Introduction
Mythimna separata (Lepidoptera: Noctuidae; commonly called the northern armyworm, oriental armyworm, or rice ear-cutting caterpillar) is a migratory, omnivorous, and intermittent explosive pest that eats gramineous crops and can damage maize, rice, and wheat (Chen et al., Reference Chen, Pan, Waqas and Liu2020; Xu Reference Xu2021; Yang et al., Reference Yang, Mu, Wang and Chen2021). The first and second instar larvae of M. separata feed on plant leaves, causing holes, and the third instar and above (referred to as armyworms) gnaw on leaf edges, causing irregular nicks (Ma et al., Reference Ma, Sha, Zhang and Wang2019b). During the period of most intensive feeding (fifth and sixth instars), the larvae consume vast amounts of food; they nibble crop leaves to the stalk, resulting in crop yield reduction over large areas, or even a complete failure of the harvest. The food intake in this period accounts for >90% of that throughout the larval stage (Zhang et al., Reference Zhang, Yu, Jiang, Sun, Hui, Zheng, Li, Wang and Wu2018; Li et al., Reference Li, Liu and Bao2020). The average number of eggs laid by this high-fecundity pest is >1000, and it can mate and lay eggs multiple times (Wei, Reference Wei2012). In addition, adult M. separata have strong flight ability (Liu et al., Reference Liu, Liu, Xin, Liu and Feng2022). Migratory pests have the characteristics of attacking suddenly and explosively, which makes them difficult to monitor, forecast, prevent, and control. Effective pest management and control of M. separata is crucial. Chemical control is still the most commonly used method, but chemical use over the years has caused the ‘3R’ problem: pesticide residue, resistance, and resurgence (Wu, Reference Wu2017; Li et al., Reference Li, Sun, Yasoob, Tian and Zhang2021). To help solve these problems, the coordinated application of other control methods, including biological control, which has a lasting and stable control effect on pests with no toxic effects on the surrounding environment and organisms, should be improved (Ma et al., Reference Ma, Ma, Ren and Ma2021; Yang et al., Reference Yang, Mu, Wang and Chen2021). At present, the most commonly used biological control methods for M. separata are application of the fungus Beauveria bassiana and the bacterium Bacillus thuringiensis (Bt).
B. bassiana is a typical entomogenous fungus (i.e. a fungus that grows on insects), which has the characteristics of a wide host range, strong adaptability, and high pathogenicity; it has become one of the most widely used entomogenous fungi in biological pest control, and does not pollute the environment (Duan et al., Reference Duan, Jia, Xu, Long, Wang, Li and Zhao2023). B. bassiana can invade and infect insects through the body wall, digestive tract, trachea, and wounds. After B. bassiana enters the insect body, the hyphae begin to reproduce and enter the haemolymph of the insect body and produce mycelia. Secondary metabolites produced by the fungal pathogen begin to gradually destroy physiological processes of the insect body and attack the host defence system (Zhuang et al., Reference Zhuang, Zhao, Pei, Sun, Wang, Chen and Yu2019; Duan et al., Reference Duan, Jia, Xu, Long, Wang, Li and Zhao2023). Following this, the mycelia begin to grow vigorously in the insect body. Finally, after the death of the infected insect, the hyphae penetrate the epidermis to produce conidia, which spread to transmit the fungus (Li and Zhang, Reference Li and Zhang2005). Conidia of entomopathogenic fungi can survive for months to years in the natural environment, and they can grow on dead insects and continue to act as a source of infection, and thus their control effect on pests lasts for a long time (Hajek and Leger, Reference Hajek and Leger1994; Scholte et al., Reference Scholte, Knols, Samson and Takken2004).
Bt is a facultative anaerobic Gram-positive bacterium that is widely distributed in soil, water, air, and vegetation. Bt and its products have proved effective in controlling Lepidoptera, Diptera, Orthoptera, Coleoptera, and other pests (Roh et al., Reference Roh, Choi, Li, Jin and Je2007). Moreover, Bt and its products are non-toxic and harmless to non-target organisms, and thus Bt has become the most widely used biological insecticide in agriculture (Cui et al., Reference Cui, Huang, He, Xu, Hu and Cui2023). Bt produces many insecticidal substances in the process of growth and metabolism, including chitinase, phosphoesterase C (α-exotoxin), thuringiin (β-exotoxin), insecticidal crystal protein (δ-endotoxin), haemolysin, enterotoxin, and nutritive phase insecticidal protein (Cao and Ni, Reference Cao and Ni2014). The insecticidal crystal protein is the main toxin component of Bt as an insecticide, and is produced by the parasporal crystal of Bt (Schnepf et al., Reference Schnepf, Crickmore, Van Rie, Lereclus, Baum, Feitelson, Zeigler and Dean1998). δ-Endotoxins are divided into two groups: Cry proteins and Cyt proteins. Cry protein is considered to be the most effective component of Bt in insect control; it specifically binds to insect midgut receptors and is inserted into the cell membrane of midgut cells, and then ion pores are formed in the membrane of the midgut epithelial cells. At this stage, insects stop feeding and the intestinal mucosa is soon destroyed and cell permeability is affected, which results in insect cell autolysis and ultimately insect death. Cyt protein is specifically toxic to insects, causes haemolysis, and seriously affects the activity and physiological function of insect cells (Aronson and Shai, Reference Aronson and Shai2001; Song et al., Reference Song, Yang and Zhao2005; Li et al., Reference Li, Ma, Li, Yu and Zhang2012). However, control of M. separata by B. bassiana and Bt is slow, and some M. separata survive after being infected with these pathogens. We speculate that there is a resistance gene in M. separata, which helps them resist or even kill these pathogens.
Antimicrobial peptides (AMPs), small polypeptides of 12–60 amino acids, exist in all organisms, protect the host from infection, and serve as a major component of the innate immune system, playing a variety of functions (Wang, Reference Wang2020). Insect AMPs are synthesised by the fat body and released into the haemolymph. AMPs have a broad spectrum of antibacterial activity and they are not prone to bacterial resistance (Xue et al., Reference Xue, Feng and Li2023). They have rapid killing activity against bacteria, viruses, parasites, fungi, and even cancer cells (Zasloff, Reference Zasloff2002). AMPs can also promote the proliferation of immune cells and strengthen the function of phagocytes in mammals (Wang, Reference Wang2023). They have high thermal stability and water solubility, and acid and alkali resistance. The primary structure is conserved; the N-terminus of the peptide chain is rich in hydrophilic basic amino acids and carries a positive charge, while the C-terminal end of the peptide chain is rich in hydrophobic amino acids, which are often amidated and neutral. AMPs have an amphiphilic molecular structure (affinity for both water and lipids), which may be a prerequisite for efficient and rapid sterilisation by AMPs (Zhang and Hua, Reference Zhang and Hua2023).
On the basis of differences in structure and function, AMPs can be divided into cecropins, defensins, proline-rich peptides, and glycine-rich peptides. The main mechanisms of action of AMPs are action on the cell wall, action on the membrane, and action on a target in the cell. When acting on bacteria, AMPs first come in contact with the cell wall and can exert an antibacterial effect by destroying the cell wall structure. AMPs can bind to lipopolysaccharides and teichoic acids in the cell wall, thereby damaging the cell wall (Kang et al., Reference Kang, Meng, Wang and Zhang2021). AMPs fuse with the lipid membrane of cells to form pores. Because of the different physicochemical properties of the AMPs, the modes of pore formation are different, but they are interrelated. Hypotheses about pore-forming methods include the bucket plate model, carpet model, ring hole model, and polymerisation model (Li et al., Reference Li, Yao, Ren, Yang, Zou, Zhang and Lin2019). After entering the cell, AMPs act on nucleic acids, affect the synthesis and replication of DNA and RNA, and affect the synthesis of proteins. They can also act on protein molecules, cause changes in the structure of proteins, and make many enzymes inactive. Some AMPs can enter mitochondria, affect the activity of enzymes in the mitochondria, and cause disorder of metabolism (Feng et al., Reference Feng, Tan and Tan2019).
Cecropin B belongs to the cecropin class of AMPs, with a positive charge and two α-helices. It was first isolated from the haemolymph of Hyalophora cecropia (cecropia moth). Studies have shown that insect cecropins can inhibit Gram-positive bacteria, Gram-negative bacteria, and some fungi, and that cecropin B is the most active cecropin (De Lucca et al., Reference De Lucca, Bland, Jacks, Grimm and Walsh1998; Hu et al., Reference Hu, Wang, Guo, Li, Wang and He2013). Cecropin B has the ability to form specific amphipathic α-helices that target non-polar lipid in cell membranes. Upon membrane targeting, ion-permeable channels form resulting in cell depolarisation, irreversible cytolysis, and finally death (Boman, Reference Boman2003).
The present study was undertaken to investigate the effect of M. separata cecropin B on B. bassiana and Bt infection in M. separata. We show that cecropin B helps M. separata survive infection by these entomopathogenic microorganisms. Importantly, this research provides a molecular target for development of ‘green’ pesticides that inhibit the synthesis of AMPs in insects, so as to enable control of these pests through biological means.
Materials and methods
Experimental insects and pathogens
M. separata were established from a culture at the experimental base of Northeast Agricultural University, Harbin, China, in 2020, and maintained in the laboratory. The larvae were fed on fresh maize leaves every day in a climate-controlled chamber at 26 ± 2°C and relative humidity of 70 ± 5% with a light/dark period of 14 h/10 h. Adults were maintained under the same conditions, and fed on 5% honey water. Three days after eclosion, egg strips were hung in the cage, and female laid eggs on the egg strips. B. bassiana (Bb170428) (Yu et al., Reference Yu, Ren, Wang and Pei2023) and B. thuringiensis strain 46 (Bt46) used in this study were provided by Northeast Agricultural University, Harbin, China.
Cloning of the gene encoding M. separata cecropin B
M. separata larvae were collected and flash-frozen in liquid nitrogen for subsequent transcriptome sequencing, which was performed by Annoroad Gene Technology Company (Beijing, China). The sequences of AMP-encoding genes were identified based on public protein databases from NCBI. The total RNAs of larvae were extracted by using TRIzol reagent (1 ml 100 mg−1, Invitrogen, Carlsbad, CA, USA), and first-strand cDNA was synthesised using PrimeScript™ RT Master Mix (TaKaRa, Beijing, China). Reaction conditions were as follows: 37°C for 15 min and 85°C for 5 s. The reaction system was as follows: 500 ng RNA, 2 μl mix, and double-distilled water (ddH2O) were added to yield a total volume of 10 μl. All primers in this research were designed using Primer Premier 5.0 software and synthesised by Jiansu Gene (Heilongjiang, China) (table 1). The gene encoding M. separata cecropin B was amplified by polymerase chain reaction (PCR) using Premix Taq (TaKaRa). PCR conditions were as follows: 94°C for 5 min; 35 cycles of 94°C for 30 s, 44°C for 30 s, and 72°C for 1 min; 72°C for 10 min. The reaction system was as follows: 1 μl cDNA, 2 μl sense and anti-sense primers (10 μmol), 25 μl mix, and ddH2O were added to yield a total volume of 50 μl. PCR products were checked by agarose gel electrophoresis and purified by using a MiniBEST DNA Fragment Purification Kit (TaKaRa). The purity and concentration of the target genes were detected using a microspectrophotometer. The target gene was linked to vector pMD™18-T at 16°C overnight (reaction system: the amount of target genes was ≥3× that of the plasmid backbone, 5 μl solution I was added, and ddH2O was added to yield a total volume of 10 μl) (TaKaRa). The resulting plasmid was transformed into Escherichia coli DH5α. Positive clones were selected by conventional blue-white screening and confirmed by colony PCR. The reaction system was as follows: 0.5 μl bacterial culture, 5 μl mix, 1 μl sense and anti-sense primers (10 μmol) (sense primer: 18-T vector universal primer; anti-sense primer: anti-sense primer of the target gene), and ddH2O was added to yield a total volume of 10 μl (Premix Taq, TaKaRa). Reaction conditions were as follows: 95°C for 15 min; 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min; 72°C for 10 min. Plasmid isolated from bacteria positive for the target band was confirmed by sequencing.
Table 1. Primer sequences used in this study
Bioinformatic analysis
The obtained sequence of MscecropinB (the gene encoding M. separata cecropin B) was registered and deposited in the NCBI database (https://www.ncbi.nlm.nih.gov/). The open reading frame was predicted and translated using ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Insect cecropin B amino acid sequences were downloaded from the NCBI database for construction of a phylogenetic tree by the neighbour-joining method with best-fitting model in MEGA 7.0 software with 1000 bootstrap replicates. The isoelectric point, molecular weight, and protein hydrophobicity were predicted using the ExPASy Compute pI/MW online tool (https://www.expasy.org/). For signal peptide prediction, SignalP 5.0 online software was used (http://www.cbs.dtu.dk/services/SignaIP/). Protein transmembrane structure was predicted by TMHMM server v2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Protein secondary structure was predicted by SOPMA (http://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl). The tertiary structure of protein was modelled using SWISS-MODEL (http://swissmodel.expasy.org/interactive).
Spatiotemporal expression pattern of MscecropinB
Total RNA was extracted from insects at different developmental stages (egg, first-day first to sixth instar larvae, prepupae, pupae, and adults) and from various tissues (foregut, midgut, hindgut, salivary gland, Malpighian tubules, integument, fat body, and haemolymph), and then reverse-transcribed into cDNA for quantitative real-time PCR (qRT-PCR). The reaction system for reverse transcription was as follows: 1000 ng RNA, 4 μl mix, and ddH2O were added to yield a final volume of 20 μl. The expression levels of MscecropinB in different M. separata developmental stages and tissues were analysed with M. separata β-actin (GenBank: GQ856238) and GAPDH (GenBank: HM055756) as reference genes. qRT-PCR system was as follows: 2 μl cDNA, 6.8 μl ddH2O, 0.6 μl sense and anti-sense primers, and 10 μl THUNDERBIRD SYBR qPCR Mix (Toyobo, Shanghai, China). The reaction conditions were as follows: 95°C for 3 min; 35 cycles of 94°C for 10 s, 56°C for 30 s, and 72°C for 30 s. The data were recorded using Bio-Rad CFX Manager 3.1 software. Melting curves were checked to assess the specificity of the qRT-PCR. The qRT-PCR was performed in three technical replicates each with three biological replicates.
Determination of LC50 values of two pathogens towards M. separata
To prepare conidia of B. bassiana cultured on potato dextrose agar, spore suspension was dissolved in sterile water containing 0.1% Tween 80, and suspensions of 1 × 109, 1 × 108, 1 × 107, and 1 × 106 spores ml−1 were prepared for later use. We used a haemocytometer to count the filtered spore suspensions. Healthy M. separata third and fourth instar larvae of the same size were treated by the spray method (Wang et al., Reference Wang, Zeng, Li, Wang and Wu2012), and spore suspensions of different concentrations were uniformly sprayed on the surface of the larvae using a 30 ml hand-held plastic aerosol sprayer (JS-0119). The blank control was sterile water containing 0.1% Tween 80. To prepare Bt46 cultured in Luria–Bertani medium, the bacterial suspension was dissolved in sterile water, and the cells were evenly dispersed. Then, bacterial suspensions of 1 × 1010, 1 × 109, 1 × 108, and 1 × 107 cells ml−1 were prepared for later use. Fresh maize leaves were treated with the bacterial suspension by the leaf soaking method (Yu et al., Reference Yu, Ren, Wang and Pei2023) for 1–2 min. They were then dried and fed to healthy M. separata third and fourth instar larvae of the same size. The blank control was fresh maize leaves soaked in sterile water.
Each group included 20 larvae, and the experiments were repeated three times. After infection, larvae were maintained at constant temperature (26 ± 2°C) and humidity (70 ± 5%), and fed fresh, untreated maize leaves. Over 10 days of continuous observation, the number of living and dead insects was counted. Regression statistical analysis was performed by using DPS7.5 software to calculate LC50 values.
Effect of insect population density on MscecropinB expression
Immediately after egg hatching, M. separata were transferred to feeding boxes (0.5 litres) at a density of 2 larvae per box, 4 larvae per box, 8 larvae per box, 16 larvae per box, and 32 larvae per box. Each experiment was repeated three times. The larvae were fed until the first day of the fourth instar whereupon total RNA was extracted, and then reverse-transcribed into cDNA for qRT-PCR analysis.
Effect of infection on MscecropinB expression
Healthy third and fourth instar larvae were treated with the LC50 of B. bassiana spore suspension or Bt46 bacterial suspension for the corresponding instar; the treatment methods were the same as described in the section ‘Determination of LC50 values of two pathogens towards M. separata’. Each group included 25 larvae and the experiment was repeated three times. The B. bassiana treatment group was observed once daily for 5 days; for Bt46 treatment, samples were collected at 12, 24, 36, 48, and 70 h. At each time point, larvae were quickly frozen in liquid nitrogen. Total RNAs were extracted, reverse-transcribed into cDNA, and then qRT-PCR was performed to detect MscecropinB expression.
Fourth instar larvae were treated with the LC50 of B. bassiana after 3 days. Bt46-treated larvae were fed after 48 h with maize leaves soaked in bacterial suspension at the LC50. Control larvae were treated with 0.1% Tween 80 in sterile water and sterile water only, respectively. The larvae were dissected to obtain the fat bodies and haemolymph. Each group contained 25 larvae, and each experiment was repeated three times. The total RNA extracted from tissues was reverse-transcribed into cDNA and then qRT-PCR was performed.
siRNA synthesis and interference efficiency measurement
A siRNA sequence for MscecropinB and a negative control siRNA were synthesised by Harbin Ruibo Xingke Biotechnology (table 2). The siRNAs were diluted into diethylpyrocarbonate-treated water to a final concentration of 20 μM. First-day fourth instar larvae were injected with 2 μl siRNA for MscecropinB or the negative control siRNA into the internode membrane of the last two to three segments of the abdomen using a 10 μl microinjector (needle tip outer diameter: 0.5 mm). The larvae were fed normally after the injection. Each group included 20 larvae, and the experiments were repeated three times. Larvae were collected 3, 6, 12, 24, 48, 72, and 96 h after injection. Total RNAs were extracted from the larvae and then reverse transcribed to cDNA for analysis of MscecropinB expression by qRT-PCR.
Table 2. Sequences of siRNAs used in this study
Effects of RNA interference (RNAi) on growth and development of M. separata
The death rate of M. separata larvae 12, 24, 48, and 96 h after interference was recorded, and the subsequent pupation rate, pupal weight, emergence rate, and egg-laying capacity of surviving larvae were observed and calculated. The pupal weight on the second day was determined using an Electronics Weighing Scales (One Over Ten-thousand Analytical Balance). Each treatment group contained 50 larvae, and the experiments were repeated three times.
Synergistic effects of pathogens and RNAi on MscecropinB expression
Healthy third instar larvae were treated with B. bassiana, and fourth instar larvae were treated with Bt46, at the LC50 values, respectively. Thirty-six hours after infection, all larvae were treated by siRNA. Controls were treated with B. bassiana or Bt46 and injected with the negative control siRNA. Each treatment group included 25 larvae, and the experiments were repeated three times. Larvae were collected 3, 6, 12, 24, 48, 72, and 96 h after the injection of siRNA for analysis of MscecropinB expression. For growth and development research, each treatment group included 50 larvae. The death rate of the treated M. separata larvae was recorded 12, 24, 48, and 96 h after interference, and the subsequent pupation rate, pupal weight, emergence rate, and egg-laying capacity of surviving larvae were observed and calculated. The pupal weight on the second day was determined by using a balance (One Over Ten-thousand Analytical Balance).
Statistical analysis
The relative expression of genes was calculated by the 2−ΔΔCt method (Livak and Schmittgen, Reference Livak and Schmittgen2001). Differences between multiple treatment groups were analysed by one-way analysis of variance, and differences between treatment and control groups were analysed using the t-test in software GraphPad Prism 9.3.0 and IBM SPSS Statistics 23. Duncan's multiple comparison method was used to analyse the significance of differences (P < 0.05). Graphs were drawn in GraphPad Prism 9.3.0 software. Simple numerical operations were performed using Microsoft Excel 2010.
Results
Cloning of MscecropinB
The gene encoding M. separata cecropin B (referred to here as MscecropinB) was amplified using fourth instar larvae as the template, as shown by polyacrylamide gel electrophoresis (fig. S1). A recombinant plasmid of MscecropinB was successfully constructed and transformed into E. coli DH5α (fig. S2). After blue-white screening, white colonies were randomly selected and checked by colony PCR detection. Colonies that presented a single band of the expected amplicon size (approximately 200 bp) were sent for verification of the MscecropinB expression plasmid by DNA sequencing (fig. S3). The amplified MscecropinB sequence was consistent with the sequence from the transcriptome.
Bioinformatic analysis of MscecropinB
The MscecropinB-encoding cDNA (GenBank: OQ914365) is 765 bp long, with an open reading frame of 192 bp. The encoded protein (63 amino acids) (fig. 1) has a predicted isoelectric point of 10.15 and molecular weight of 6.79 kDa. Amino acids 1–22 at the N-terminus form a predicted signal peptide. Residues 7–24 form a predicted transmembrane domain. In the predicted secondary structure of MscecropinB, α-helix accounts for 63.49%, random coil for 20.63%, extended chain for 12.70%, and β-angle for 3.17%. The tertiary structure of MscecropinB was predicted based on the secondary structure of Bombyx mori cecropin E (PDB: Q308S4.1.A) as a template; the confidence was 74.9%, and the sequence coverage was 98%. As shown in fig. S4, MscecropinB is predicted to have two α-helices, which is consistent with cecropin structure studies. Multiple-sequence alignment with cecropin B sequences from other insects (fig. S5) showed that they are highly conserved. In a phylogenetic tree constructed for MscecropinB and some insect cecropin B sequences, MscecropinB was clustered with the cecropin B of Trichoplusia ni (ABV68872.1) and Spodoptera exigua (AKJ54503.1) (fig. 2), indicative of a relatively close relationship. The next closest relationships were with the cecropins B from Danaus plexippus plexippus (XP_032523475.1), H. cecropia (AAA29184.1), and Antheraea pernyi (QDH09591.1), which are again closely related species to M. separata. M. separata cecropin B was distant in the tree from cecropins B from mosquitoes and fruit flies.
Figure 1. cDNA and peptide sequences of M. separata cecropin B (MscecropinB). The signal peptide is shaded in grey, the transmembrane domain is underlined, and the two predicted helices are boxed.
Figure 2. Phylogenetic analysis of cecropin B sequences from Lepidoptera and Diptera. Ap: A. pernyi; Hc: H. cecropia; Dp: D. plexippus; Bm (XP028043560.1): Bombyx mandarina; Bm (AAC60501.1): B. mori; Se: S. exigua; Tn: T. ni; Aa (AAD37702.1): Aedes albopictus; Aa (XP050087620.1): Anopheles aquasalis; Db: Drosophila busckii; Ds (XP016938596.1): Drosophila suzukii; Dm: Drosophila melanogaster; Ds (EDW53499.1): Drosophila sechellia; Ds (EDX14991.1): Drosophila simulans.
Spatiotemporal expression pattern of MscecropinB
qRT-PCR was used to analyse the expression of MscecropinB in M. separata at different developmental stages. The results (fig. 3A) (F(9, 80) = 9943.4358; P < 0.05) showed that MscecropinB was expressed in all developmental stages, with the highest expression in the prepupal stage, followed by the pupal stage. The lowest MscecropinB expression level was in the egg stage. The MscecropinB expression level in the prepupal stage was 123.34 times that in the egg stage, and 1.64 times that in the pupal stage. In the larval stage, the highest expression level was in the fourth instar, 15.63 times that in the egg stage.
Figure 3. Spatiotemporal MscecropinB expression pattern in different developmental stages (a) and in diverse tissues (b) of M. separata. Mg, midgut; Sg, salivary gland; Ig, integument; Mt, Malpighian tubules; Fg, foregut; Fb, fat body; Hg, hindgut; Hl, haemolymph. Different lowercase letters above the columns indicate significant differences (P < 0.05).
To explore the expression patterns of MscecropinB in different tissues, we dissected fourth instar larvae and obtained eight tissues: foregut, midgut, hindgut, salivary gland, Malpighian tubules, integument, fat body, and haemolymph. MscecropinB was most highly expressed in the fat body, followed by the haemolymph; the lowest expression was in the foregut (fig. 3B) (F(7, 64) = 2641.6824; P < 0.05). MscecropinB expression in the fat body was 46.63 times that in the foregut, and 1.43 times that in the haemolymph.
Determination of LC50 of two pathogens towards M. separata
Larvae were treated with four different concentrations of B. bassiana or Bt46 suspension for 10 days, and the mortality rate was observed for each treatment (fig. 4). The LC50 value of B. bassiana towards third instar M. separata was 1.07 × 107 spores ml−1, the virulence equation was y = 2.3678 + 1.3363x (r = 0.9422, y: mortality, x: concentration). The LC50 of B. bassiana towards fourth instar M. separata was 1.08 × 108 spores ml−1 (y = 1.7174 + 1.0937x, r = 0.9528). The LC50 of Bt46 towards third instar M. separata was 1.24 × 108 cells ml−1 (y = 1.7583 + 1.0574x, r = 0.9411). The LC50 of Bt46 towards fourth instar M. separata was 1.13 × 109 cells ml−1 (y = 0.4551 + 1.1298x, r = 0.9448).
Figure 4. Third instar M. separata larvae infected by B. bassiana and fourth instar larvae infected by Bt46.
The effect of population density on expression of MscecropinB
The expression level of MscecropinB was significantly impacted by the population density. As shown in fig. 5 (F(4, 41) = 1074.757; P < 0.05), when M. separata were exposed to high-density conditions, the expression of MscecropinB was significantly increased. The expression of MscecropinB at 32 larvae per box was 11.63 times that for 2 larvae per box. However, there was no significant difference in larval MscecropinB expression in several low-density conditions, indicating that there is a threshold insect population density for the regulatory effect of population density on MscecropinB expression.
Figure 5. Expression of MscecropinB under different conditions of larval density. Different lowercase letters above the columns indicate significant differences (P < 0.05).
Pathogens upregulated the expression of MscecropinB
There were significant differences in the expression of MscecropinB at different times after infection of third and fourth instar M. separata with the LC50 of B. bassiana (fig. 6A, B) (P < 0.05). During the second day after B. bassiana infection, the MscecropinB expression in third instar larvae was 9.49 times that in control. The expression levels on the third, fourth, and fifth days of infection were 3.52, 3.26, and 2.58 times those in the control, respectively. In fourth instar larvae, MscecropinB showed significant differential expression compared with controls from the first day of infection. The expression reached 9.91 and 7.36 times that in the controls on days 3 and 4, respectively.
Figure 6. Expression of MscecropinB in third (a) and fourth (b) instar M. separata infected with B. bassiana. Healthy third and fourth instar larvae were treated with B. bassiana at the LC50 values. Controls were treated with sterile water containing 0.1% Tween 80 (*P < 0.05; **P < 0.01; ns, P > 0.05).
There were significant differences in the expression of MscecropinB at different times after infection of third and fourth instar M. separata with the LC50 of Bt46 (fig. 7A, B) (P < 0.05). At 36 and 48 h after Bt46 infection, the MscecropinB expression in third instar larvae was 7.63 and 8.88 times that in the controls, respectively. In fourth instar larvae, the MscecropinB expression was significantly different compared with controls 12 h after infection. The expression reached 5.40 and 9.66-times that in controls at 36 and 48 h, respectively.
Figure 7. Expression of MscecropinB in third (a) and fourth (b) instar M. separata infected with Bt46. Healthy third and fourth instar were treated with Bt46 at the LC50 values. Controls were treated with sterile water (*P < 0.05; **P < 0.01; ns, P > 0.05).
Because different pathogens take effect at different times, fourth instar larvae treated with the LC50 of B. bassiana (3 days) and Bt46 (48 h) were dissected to obtain the fat bodies and haemolymph. Compared with controls, qRT-PCR showed that MscecropinB expression in the fat body and haemolymph increased 5.62- and 2.20-fold after B. bassiana infection, and 2.63- and 2.19-fold after Bt46 infection (fig. 8) (P < 0.05).
Figure 8. Expression of MscecropinB in fat body and haemolymph after infection by pathogens (*P < 0.05; **P < 0.01; ns, P > 0.05).
RNAi efficiency
To confirm the efficiency of RNAi, fourth instar larvae were injected with equal amounts of siRNA for MscecropinB or negative control siRNA. The MscecropinB expression level was analysed 3, 6, 12, 24, 48, 72, and 96 h after injection. At 6, 12, and 24 h, the expression of MscecropinB decreased by 80.01, 96.52, and 81.09%, respectively, in the treatment group compared with the negative control group (fig. 9) (P < 0.05). There was no significant interference effect at 3, 48, or 96 h. Therefore, the time of maximal interference efficiency was about 12 h after injection.
Figure 9. Efficiency of RNAi with MscecropinB (*P < 0.05; **P < 0.01; ns, P > 0.05).
Effects of RNAi on growth and development of M. separata
The silencing of MscecropinB had no effect on the mortality of the fourth instar M. separata for up to 96 h (table 3). The RNAi-treated M. separata were further observed for pupation rate, pupal weight, emergence rate, and single female egg production. The interference with MscecropinB had no effect on the pupation rate or pupal weight, but the emergence rate has decreased by 22.22%, and the single female egg production has decreased by about 272 eggs.
Table 3. Mortality, growth, and development of RNAi-treated M. separata
Note: Data are presented as the mean ± SE. SiNC, negative control RNAi.
*P < 0.05, **P < 0.01 (t-test).
Synergistic effect of pathogens and RNAi on MscecropinB
qRT-PCR results showed that RNAi treatment significantly suppressed MscecropinB expression 3–24 h after interference in B. bassiana-infected third instar larvae, and 3–48 h after interference in Bt46-infected fourth instar larvae (fig. 10) (P < 0.05). Comparing the B. bassiana-infected group with the control group (infected, but not subjected to siRNA), the MscecropinB expression levels at 3, 6, 12, and 24 h were decreased by 69.92, 82.22, 80.66, and 90.16%, respectively. In the Bt46 plus RNAi treatment group, the MscecropinB expression levels at 3, 6, 12, 24, and 48 h were decreased by 47, 74.61, 85.76, 90.35, and 50.69%, respectively, compared with controls (infected, but not subjected to siRNA). It can be seen that in infected larvae, the RNAi was most effective at about 24 h after injection.
Figure 10. Analysis of MscecropinB expression after pathogen infection and RNAi treatment. Infection with (a) B. bassiana and (b) Bt46 (*P < 0.05; **P < 0.01; ns, P > 0.05). Healthy third instar larvae were treated with B. bassiana, and fourth instar were treated with Bt46 at the LC50 values, respectively. Thirty-six hours after infection, all larvae were treated by siRNA. Controls were treated with B. bassiana or Bt46 and injected with negative control siRNA.
Effect of RNAi of MscecropinB on susceptibility of M. separata to pathogens
Changes in physiological parameters were observed in pathogen plus RNAi-treated third and fourth instar M. separata. The mortality rates in both treatment groups (B. bassiana + siRNA or Bt46 + siRNA) were significantly higher than that of the control group (infected but not treated by siRNA) after interference for 24 h (table 4). After 96 h of interference, the death rate in the B. bassiana group was 19.34% higher than that in the control group; the death rate in the Bt46 group was 22% higher than that in the control. The pupation rate, emergence rate, and single female egg production were also significantly influenced by RNAi treatment of M. separata infected with either B. bassiana or Bt46, while the pupal weight was unaffected. The pupation rate in the B. bassiana plus RNAi-treated group was 59.17% lower, the emergence rate was 31.11% lower, and the single female egg production was about 397 eggs per female lower than those in the control group (all P < 0.05). In the Bt46 plus RNAi-treatment group, the pupation rate was 40.83% lower, the emergence rate was 18.89% lower, and the single female egg production was about 347 eggs per female lower than those in the control. The results showed that interference with MscecropinB expression increased the sensitivity of M. separata to B. bassiana and Bt46, and the synergism between RNAi and pathogen infection markedly affected the growth and development of M. separata.
Table 4. Mortality of M. separata treated with pathogens and RNAi
Note: Data are presented as the mean ± SE. SiNC, negative control RNAi.
*P < 0.05, **P < 0.01 (t-test).
Discussion
AMPs are important effector molecules of insect innate immunity and are involved in many defence processes, such as neutralising endotoxins, regulating immune responses, and killing bacteria, viruses, and fungi (Singh et al., Reference Singh, Papareddy, Kalle, Schmidtchen and Malmsten2014). AMPs show broad-spectrum antibacterial activities. In this experiment, a new M. separata AMP, cecropin B, was discovered and the encoding gene was successfully cloned. Bioinformatic analysis showed that MscecropinB is a positively charged small peptide with amphiphilicity and two amphiphilic α-helices, which corresponds with previous structure research (Brogden et al., Reference Brogden, De Lucca, Bland and Elliott1996; Schittek et al., Reference Schittek, Hipfel, Sauer, Bauer, Kalbacher, Stevanovic, Schirle, Schroeder, Blin and Meier2001). The positive charge and amphiphilic nature of AMPs endow them with capacity to efficiently and quickly participate in immune reactions. They act on the cell membrane or cell wall of pathogenic microorganisms, form pores to destroy the ion balance, and kill the microorganism (Singh et al., Reference Singh, Papareddy, Kalle, Schmidtchen and Malmsten2014; Holdbrook et al., Reference Holdbrook, Singh, Choong, Petrlova, Malmsten, Bond, Verma, Schmidtchen and Saravanan2018; Li et al., Reference Li, Yao, Ren, Yang, Zou, Zhang and Lin2019; Zhang and Hua, Reference Zhang and Hua2023). MscecropinB has a signal peptide sequence and transmembrane domain, indicating that it is a secreted protein. Tissue-specific expression analysis revealed that the highest expression of MscecropinB was in the fat body, followed by the haemolymph. Studies have shown that several classes of AMPs are synthesised in fat bodies, enter the haemolymph of insects, and react to pathogens throughout the body (Uvell and Engstrom, Reference Uvell and Engstrom2007; Hwang et al., Reference Hwang, Lee, Kim, Bang, Yun and Kim2009). Our results show that MscecropinB demonstrates these characteristics.
Previous studies have found that the AMP gene CPAlo-13768 is highly expressed in adult and fourth instar larvae of Conogethes punctiferalis (yellow peach moth), and highly expressed in the fat body (Dong, Reference Dong2021) which is consistent with MscecropinB expression patterns. Numerous field studies have shown that fourth instar M. separata larvae show significant resistance to insecticides and pathogens (Dong et al., Reference Dong, Liu, Yue, Qiao, Chu, Wang and Zhang2014; Zhao et al., Reference Zhao, Su, Li, Li, Xu, Cheng, Wang and Wu2018). Analysis of the expression pattern of MscecropinB in the larval stage in the present study suggests that the high tolerance of fourth instar M. separata to insecticides and pathogens is most likely associated with xenobiotic metabolism. In this research, MscecropinB was highly expressed during the prepupal stage followed by the pupal stage, suggesting MscecropinB has a potential function in increasing innate immune responses and adaptability to external adverse effects (Ma et al., Reference Ma, Abbas, Zhang, Hu, Xu, Lang, Kausar, Yang and Cui2019a).
The expression of MscecropinB was upregulated by increased M. separata population density. Yuan (Reference Yuan2022) studied cAMP response element-binding protein (CREB) in M. separata and found that with the gradual increase of larval density, the expression level of MsCREB-B increased significantly. After RNAi with MsCREB-B, the expression levels of AMPs (cecropin, lebocin, and gloverin) were significantly downregulated, as was the survival rate of the treatment group after Bt infection. Jing (Reference Jing2021) found that the expression levels of cecropin and defensin were significantly higher in larvae raised at high density. After RNAi with AMPs, the larval survival rate after Bt infection decreased significantly. In summary, M. separata larvae exhibit typical density-dependent disease resistance. High density induces upstream genes that affect the expression of AMPs, enhancing the disease resistance of M. separata.
Previous research has found that the expression of Mdatta2 in Musca domestica (housefly) larvae was significantly upregulated 3 and 6 h after stimulation by E. coli and Staphylococcus aureus, which proved that Mdatta2 plays a role in the immune response to these bacteria (Liu et al., Reference Liu, Sun, Tang and Wang2011). In the present study, B. bassiana and Bt46 were used to infect third and fourth instar M. separata larvae, respectively. The expression of MscecropinB was significantly upregulated after infection by B. bassiana or Bt46. Comparing the regulation of MscecropinB by B. bassiana in third and fourth instar larvae, MscecropinB expression was significantly upregulated on the first day of infection in fourth instar larvae, but a day later in third instar larvae. The expression analysis indicated that the immune response in fourth instar larvae was stronger and faster than that in third instar larvae. The difference in the regulatory effect between the two instars decreased over an observation period of 5 days. The response trends of MscecropinB were similar between Bt46 and B. bassiana treatment, in terms of timing. It takes time for pathogens to enter the insect body from the insect wall (Ferreira and de Freitas Soares, Reference Ferreira and de Freitas Soares2023). MscecropinB expression responded more rapidly in fourth instar larvae than in the third instar, and thus the fourth instar M. separata had stronger resistance to the pathogens than the third instar larvae. These results indicate that MscecropinB plays an important role in the immune process of M. separata against B. bassiana and Bt46.
Chu (Reference Chu2022) found that the kinases Spätzle and Relish are key in Toll and immune deficiency signalling pathways of AMP secretion. After 6 h of interference with Spätzle and Relish, the mortality rate of Monochamus alternatus (Japanese pine sawyer) after B. bassiana infection was significantly increased, and the relative expression levels of cecropin and defensin were significantly decreased. The effect of RNAi on gene expression is time-sensitive; in the present study, the maximal period of interference efficiency with MscecropinB was about 12 h after injection. After an extended period, the expression levels of the genes that have been interfered with return to normal levels. We found that interference with MscecropinB directly impacted the emergence rate and egg production of M. separata. It might be that interference with MscecropinB weakened the resistance of M. separata to the external environment. AMPs are important molecules in insect innate immunity and participate in defence processes. Insects lack adaptive immunity and their survival depends on the production of AMPs, which enables them to establish strong defence mechanisms to combat infections (Zhao et al., Reference Zhao, Zhang, Pan and Jiang2019). Interference with MscecropinB significantly increased the mortality rate of M. separata after B. bassiana or Bt46 infection. The pupation rate, emergence rate, and single female egg production were also significantly reduced in the treatment group. This indicates that MscecropinB plays an important role in the resistance of M. separata to infection by B. bassiana or Bt46. Controlling this pest by inhibiting the synthesis of MscecropinB combined with pathogen treatment will have excellent results.
Conclusions
MscecropinB is a small alkaline peptide. The encoding gene was expressed in all developmental stages of M. separata; the highest expression level was in the prepupal stage. In fourth instar larvae, the highest expression level was in the fat body among the tested tissues. Infection of M. separata with B. bassiana or Bt46 induced the expression of MscecropinB. After MscecropinB silencing using RNAi, M. separata showed increased sensitivity to B. bassiana and Bt46, which improved the insecticidal efficiency of these pathogens. The rates of pupation, emergence, and single female egg laying were also significantly reduced by RNAi with MscecropinB following B. bassiana or Bt46 infection. The results showed that MscecropinB plays an important role in the immune processes of M. separata defending against infection by B. bassiana and Bt46. Our findings indicate that the biocontrol effect of entomopathogenic microorganisms can be improved by changing the internal environment of the target pest. This article promotes the development of green pesticides for pest control that inhibit the synthesis of AMPs in insects.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485324000130.
Acknowledgements
This research was funded by the National Key Research and Development Program of China (2023YFD1401000). We thank Professor Yu Hongchun of Northeast Agricultural University for his gift of Beauveria bassiana and Bacillus thuringiensis. We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the language of a draft of this manuscript.
Author contributions
Yi-Xiao Wang: conceptualisation, resources, software, data curation, methodology, supervision, writing – original draft, and writing – review and editing. Hong-Jia Yang: methodology, data curation, software, and resources. Wei-Jia Zhang: data curation, investigation, and writing – review and editing. Xiao-Hui Zhao, Meng-Yao Cui and Jin-Bo Zhang: validation, resources, and data curation. Xin-Xin Zhang: conceptualisation, methodology, supervision, and writing – review and editing. Dong Fan: conceptualisation, methodology, supervision, writing – review and editing, and funding acquisition. All authors have read and agreed to the published version of the manuscript.
Competing interests
None.
