritrophic membrane was pulled to remove all food particles in the middle intestine. After three rinses with RNase-free water, three samples for each treatment were collected in a 1.5 ml RNase-free tube and stored at -80 °C for RNA isolation. The experiment was performed with two replicates of each treatment for RNA-Seq.
RNA isolation and cDNA library preparation
Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol followed by ethanol/isopropanol precipitation and suspended in a final volume of 30 μl with RNase-free water. After extraction, the total RNA quality of different treatments were measured with NanoDrop 2000 spectrophotometer (Thermo Scientific) and in a 1.5% agarose gel. RNA integrity and concentration were subsequently analyzed using 2100 RNA Nano 6000 Assay Kit (Agilent Technologies, CA, USA).
Following the Illumina protocols, 2 µg RNA of different treatment was used for cDNA library construction, and each treatment was replicated two times. Following the use of oligo (dT) magnetic beads, fragmentation of mRNA, generation of the first strand cDNA with a random hexamer primer and synthesis of second strands, cDNA end repair and adenylation at the 3’ end, and adapter ligation and cDNA fragment enrichment were performed. Then, the cDNA products were purified and amplified by PCR to create the final cDNA library.
RNA-Seq and de novo assembly
Paired-end sequencing was conducted on an Illumina HiSeq 2000 (Illumina Inc., USA) at Anoroad Genomics Co., Ltd. (Beijing, China). After sequencing, the raw reads were filtered to generate clean reads. First, the reads with adapters were removed, and then the reads with > 5% unknown bases (N) were discarded. Finally, the low-quality reads (with > 50% of nucleotides for which the Phred Quality Score Q was less than or equal to 19) were filtered out.
The clean reads of each treatment were loaded to Trinity (Trinity Release v2.4.0) [41] for de novo assembly under the paired-end mode with default parameters. Based on the filtered clean data, the full-length transcript sequence was assembled with Trinity, and each gene was taken based on the transcript sequence. The longest transcript sequence was recorded as ‘Unigene’.
RPKMs (Reads Per Kilobase Million Mapped Reads) [42] were used in DESeq2 [43] to compare the differences of gene expression between the nematodes and sterile water treat. Genes with |log2 Fold Change| ≥ 1 and q < 0.05 were selected as significantly DEGs. Then, DEGs were further annotated by Gene Ontology (GO) function (http://www.geneontology.org/).
Identification of immune-related genes in Holotrichia parallela
To identify the immune-related genes involved in the process of H. parallela larvae defence against nematode infection, we query with the terms that are reported as the immune relative genes from the differential gene expression data that obtained above. The terms mainly include ‘immune’, ‘recognition protein’, ‘haemocyte’, ‘glutactin’, ‘antimicrobial’, ‘lysozyme’, ‘lipopolysaccharide’, ‘lectins’, ‘peptidoglycans’ etc. All candidate immune genes were manually verified with the BLASTx program at the National Center for Biotechnology Information (NCBI: https://www.ncbi.nlm.nih.gov/).
Bioinformatics analysis of three potentialcandidateimmune-related genes
The deduced amino acid sequences of three potential candidate immune-related genes (PGRP-SC1, PPAE-I and GST) were obtained using the Translate tool provided by the Swiss Institute Bioinformatics (https://web.expasy.org/translate/). The signal peptide and transmembrane domain were analyzed by SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP) and TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). In addition, domain analysis of the retrieved protein sequences were executed by NCBI Conserve-Domain Tool (https://www.ncbi.nlm.nih.gov/cdd) and PROSITE (http://au.expasy.org/prosite/). The 3D structures were obtained using templates of Bumblebee PGRP-SA (5xz4.1.A, 34.76% identity), Holotrichia diomphalia PPAF-I (2olg.1.A, 66.67% identity) and Nilaparvata lugens GST S2 (5h5l.1.A, 40.30% identity), respectively, employing online Swiss-model software. Pro-CHECK (http://servicesn.mbi.ucla.edu/PROCHECK/) was used to evaluate the rationality of the 3D structure of the constructed model. The generated PDB files were then visualized with Pymol Molecular Graphics System.
Phylogenetic analysis of PGRP-SC1 from H. parallela and 53 PGRPs from 12 other insect species; PPAE-I from H. parallela and 26 SPs from 12 other insect species; GST from H. parallela and 26 GST from 12 other insect species, which were used to construct a phylogenetic tree using MEGA5.0. Phylogenetic analysis were conducted by the neighbour-joining method with p-distance under the default parameters. Bootstrap values were obtained by the bootstrap method using 1000 repetitions. The species name, gene name, abbreviation and GenBank accession involved in phylogenetic analysis are listed in Additional files 1.
Spatiotemporal analysis of PGRP-SC1, PPAE-I and GST
To determine the expression profiles of these genes in different development stages and tissues, we collected eggs (5 days old, ~60 mg), first instar larvae (10 days old, ~60 mg), second instar larvae (18 days old), third instar larvae (28 days old) and various tissues (hemolymph, malpighian tubule, midgut and fat body) of the first instar larvae for RNA isolation. Total RNA samples of each treatment were individually extracted using TRIzol Reagent (TIANGEN, Biotech (Beijing) Co., Ltd., China) and each treatment repeat three times. First-strand cDNA was synthesized from 1 μg of total RNA following the instructions of TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech (Beijing) Co., Ltd, China). The cDNA products were diluted 10-fold for use as template in qRT-PCR.
qRT-PCR was performed with Tip Green qPCR SuperMix (TransGen, China) on an ABI Prism 7500 Fast Detection System using a GO Taq®qPCR kit (Invitrogen, USA) according to the manufacturer’s instructions. Gene-specific primers designed for target genes are listed in Additional files 2. qRT-PCR reactions were performed in a 20 μl mixture containing 2 μl of cDNA, 10 μl of Tip Green qPCR SuperMix, 0.4 μl of each primer (10 μM), and 7.2 μl of nuclease-free water. The optimized qRT-PCR programme conditions were 94 ºC for 30 sec followed by 40 cycles of 94 ºC for 5 sec and 60 ºC for 34 sec. After the cycling protocol, melting curves were obtained by increasing the temperature from 60 to 95 °C (0.2 °C sec-1) to denature the double-stranded DNA. H. parallela DAPDH was used as an internal standard to normalize the expression level. Each qRT-PCR experiment was performed with three biological replicates and four technical replicates. The relative expression level of target genes was calculated with the 2-ΔΔCt method [44].
qRT-PCR validation
To confirm the transcriptome results, these immune-related genes (i.e., PGRP-SC1, PPAE-I and GST) of H. parallela responding to nematode infection were selected for qRT-PCR analysis. Fresh total RNA was obtained as described in RNA-Seq and qRT-PCR was performed as described above.
RNA interference to Holotrichia parallela larvae
dsRNA synthesis: RNAi was used to study the functions of PGRP-SC1, PPAE-I, and GST involved in the immune defence system of H. parallela against H. beicherriana LF infection. Specific primers (Additional files 2) with T7 promoter sequences were designed to synthesize dsRNA of PGRP-SC1, PPAE-I, GST and GFP using the T7 RiboMAX™ Express RNAi kit (Promega, WI, USA) according to the manufacturer’s instructions. The size of the dsRNA products was confirmed by electrophoresis on 1.2% agarose gel, and the final concentration of dsRNA was adjusted to 1 μg/μl.
In vivo RNAi: For in vivo RNAi, 1.5 μg of each dsRNA, dsGFP and the same volume of RNase-free water were injected into the haemocoel of H. parallela second instar larvae (18-20 days old) through the abdomen (WPI microinjection system, USA). After injection, the larvae were kept individually in the holes of six-well bioassay trays and fed germinated wheat seeds. The six-well bioassay trays were transferred into a dark incubator under the following conditions: 25 °C and 80% RH. The midgut samples (n = 3 for each treatment) from challenged or control groups (dsGFP-injected and water-injected) was collected after treatment for 24, 48 and 72 h, respectively, and the experiment was performed with three biological replicates. The qRT-PCR process was executed as described above.
Susceptibility of Holotrichia parallela larvae to Heterorhabditis beicherriana LF after RNAi
Susceptibility of H. parallela larvae to H. beicherriana LF after RNAi of PGRP-SC1, PPAE-I and GST was conducted by determining the pathogenicity change of H. beicherriana LF on H. parallela larvae. After injection following our previous procedures, the larvae (18-20 days old) were kept individually in the holes of six-well bioassay trays, fed germinated wheat seeds and then transferred into a dark incubator as described above. Forty eight hours later, two concentrations (100 and 200 IJs/grub) of H. beicherriana LF were added to each hole of six-well bioassay trays. Each treatment containing 36 individuals (grub) with three replicates. The grub mortality was monitored after 24 h, and the infected grubs were marked as inactive and red.
Statistical analysis
Data statistics were performed using GraphPad Prism software version 8.0. All proportions were transformed by arcsine square root transformation in Microsoft Excel 5.0 before analysis [45]. The significance levels of spatiotemporal analysis were analyzed using one-way analysis of variance (ANOVA) followed by Tukey significant difference test (P < 0.05). The significance levels of in vivo RNAi and white grubs mortality were analyzed using multiple t-test at the significance levels set at *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant.