Introduction
It is well known that termite is a social insect; some species of which are important worldwide pests. They have the characteristics of strong survival ability, long population life, hidden lifestyle, and wide range of harm, and they often cause serious harm to housing, water conservancy, transportation, communication, warehousing, agriculture, and forestry (Huang et al., Reference Huang, Zhu, Ping, He, Li and Gao2000; Zhang et al., Reference Zhang, Ji, Liu, Cao, Yang, Liu, Ji, Soleymaninejadian and Wang2015). According to 2010 data, the global economic impact of termites is estimated at $40 billion (Rust and Su, Reference Rust and Su2012).
Reticulitermes chinensis Snyder is a widely distributed subterranean termite in China. As an important pest in forestry and construction, R. chinensis nest in soil and wood structures and feed on wood and its products (Huang et al., Reference Huang, Zhu, Ping, He, Li and Gao2000; Liu, Reference Liu2003). Chemical control is an important means to control termites. Chemical control has quick and good effect, but long-term use of chemical insecticides has caused many adverse effects. So biological control has been paid more and more attention (Verma et al., Reference Verma, Sharma and Prasad2009; Lin, Reference Lin2015). The use of pathogenic microorganism is an important pest control method, which has been studied with a variety of termites. As for R. chinensis, there are also some studies related to pathogenic microorganisms, such as Wang (Reference Wang2014) studied the lethal effect of Metarhizium anisopliae (Metschn.) Sorokin against R. chinensis, and the changes in genes and enzymes in R. chinensis infected with M. anisopliae. Tan (Reference Tan2022) studied the differences in lethality of four different fungal strains against R. chinensis.
Our research group isolated a Serratia marcescens Bizio strain SM1 (hereinafter referred to as SM1) from the disease insects and found that the strain has insecticidal activity to R. chinensis (Jiang et al., Reference Jiang, Lu, Zhang and Tang2023). As a biocontrol bacterium, S. marcescens can be used in the control of some pests, plant pathogenic fungi, and nematodes (Sezen et al., Reference Sezen, Yaman and Demirbag2001; Someya et al., Reference Someya, Nakajima, Hirayae, Hibi and Akutsu2001; Hegazy et al., Reference Hegazy, Salama, El-Ashry and Othman2019). In terms of insecticidal ability, S. marcescens has been shown to kill a wide variety of insects as an insect pathogen. Kwak et al. (Reference Kwak, Han, Nam, Choi, Lee, Choi and Park2014) verified the pathogenicity and toxicity of S. marcescens by injecting and orally treating Protaetia brevitarsis seulensis (Kolbe) larvae with bacterial suspension of S. marcescens. Mohan et al. (Reference Mohan, Selvakumar, Sushil, Bhatt and Gupta2011) isolated the S. marcescens strain SRM from the flowers of summer squash and found that the strain was pathogenic against Helicoverpa armigera (Hübner). After infection with S. marcescens, the egg production and eclosion rate of Heliothis virescens (Fabricius) adults decreased compared with the control group, while the larval mortality increased, and the female pupae became smaller and had a higher mortality (Inglis and Lawrence., Reference Inglis and Lawrence2001). In addition, S. marcescens strain TC-1 had been isolated and found to have larvicidal activity against Anomala corpulenta Motschulsky, Plutella xylostella (Linnaeus), Spodoptera exigua (Hübner), H. armigera, and Bombyx mori (Linnaeus) (Tao et al., Reference Tao, Wang, Pang, Zheng, Ayra-Pardo, Huang, Xu, Liu, Li, Wei, Wang, Niu and Li2022). S. marcescens is a biocontrol bacterium with great potential.
SM1 is a potential biocontrol bacterium against R. chinensis, and we have previously identified the immune defence mechanism of R. chinensis against SM1 by second-generation transcriptome sequencing technology (Luo et al., Reference Luo, Wang, Tang and Feng2022), but the pathogenic mechanism of SM1 to R. chinensis has not been reported. Single-molecule real-time (SMRT) sequencing technology is a third-generation high-throughput sequencing technology, which is suitable for de novo genomic sequencing and high-quality assemblies of small genomes. SMRT sequencing technology can detect epigenetic modifications directly, and has the characteristics of being polymerase chain reaction (PCR)-free, having a high speed, and having long read lengths (Liu et al., Reference Liu, Wang and Yu2015). Therefore, we intended to use SMRT sequencing technology to clarify the following questions: (1) whether SM1 has a destructive effect on the metabolism of R. chinensis; (2) whether SM1 can destroy the immunity of R. chinensis; (3) whether SM1 affects the signal transduction pathways of R. chinensis; (4) whether SM1 affects the pathways related to genetic information of R. chinensis. By clarifying the above questions, we clarified the pathogenic molecular mechanism of SM1 to R. chinensis.
Materials and methods
Insects and bacteria
R. chinensis populations were collected in Nanjing, Jiangsu province, China. Each R. chinensis colony was kept in separate plastic boxes (20 cm × 15 cm × 15 cm). In our laboratory, the colonies were set in 25 ± 1°C with 90 ± 5% relative humidity and dark conditions (0 h light:24 h dark). Healthy workers in the colonies were selected for the experiment.
We placed SM1 on solid bacterial basal medium and cultured it at 30°C for 12 h. After the generation of single colonies, we placed a single colony in 50 ml seed culture medium, and cultured it in a shaker at 30°C and 200 r min−1 for 12 h. Then, we added an appropriate amount of seed solution into 200 ml fermentation medium and cultured in a shaker at 30°C and 200 r min−1 for 36 h. Bioassays were performed using SM1 fermentation medium with a concentration of 1.52 × 1010 cells ml−1.
Sample processing
We placed 20 healthy third-instar worker termites into each Petri dish with a diameter of 20 cm and starved them for 12 h. In the treatment group (SM_RC), 1 μl SM1 fermentation medium was dropped on the pronotum of R. chinensis, and 1 μl sterile fermentation medium was dropped on the same locations of R. chinensis in the control group (RC). After 20 h, ten live R. chinensis were deposited at −80°C respectively for subsequent experiment. Three replicates were set in the treatment group and the control group.
RNA sample preparation
Total RNA of the samples was extracted by the TRIzol method. The purity and concentration of RNA was determined by Nanodrop 2000 spectrophotometer, and agarose gel electrophoresis was used to detect the integrity of RNA.
Library preparation and SMRT sequencing
Full-length cDNA of mRNA was synthesised by Clonetech SMARTer™ PCR cDNA Synthesis Kit. Primer with Oligo dT was used to pair the A–T bases with the structure of poly-A at the 3′ end of mRNA, and primer was added to the end of reverse-synthesised full-length cDNA. The full-length cDNA was amplified by PCR, purified by SMRTbell cleanup beads (PB) magnetic beads, and quantified by Qubit 3.0. The end of the full-length cDNA was repaired and attached to the SMRT dumbbell adapter, and the unattached fragments were digested by exonuclease. After purification by PB magnetic beads, the sequencing library was obtained. Accurate quantification was performed using Qubit 3.0 and library size was detected using Agilent 2100. The full-length transcriptome sequencing was performed by using a PacBio sequencer after the test results met the requirements.
Sequel data output and quality control
The original sequencing output data were preprocessed by using SMRTLink. The main parameters of SMRTLink were minimum subread length = 50, maximum subread length = 15,000, minimum number of passes = 3, minimum predicted accuracy = 0.99. The Iso-Seq analysis process was used to obtain full-length transcript sequences. The single-molecule polymerase reads were separated to obtain subreads, and circular consensus sequence (CCS) was formed by the self-correction of the subreads obtained from the same polymer reads. The 3′ poly-A sequences, primer sequences, and chimeric sequence of CCSs were detected to classify CCSs and find full-length non-concatemer (FLNC) sequences. The iterative clustering and error correction tool of SMRTLink software was used to cluster and eliminate redundancy of the FLNC sequences and then the arrow algorithm in SMRTLink was used to make further correction to obtain the polished transcripts. Finally, cd-hit software was used for clustering and redundancy removal (Li and Godzik, Reference Li and Godzik2006).
Functional annotation of transcripts
Isoform sequences were aligned to gene ontology (GO), Swiss-Prot Protein Sequence Database (Swiss-Prot), Clusters of Orthologous Groups/Eukaryotic Orthologous Groups (COG/KOG), NCBI nonredundant protein sequences (NR), and Kyoto Encyclopedia of Genes and Genomes (KEGG) by diamond blastx to obtain protein IDs with high sequence similarity and the protein functional annotation information of the isoforms (Buchfink et al., Reference Buchfink, Xie and Huson2015). The annotation status of all transcripts was statistically summarised, and the BLAST function of the NCBI website (https://www.ncbi.nlm.nih.gov/) was used to further verify the transcripts with contradictory or ambiguous annotations.
Digital gene expression library preparation and analysis
Using bowtie2 software, the clean reads of each sample were compared with the isoforms that had redundancy removed (Langmead, Reference Langmead2010). RSEM was used to count the results of bowtie2 and perform fragments per kilobase per million bases (FPKM) conversion (Li and Dewey, Reference Li and Dewey2011). When the differential expression analysis was performed by DESeq2 (Love et al., Reference Love, Huber and Anders2014), the P-value threshold of multiple tests was determined by the false-discovery rate (FDR) method, and an FDR threshold of <0.05 and an absolute value of |log2 fold change (FC)| > 1 were set. Then, KEGG pathway enrichment analysis and GO enrichment analysis were performed.
Results
Overview of the full-length transcriptome database
Sequencing data output and transcript clustering analysis
The full-length transcriptome sequencing was performed by using a PacBio sequencer after the test results met the requirements, and 145 Gb of original data were generated and stored in the NCBI Sequence Read Archive with accession number SRR24891407. The number of polymerase reads was 690,759, and 80 Gbp of subreads remained after removing the adapter sequence of polymerase reads. A total of 537,340 sequences were CCSs, and a total of 357,749 FLNC were obtained by CCS classification. A total of 27,570 polished isoform sequences were assembled from FLNC, and a total of 26,097 isoforms were obtained by cd-hit-est software for sequence clustering and elimination of redundancy (table 1).
Table 1. Statistics of sequencing data and transcript clustering data
Functional annotation of transcripts
In this study, 3808 transcripts were not annotated, and 22,289 transcripts were annotated, and 5256 transcripts were annotated by all databases. A total of 22,148 transcripts were annotated in NR database, 7765 in GO database, 13,168 in KEGG database, 16,696 in KOG database, and 19,180 in Swiss-Prot database.
In the NR annotation, 44.6% of the R. chinensis sequences were aligned to Cryptotermes secundus (Hill), followed by Zootermopsis nevadensis Hagen (26.0%), Coptotermes formosanus (5.9%), Blattella germanica Linnaeus (1.6%), Reticulitermes flavipes (Kollar) (1.2%), and others (20.7%) (fig. 1).
Figure 1. NR classification of all R. chinensis unigenes.
In the GO annotation, the transcriptome was divided into three major functional processes including 52 terms. The most abundant terms in the biological process (BP) category were cellular process (3841), metabolic process (3048), and single-organism process (2508). In the cellular component (CC) category, cell (3564), cell part (3489)s and organelle (2689) had the most transcripts. The transcripts of binding (4082) and catalytic activity (3836) were far more numerous than other subcategories in the molecular function (MF) category (fig. 2).
Figure 2. GO functional classifications of R. chinensis transcripts. MF, BP, and CC are represented in green, red, and blue, respectively. The x-axis and y-axis represent the number of transcripts and GO categories, respectively.
A total of 13,168 transcripts were annotated to 287 KEGG pathways on 34 smaller branches on the five major branches. The terms with the largest number of transcripts in five major branches, including environmental information processing, organismal systems, metabolism, cellular processes, and genetic information processing, were signal transduction (2106), endocrine system (1427), carbohydrate metabolism (1031), transport and catabolism (1730), and folding, sorting, and degradation (1132), respectively (fig. 3).
Figure 3. KEGG classification of R. chinensis transcripts. Genetic information processing, organismal systems, environmental information processing, cellular processes, and metabolism are represented in green, orange, blue, red, and purple, respectively. The x-axis and y-axis represent the number of transcripts and KEGG pathway categories, respectively.
A total of 16,696 transcripts were divided into 26 KOG groups. The three largest groups were general function prediction only (2563), posttranslational modification, protein turnover, chaperones (2027), and signal transduction mechanisms (1924) (fig. 4).
Figure 4. Eukaryotic orthologous groups of protein (KOG) annotation of R. chinensis transcripts. The x-axis and y-axis represent KOG categories and the number of transcripts, respectively.
Differentially expressed genes in R. chinensis in response to SM1 infection
In order to explore the mechanism of SM1 infection, we used PacBio Sequel sequencing to identify differentially expressed genes (DEGs) in R. chinensis. The combined data from three biological replicates were used to compute FPKM values, and the results were compared between the replicate RC and SM_RC groups. DEGs were significantly different when FDR < 0.05 and |log2 FC| > 1 (fig. 5A). A total of 230 DEGs were identified from 22,289 unigenes, including 103 downregulated DEGs and 127 upregulated DEGs (fig. 5B).
Figure 5. Overview of DEGs. (A) Comparison of DEGs between the RC library and SM_RC library. Red, blue, and black spots represent significantly upregulated genes, significantly downregulated genes, and genes with no significant difference in expression, respectively. (B) Heatmaps illustrating differences in normalised log signal intensity for the identified R. chinensis genes. R. chinensis treatment groups were labelled as SM_RC1, SM_RC2, and SM_RC3, and control groups were labelled as RC1, RC2, and RC3. Red and blue indicates genes expressed at high levels and genes expressed at low levels, respectively. The colours from red to blue indicate gradual decrease in expression.
The functions of 61 DEGs in R. chinensis were divided into 30 groups in GO enrichment analysis. The most abundant terms in the BP category were cellular process (29), metabolic process (22), and single-organism process (19). In the CC category, cell (27), cell part (27), membrane (17), and organelle (17) had the most transcripts. The transcripts of binding (30) and catalytic activity (24) were far more numerous than other subcategories in the MF category (fig. 6).
Figure 6. GO functional classification of DEGs in R. chinensis. MF, BP, and CC are represented in green, red, and blue, respectively. The x-axis and y-axis represent the number of transcripts and GO categories, respectively.
Mapping DEGs to typical KEGG pathways identified biological pathways that responded to SM1 processing, 80 DEGs were assigned into 104 KEGG pathways (Supplementary table S1). P-values of pathways <0.05 indicated highly enriched pathways (table 2).
Table 2. Highly enriched KEGG pathways of DEGs in the R. chinensis transcriptome
Transcriptomic response of R. chinensis to SM1 infection
SM1 affected the expression of metabolism-related genes of R. chinensis
Metabolism is a considerable component of life activities. By searching the KEGG database, we screened the genes related to metabolic pathways of R. chinensis, which could be divided into nine categories: carbohydrate metabolism, xenobiotics biodegradation and metabolism, energy metabolism, nucleotide metabolism, lipid metabolism, metabolism of terpenoids and polyketides, amino acid metabolism, glycan biosynthesis and metabolism, and metabolism of cofactors and vitamins. In R. chinensis infected with SM1, 16 genes related to metabolic pathways were downregulated (fig. 7A) (Supplementary table S2). The pathway related to carbohydrate metabolism had the largest number of downregulated genes, with six downregulated genes, followed by amino acid metabolism and energy metabolism, with four downregulated genes respectively. Downregulated genes related to carbohydrate metabolism include UTP-glucose-1-phosphate uridylyltransferase (UGP), inositol-3-phosphate synthase (ISYNA), 2-oxoglutarate dehydrogenase (OGDH), phosphoglycerate mutase (PGAM), pyruvate kinase (PK), and β-glucuronidase (GUSB). UGP plays central roles in carbohydrate interconversion and glycogen synthesis. ISYNA is involved in the formation of inositol compounds. OGDH is involved in the citrate cycle in carbohydrate metabolism. PGAM is a protease that plays an important role in glycolysis and gluconeogenesis. PK is an enzyme that regulates glycolysis. The activity of animal GUSBs often exhibit changes in various physiological and pathological processes. In addition to carbohydrate metabolism, GUSB was not only related to xenobiotics biodegradation and metabolism, glycan biosynthesis and metabolism, and metabolism of cofactors and vitamins, but also located in immune-related pathway. In addition to OGDH and PGAM, downregulated genes in amino acid metabolism included arginine kinase (AK) and ornithine decarboxylase (ODC). AK is a phosphotransferase in invertebrates, and ODC is important for polyamine biosynthesis. The four downregulated genes related to amino acid metabolism were located in arginine-, glycine-, proline-, serine-, lysine-, tryptophan-, and threonine-related metabolic pathways. In addition to PGAM, one NADH dehydrogenase (ubiquinone), one beta subcomplex subunit 8 (NDUFB8) gene, and 2V-type proton ATPase (ATPeV1B and ATPeV0C) genes were downregulated in the energy metabolism pathway. NDUFB8 is associated with oxidative phosphorylation. As a class of transporters, ATPeV1B and ATPeV0C located in the energy metabolism pathway were also present in immune-related pathways. In addition, we found that in the metabolism of terpenoids and polyketides, the expression of juvenile hormone epoxide hydrolase (JHEH), which is essential for juvenile hormone metabolism, was downregulated. All in all, SM1 downregulated some metabolism-related genes in R. chinensis and negatively regulated metabolic pathways.
Figure 7. Heatmap analysis of downregulated genes in R. chinensis infected with SM1. RC1, RC2, and RC3 are the control groups. SM_RC1, SM_RC2, and SM_RC3 are the treatment groups. (A) Metabolism, (B) immune, (C) signal transduction, and (D) genetic information.
SM1 negatively affects the immune response in R. chinensis
By screening the annotations of genes, we found that some immune-related genes of R. chinensis were downregulated after infection with SM1 (fig. 7B) (Supplementary table S2). We found that three pattern recognition receptor (PRR) genes were downregulated, and they were lipopolysaccharide binding protein (LBP), galectin-3 (Gal-3), and apolipophorin-III (apoLp-III). Regarding cellular immunity-related genes, we found downregulated genes in endocytosis, lysosome, and phagosome pathways. ATPeV0C and ATPeV1B both had one downregulated gene in the phagosome pathway. In the lysosome pathway, one gene each of cathepsin F (CTSF), lysosomal-associated transmembrane protein (LAPTM), insulin-like growth factor 2 receptor (IGF2R), ATPeV0C, and GUSB was downregulated. Cathepsin is associated with various physiological processes. LAPTMs regulate lysosomal function. IGF2R is a multifunctional binding protein, and IGF2R gene was not only related to lysosome, but also located in the endocytosis pathway in our data. In addition, downregulated genes located in the endocytosis pathway also included one ADP-ribosylation factor (ARF) gene. ARF is involved in regulating membrane trafficking pathways. Insect hexamerin is primarily considered a storage protein, but this protein is also involved in other biological functions. In humoral immunity, two hexamerin (Hex-1 and Hex-2) genes were downregulated. In addition, our results showed that transferrin (Tf1 and Tf2) had two downregulated genes. Besides transporting iron, this protein has other physiological functions.
SM1 downregulated genes associated with multiple signal transduction pathways in R. chinensis
We found the presence of DEGs in nine signal transduction pathways, and six signal transduction pathways were found to have downregulated genes after R. chinensis was infected by SM1. The six signal transduction pathways were phospholipase D (PLD) signalling pathway, mammalian target of rapamycin (mTOR) signalling pathway, mitogen-activated protein kinase (MAPK) signalling pathway, cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signalling pathway, cyclic adenosine monophosphate (cAMP) signalling pathway, and calcium signalling pathway. A total of six downregulated genes were found in these six signal transduction pathways (fig. 7C) (Supplementary table S2). Both cAMP signalling pathway and cGMP-PKG signalling pathway had two downregulated genes, and one gene was downregulated in other pathways, respectively.
SM1 downregulated genes in pathways related to genetic information in R. chinensis
In this study, we found DEGs in eight pathways related to genetic information processing in R. chinensis infected with SM1, among which six pathways had downregulated genes. The six pathways were basal transcription factors, RNA degradation, RNA transport, ribosome biogenesis in eukaryotes, ribosome and protein processing in endoplasmic reticulum, and there are six downregulated genes in these pathways (fig. 7D) (Supplementary table S2). RNA degradation pathway had the highest number of downregulated genes, with two downregulated genes, while other pathways had only one downregulated gene each.
Discussion
In our study, SM1 infection had different effects on nine categories metabolic pathways of R. chinensis, among which carbohydrate metabolism had the most downregulated genes, followed by amino acid metabolism and energy metabolism. A similar situation also appeared in H. armigera infected with nucleopolyhedrovirus (NPV), most of the downregulated genes were enriched in the gene cohorts of carbohydrate, amino acid and energy metabolism, and the downregulated proteins participated in carbohydrate, amino acid, and energy metabolism pathways (Xing et al., Reference Xing, Yuan, Wang, Lin, Shen, Hu and Zou2017). In studies of arthropods such as Eriocheir sinensis (H. Milne Edwards), S. exigua, and B. mori, some genes related to carbohydrate metabolism were downregulated after infection with pathogenic microorganisms, which suggested that pathways related to carbohydrate metabolism were negatively affected to some extent (Huang et al., Reference Huang, Cheng, Xu, Cheng, Fang and Xia2009; Ding et al., Reference Ding, Pan, Huang, Jiang, Chen, Zhu, Wang and Xu2018; Li et al., Reference Li, Yu and Huang2018). With regards to energy metabolism, in Epiphyas postvittana (Walker) and Musca domestica Linnaeus, some genes involved in energy metabolism were downregulated after infection by pathogenic microorganisms, indicating that their energy metabolism was disrupted (Gatehouse et al., Reference Gatehouse, Markwick, Poulton, Ward, Young, Wilson, Dellow, Sneddon, Gatehouse, Simpson, Janssen, Bishop, Schaffer and Christeller2007; Tang et al., Reference Tang, Li, Yang, Yu, Wang, Liu and Huang2014). In the study of Ceracris kiangsu Tsai, when energy metabolism was disrupted and related supply and demand was unbalanced, the cell activities were affected, and eventually the body died (Zhao et al., Reference Zhao, Luo, Chen, Wang and Yang2004). In organisms, amino acids not only participate in the synthesis of nucleic acids, proteins and enzymes, but also play roles in immune regulation. Different amino acids have different functions in organisms (Li et al., Reference Li, Yin, Li and Kim2007). The changes of genes related to amino acid metabolism will undoubtedly affect the normal function of the organism. In the study of Scylla paramamosain Estampador, Cheng et al. (Reference Cheng, Ma, Liu, Deng, Jiang, Feng and Guo2022) found that the levels of some amino acids were changed by mud crab reovirus infection and some genes in amino acid metabolism pathways were affected, which could have an impact on the regulation of oxidative stress and metabolic processes. In this study, we found that JHEH genes were downregulated. After JHEH gene expression was inhibited by RNAi technology, the survival rate of Apolygus lucorum (Meyer-Dur) nymphs decreased significantly, and some nymphs showed difficulty in moulting (Tusun et al., Reference Tusun, Li, Liang, Yang, Yang and Wang2017). After JHEH of Mythimna separata (Walker) larva was treated with RNAi technology, the emergence rate of M. separata was reduced (Li et al., Reference Li, Yang, Zhang, Zhang, Hu and Fan2021). In conclusion, SM1 infection affected the expression of genes related to metabolism in R. chinensis, and negatively regulated its metabolism.
PRRs are a class of receptors that recognise and bind to pathogen-associated molecular patterns (PAMPs) that are specific to the pathogen but not present in the host when insects are infected with pathogenic microorganisms, thus activating relevant immune pathways in insects (Akira et al., Reference Akira, Uematsu and Takeuchi2006; Kumar et al., Reference Kumar, Kawai and Akira2011; Brubaker et al., Reference Brubaker, Bonham, Zanoni and Kagan2015; Stokes et al., Reference Stokes, Yadav, Shokal, Smith and Eleftherianos2015). Lipopolysaccharide is a special structure of Gram-negative bacteria, which can be recognised by LBP as PAMP. LBP has been reported in some insects, including B. mori, Galleria mellonella (Linnaeus), and Periplaneta americana (Linnaeus) (Jomori et al., Reference Jomori, Kubo and Natori1990; Dunphy and Halwani, Reference Dunphy and Halwani1997; Koizumi et al., Reference Koizumi, Morozumi, Imamura, Tanaka, Iwahana and Sato1997). Studies on B. mori had found that LBP can remove bacteria by promoting the formation of haemocyte nodules (Koizumi et al., Reference Koizumi, Imai, Morozumi, Imamura, Kadotani, Yaoi, Iwahana and Sato1999). Chen et al. (Reference Chen, Zhang, Chen, Zeng and Li2023) found that LBP regulates the immune response of C. formosanus to Gram-negative bacteria by affecting the immune deficiency (IMD) pathway. They also found that C. formosanus treated with both Gram-negative bacteria and the dsRNA of LBP had a higher mortality rate than C. formosanus treated with only Gram-negative bacteria or dsRNA. Gal has been found to have an affinity for galactoside, and has functions of inducing apoptosis, immune regulation, and parasitic immune evasion in different organisms (Advedissian et al., Reference Advedissian, Deshayes and Viguier2017; Lu et al., Reference Lu, Tian, Yang, Yuan, Ehsan, Liu, Yan, Xu, Song and Li2017; Xue et al., Reference Xue, Liu, Zhao, Zhang, Guan, Cheng, Zhou and Tai2017). Among the arthropods, the Gal-4 of B. mori showed obvious binding to fungi, Gram-positive bacteria and Gram-negative bacteria, and Gal of Apis cerana Fabricius had binding reactions with Chinese sacbrood virus and chronic bee paralysis virus. After the Gal gene was silenced, the number of bacteria in the haemolymph of Marsupenaeus japonicus (Bate) increased, and the survival rate of Fenneropenaeus merguiensis (de Man) against pathogenic microorganisms decreased (Shi et al., Reference Shi X, Wang, Xu, Zhang, Zhao, Vasta and Wang2014; Wang et al., Reference Wang, Wang and Lü2014; Yue et al., Reference Yue, Ma, Liu, Jiang, Fei, Zhang, Sun, Li and Ma2018; Praparatana et al., Reference Praparatana, Maskaew, Thongsoi, Runsaeng and Utarabhand2022). Studies have shown that apoLp-III is a multifunctional protein involved in immune response and lipid transport. The study on Antheraea pernyi (Guerin-Meneville) found that apoLp-III shows important role for binding of PAMPs, activation of prophenoloxidase, and production of antimicrobial peptides (AMPs) (Wen et al., Reference Wen, Wang, Shang, Huang, Li, Wu, Zhang and Zhang2016). Injection of purified BmApoLp-III into B. mori larvae infected with Beauveria bassiana (Bals.-Criv.) Vuill. delayed the onset and death of the larvae. In contrast, silencing BmApoLp-III gene with RNAi led to early onset and death of B. mori larvae (Wu et al., Reference Wu, Lin, Zhao, Su, Li, Zhang and Guo2021). These three PRRs all play important roles in insect immunity, but in R. chinensis infected with SM1, these three PRRs were downregulated, which to some extent reflects the negative effects of SM1 on R. chinensis immunity.
Unlike vertebrates, insect immune systems do not have acquired immunity and rely on non-specific innate immunity to fight pathogens, including cellular immunity and humoral immunity (Lemaitre and Hoffmann, Reference Lemaitre and Hoffmann2007). Cellular immunity is mediated by haemolymphatic cells of insects and consists mainly of nodulation, phagocytosis, and encapsulation (Kojour et al., Reference Kojour, Han and Jo2020). Endocytosis is an important way for organisms to remove pathogens (Ferreira and Boucrot, Reference Ferreira and Boucrot2018; Kaksonen and Roux, Reference Kaksonen and Roux2018). IGF2R, also called cation-independent mannose-6-phosphate receptor, is a multifunctional receptor that performs a variety of tasks essential for normal cell function (Ghosh et al., Reference Ghosh, Dahms and Kornfeld2003). In our results, one IGF2R and one ARF were downregulated in the endocytosis pathway of R. chinensis infected with SM1. Ma et al. (Reference Ma, Zhang, Ruan, Shi and Xu2010) found that ARF1 gene may be important in innate immunity of M. japonicus. V-ATPase is a class of transporters that are important for cellular processes and plasma membrane proton transport (Wagner et al., Reference Wagner, Finberg, Breton, Marshansky, Brown and Geibel2004; Beyenbach and Wieczorek, Reference Beyenbach and Wieczorek2006; Forgac, Reference Forgac2007; Hinton et al., Reference Hinton, Bond and Forgac2009). In addition to endocytosis, V-ATPase plays roles in many aspects, including amino acid transport, intracellular pH homoeostasis, intracellular waste disposal, neurotransmitter uptake, and protein degradation (Forgac, Reference Forgac1989; Harvey, Reference Harvey1992; Zhao et al., Reference Zhao, Benlekbir and Rubinstein2015; McGuire et al., Reference McGuire, Stransky, Cotter and Forgac2017). After RNA interference on V-ATPase genes of arthropods such as Tribolium castaneum (Herbst), Acyrthosiphon pisum (Harris), Cimex lectularius Linnaeus, and Neoseiulus californicus (McGregor), their physiological activities and survival were severely affected (Basnet and Kamble, Reference Basnet and Kamble2018; Cao et al., Reference Cao, Gatehouse and Fitches2018; Ghazy and Suzuki, Reference Ghazy and Suzuki2022). E. sinensis increased mortality and decreased moulting rate after RNA interference with the ATPeV1B gene (Hou et al., Reference Hou, Chen, Yang, Yue, Wang, Han and Wang2020). In R. chinensis infected with SM1, ATPeV0C and ATPeV1B each had one downregulated gene in the phagosome pathway. Degradation and recycling of cellular waste is an important function of lysosome. Substances reach lysosomes through endocytosis, phagocytosis, or autophagy, and then are degraded by lysosomal hydrolases (Saftig and Klumperman, Reference Saftig and Klumperman2009; Ballabio, Reference Ballabio2016). In addition to ATPeV0C and IGF2R, LAPTM, GUSB, and CTSF were also downregulated in the lysosome pathway of R. chinensis infected with SM1. In other arthropod studies, these three genes had also been linked to immunity (Lanz et al., Reference Lanz, Tsutsumi and Aréchiga1993; Liu et al., Reference Liu, Xin, Zhu, Wang, Zhang, Jiang, Zhang, Zhou, Liu and Tang2018; Yang et al., Reference Yang, Ma, Dong, Li, Ren, Zhang, Wu, Wang, Liu and Zhang2021). Humoral immunity consists mainly of melanisation and production of AMPs (Zänker, Reference Zänker2010). Phenoloxidase (PO) is a key enzyme for melanism, and the study of Neoaliturus haematoceps (Mulsant and Rey) found that Hex was necessary for PO to obtain optimal activity (Cotter et al., Reference Cotter, Myatt, Benskin and Wilson2008; Eliautout et al., Reference Eliautout, Dubrana, Vincent-Monégat, Vallier, Braquart-Varnier, Poirié, Saillard, Heddi and Arricau-Bouvery2016). In this study, two Hex genes were downregulated after R. chinensis infection with SM1. To sum up, SM1 infection had negative effects on both cellular and humoral immunity of R. chinensis.
Tf is a glycoprotein with multiple functions. It plays an important role in immunity, iron transport, and oxidative stress prevention in insects (Geiser and Winzerling, Reference Geiser and Winzerling2012). Studies have found that Tf, as an immune-related gene, was upregulated by pathogenic microorganism infection in a variety of insects (Yoshiga et al., Reference Yoshiga, Georgieva, Dunkov, Harizanova, Ralchev and Law1999; Valles and Pereira, Reference Valles and Pereira2005; Yun et al., Reference Yun, Lee, Kwon, Hwang, Kim, Kang, Lee, Ding, You and Goo2009; Brummett et al., Reference Brummett, Kanost and Gorman2017). But in some cases, Tf was downregulated after insects were infected with pathogenic microorganisms, such as Aedes aegypti (Linnaeus) infected with Sindbis virus and Spodoptera littoralis Boisduval infected with NPV (Kim and Muturi, Reference Kim and Muturi2013; Hamama et al., Reference Hamama, Hussein, Fahmy and Fergani2016). These may be because Tf will be induced as an immune-related gene to respond to the infection of pathogenic microorganisms on the one hand, and the infection of pathogenic microorganisms will cause damage to insects on the other. This may depend on the time, dose, and microorganism species of infection. When Tf expression is inhibited, insect immunity will be affected. For example, after Tf inhibition, P. xylostella showed increased susceptibility to pathogenic microorganisms, and the production of blood nodules was inhibited (Kim and Kim, Reference Kim and Kim2010; Xu et al., Reference Xu, Hao, Wang, Li, Guo and Dang2020). After R. chinensis infection with SM1, Tf expression was downregulated. This also represents the negative effect of SM1 infection on R. chinensis immunity.
In terms of signal transduction pathways, we found downregulated genes in six pathways, each of which is important for the normal physiological activities of insects. Calcium signalling pathway is related to cold sensitivity and diapause regulation in insects (Teets et al., Reference Teets, Yi, Lee and Denlinger2013; Zhao et al., Reference Zhao, Zhao, Sun, Zou, Yang, Han, Zhu, Yin and Hong2017; Dong et al., Reference Dong, Chen, Clarke and Niu2019). Studies on a variety of insects have found that the cAMP signalling pathway is involved in important physiological activities such as memory, immunity, heat stress, moulting, and cell death in insects (Venkatesh et al., Reference Venkatesh, Siddhartha, Joshi, Patel and Hasan2001; Kimura et al., Reference Kimura, Kodama, Hayasaka and Ohta2004; Armstrong et al., Reference Armstrong, Shoemaker, Money and Robertson2006; Matsumoto et al., Reference Matsumoto, Hatano, Unoki and Mizunami2009; Sajjadian and Kim, Reference Sajjadian and Kim2020). Regarding the cGMP-PKG signalling pathway, researchers have found that PKG signalling plays a role in neuronal plasticity of motor, sensory, and cognitive functions, including response to pheromones, appetitive learning, stress response, aggression, and phototaxis (Lucas and Ben-Shahar, Reference Lucas and Ben-Shahar2021). MAPK regulates important cellular processes such as external stress, immune defence, apoptosis, cell proliferation, and intracellular metabolism, and the MAPK signalling pathway is involved in reproduction, drug resistance, immunity, moulting, and other aspects of insects (Covi et al., Reference Covi, Chang and Mykles2012; Yang et al., Reference Yang, Deng, Wei, Yang, Zhao, Yin, Du, Guo, Xia, Yang, Xie, Wang, Wu, Yang, Zhou, Nauen, Bass and Zhang2020; Zhang et al., Reference Zhang, Tettamanti, Bassal, Heryanto, Eleftherianos and Mohamed2021; Huang et al., Reference Huang, Tian, Zhao, Zhu, Liu and Wang2022). As for the PLD signalling pathway, PLD is an important enzyme in the production of lipid second messenger phosphatidic acid, involved in a variety of basic cellular processes, and modulated a variety of cellular responses (Weernink et al., Reference Weernink, de Jesús and Schmidt2007). The mTOR signalling pathway is also a multifunctional pathway involved in autophagy, moulting, hypoxia adaptation, and other aspects of insects (Covi et al., Reference Covi, Chang and Mykles2012; Zhang et al., Reference Zhang, Zhang, Yang, Wang, Li, Wang, Chen and Yuan2017; Li et al., Reference Li, Xiao, Li and Tian2022). The downregulation of genes in these signal transduction pathways represents the interference of SM1 infection on R. chinensis signal transduction pathways, and further affects various physiological activities of R. chinensis.
Ribosomes are basic macromolecular machines that play an important role in translation mechanism, converting encoded information in mRNA into protein (Thomson et al., Reference Thomson, Ferreira-Cerca and Hurt2013). RPL31 is a component of the large ribosome subunit and may be involved in the normal function of the chaperone complex (Peisker et al., Reference Peisker, Braun, Wölfle, Hentschel, Fünfschilling, Fischer, Sickmann and Rospert2008). In R. chinensis infected with SM1, RPL31 gene expression level in the ribosome pathway was downregulated. Ribosome biogenesis is the basic process that provides cells with the molecular factories that produce cellular proteins (Kressler et al., Reference Kressler, Hurt and Baβler2010). In our results, there is a downregulated gene, 5′–3′ exoribonuclease 1 (XRN1), in the ribosome biogenesis in the eukaryote pathway of R. chinensis infected with SM1, which is also located in the RNA degradation pathway. RNA degradation systems in organisms have demonstrated powerful efficacy in removing defective or no longer required RNA and RNA–protein complexes (Houseley and Tollervey, Reference Houseley and Tollervey2009). In this study, RNA degradation pathway had two downregulated genes. The gene downregulated in the RNA transport pathway is eukaryotic translation initiation factor 3 subunit F (EIF3F). Eukaryotic translation initiation factor 3 (EIF3) is a large multisubunit protein that plays a central role in translation initiation, and EIF3F may play a regulatory role in EIF3 (Mayeur, Reference Mayeur2001). One gene of transcription initiation factor TFIID subunit 12 (TAF12) was downregulated in the basal transcription factors pathway of R. chinensis infected with SM1. TAF12 is one of the subunits of TFIID, and the general transcription factor TFIID is very important for the initiation of mRNA gene transcription (Sanders et al., Reference Sanders, Garbett and Weil2002). The endoplasmic reticulum is a subcellular organelle. The unfolded protein enters the endoplasmic reticulum and uses the protein chaperones and catalysts of protein folding to form the final suitable conformation (Malhotra and Kaufman, Reference Malhotra and Kaufman2007). In our results, one gene in the protein processing in the endoplasmic reticulum pathway was downregulated. All these pathways are related to genetic information processing, and genetic information of organisms is associated with various life activities. In this study, SM1 infection downregulated some of the genes in these pathways, which may indicate the influence of SM1 infection on the genetic information processing of R. chinensis and on other life activities directly or indirectly.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S000748532300072X.
Acknowledgements
This research was supported by The Natural Science Foundation of the Jiangsu Higher Education Institutions of China [grant number 20KJA220003], Postgraduate Research & Practice Innovation Program of Jiangsu Province [grant number KYCX23_1223], and the Priority Academic Program Development Fund of Jiangsu Higher Education Institutions. No additional external funding was received for this study.
Competing interests
None.
