Introduction
Protease inhibitors exist widely in all organisms and play an important role in physiological processes such as growth and development (Gubb et al., Reference Gubb, Sanz-Parra, Barcena, Troxler and Fullaondo2010). Protease inhibitors can be divided into four categories: serine protease inhibitors (SPIs), cysteine protease inhibitors, metalloproteinase inhibitors and aspartic protease inhibitors (Getti and Peter, Reference Getti and Peter2002), among which SPIs is a class of structurally conserved enzyme activity regulators, which can inhibit target enzymes to participate in important metabolic processes of life, such as anticoagulation, reproduction, inhibition of excessive phagocytosis, tissue remodelling, antibacterial and immune responses (Zhao et al., Reference Zhao, Tao and Pan2016). Insects produce innate immune response when pathogens infect, which requires a variety of serine proteases (SPs) to ensure the transmission and expansion of immune signals. SPIs regulate the activity of SPs to make the immune response rapid and intense, and limit it to a certain extent and range to protect from parasitic organisms (Irving et al., Reference Irving, Pike, Lesk and Whisstock2000; Ferrandon et al., Reference Ferrandon, Imler, Hetru and Hoffmann2007).
Kazal-type serine protease inhibitor (KaSPI) is the most conservative class of SPIs, and its extensive biological functions make it a research hotspot (Laskowski and Kato, Reference Laskowski and Kato1970; Rimphanitchayakit and Tassanakajon, Reference Rimphanitchayakit and Tassanakajon2010; Hoef et al., Reference Hoef, Breugelmans, Spit, Simonet, Zels and Broeck2013). KaSPI has been identified from many insects, including Drosophila melanogaster (Niimi et al., Reference Niimi, Yokoyama, Goto, Beck and Kitagawa1999), Apis cerana (Kim et al., Reference Kim, Lee, Zou, Wan, Choi, Yoon, Kwon, Je and Jin2013), Aedes aegypti (Torquato et al., Reference Torquato, Lu, Martins, Tanaka and Pereira2017), Pachycrepoideus vindemiae (Yang et al., 2020), etc. It was expressed in the foregut and midguts of Bombyx mori, and was positively regulated by 20-hydroxyecdysone (20E) (Zheng et al., Reference Zheng, Chen and Nie2007; Gan et al., Reference Gan, Liu and Li2016). In addition, the messenger RNA (mRNA) levels of KaSPI in B. mori (Zhao et al., Reference Zhao, Dong, Duan, Wang, Wang, Li, Xiang and Xia2018) and Antheraea pernyi (Wang et al., Reference Wang, Qiu, Qian, Zhu and Liu2014) were up-regulated under the attack of bacteria and virus, but the degree and time of up-regulation were different. Interestingly, the KaSPI gene may be involved in the reproductive process of insects. The inhibitor gene Greglin was isolated from the yolk membrane of Locusta migratoria. The study showed that the inhibitor was induced by the moulting hormone, and was expressed at a very high level during vitellogenesis. Knockout of Greglin in adults could accelerate the degradation of SP substrate and significantly reduce the level of Greglin protein in haemolymph and ovary. This hinders the maturation of oocytes and regulates the reproductive process of insects (Guo et al., Reference Guo, Wu, Yang, Cai, Zhao and Zhou2019).
Mythimna separata seriously endangers food security of its migratory, clustering, polyphagous and fulminant characteristics. In this study, the cDNA sequence of the MsKaSPI was cloned and the effects of MsKaSPI silencing on the growth and development of M. separata and the sensitivity to B. bassiana were studied. By exploring the role of KaSPI in insect growth, development and pathogen infection, this research lays the foundation for the follow-up study of the physiological functions of KaSPI in insects and the use of pathogenic microorganisms for biological control of agricultural pests.
Materials and methods
Experimental insect and strain
Adults of M. separata were collected from a light trap at the experimental field of Xiangfang Experimental Base of the Northeast Agricultural University, Harbin, China. The adults were raised in net cages and routinely fed with 5% honey water. After copulation, the females laid eggs in folded plastic ropes. Newly hatched larvae were transferred to plastic boxes and fed with fresh corn leaves in an artificial climate box at 25 ± 2°C and 70% relative humidity, with a photoperiod of 14:10 (light: dark). Beauveria bassiana was provided by the Agricultural Insect and Pest Control Group, Northeast Agricultural University. It was activated on potato dextrose medium (PDA) medium for 1–2 generations, inoculated on PDA medium and cultured in 25°C incubator for 15 days. After the strains are fully sporulated, they are stored in a refrigerator at 4°C for later use.
Acquisition and sequence analysis of MsKaSPI
The first to sixth-instar larvae, pupae and adults of M. separata were collected in a 1.5 ml Eppendorf tube, and transcriptional sequencing was carried out by Annuoyoda Genome Technology Company (Beijing, China). MskaSPI of M. separata was screened from transcriptome database. After homology comparison on NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), full-length primers (table S1) were designed using Primer Premier 5.0. Polymerase chain reaction (PCR) amplification procedure was as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 4 min, and a final extension at 72°C for 10 min. PCR products were purified and sequenced for confirmation by comparing with the transcriptome sequences. The MsKaSPI and other insect KaSPIs from NCBI database were used to construct a phylogenetic tree by using MEGA 5.1. The neighbour-joining method with arithmetic averages was used, and a bootstrap analysis of 1000 replications was performed to evaluate the branch strengths of the phylogenetic tree.
Analysis of MsKaSPI expression patterns
Total RNAs were extracted from eggs, each instar larvae, pupae, female and male adults of M. separata as well as seven different tissues of third-day third-instar larvae using Trizol® reagent (Invitrogen, Carlsbad, CA, USA). The extracts were treated with 1% diethylpyrocarbonate to prevent ribonuclease contamination. An Implen NanoPhotometer® P300 was used to determine the RNA concentration, and placed in an −80°C refrigerator for later use. Complementary DNA (cDNA) was synthesised using ReverTra Ace qPCR RT Kit (TOYOBO, Shanghai, China) according to the manufacturer's instructions.
The expression patterns of MsKaSPI at different developmental stages and in different tissues were analysed by reverse-transcription quantitative real-time PCR (RT-qPCR). β-actin and GAPDH were used as reference genes. The primers were listed in table S1. The qRT-PCR procedure was as follows: initial denaturation of the cDNA at 94°C for 1 min, followed by 40 cycles of 95°C for 30 s, 59°C for 30 s and 72°C for 10 min. A melting curve was constructed to confirm the amplification specificity of qRT-PCR.
Induction of MsKaSPI by 20-hydroxyecdysone
20E solutions were obtained at concentrations of 5, 10, and 20 μg μl−1 by dissolving 5, 10, and 20 mg 20E (A506554; Sangon Biotech, Shanghai, China) in 100 μl 1% dimethylsulfoxide (DMSO) then adding double-distilled water to 1 ml, respectively. The concentration was set according to Beckstead et al.'s (Reference Beckstead, Lam and Thummel2007) and Li et al.'s (Reference Li, Ren and Yan2016) study. For each concentration, three replicates with each 45 larvae were performed. The larvae in the experimental groups were injected with 2 μl of 20E at different concentrations using 10 μl Micro Sample Syringe (F519160; Sangon Biotech), while an equal volume of 0.1% DMSO solution was injected in the control group. After 6, 12, 24, 48 and 72 h, larvae were sampled and total RNAs were extracted. The expression levels of MsKaSPI injected with different 20E concentrations were quantified using qRT-PCR.
Infection of B. bassiana and determination of MsKaSPI expression response
We collected the spores of B. bassiana cultured on the PDA medium, washed the conidia with 0.1% Tween-80 sterile water, stirred evenly with a magnetic agitator and filtered the hyphae with four layers of gauze. Then, the spore suspensions with concentrations of 4 × 109, 4 × 108, 4 × 107, 4 × 106 and 4 × 105 spore ml−1 were prepared for use, respectively. The third-day third-instar larvae of M. separata were treated by spray method. Five millilitres of each spore suspensions were evenly sprayed on the surface of third-instar larvae, and reared in culture box containing absorbent paper. The fresh feed and absorbent paper in the box were replaced every 24 h, and 0.1% Tween 80 sterile water was used as the control group. We observed continuously for 15 days and calculated the LC50 and the spore suspension with the LC50 value was 4.75 × 108 spores ml−1 according to the pre-experiment. Eighteen insects were retained in each treatment; after 3, 6, 12, 24, 48 and 72 h, half of the larvae were collected to detect the expression of MsKaSPI treated with B. bassiana at different time points by qRT-PCR, and the other half were used for the determination of the MsKaSPI protein content in subsequent infection.
Functional analysis of MsKaSPI by RNA interference
For RNA interference, specific primers of siMsKaSPI (accession No: MN931651) and the negative control (siNC) were designed and synthesised by Shanghai Jima Company (shown in table S1). When designing siMsKaSPI and siNC, do not target the 5′ and 3′ non-coding regions, and the GC content of the sequence should be around 30−60%. Starting from the AUG start codon of the transcript (mRNA), search for the ‘AA’ binary sequence and record its 19 base sequences at the three ends as potential siRNA targets. Both the sense and antisense chains are designed using these 19 bases (excluding AA repeats). Compare the selected sequences in the public database to ensure that the target sequence has no homology with other genes using BLAST (www.ncbi. nlm. nih. gov/BLAST/). Selecting suitable target sequences for siRNA synthesis can directly provide 21 base sequences starting with AA. The negative control (siNC) is a gene sequence that has the same composition as the selected siRNA sequence, but has no significant homology with the target gene and has no biological effect in M. separata.
The third-day third-instar larvae of M. separata were selected, and 2 μl of 20 μmol l−1 siMsKaSPI was injected into the 2–3 segments on the abdomen side of larvae M. separata, while the control group was injected with the same amount of siNC. After injection, the larvae were reared as above and collected at 3, 6, 12, 24 and 48 h. These RNA interference (RNAi)-treated larvae were used for interference efficiency detection, determination of MsKaSPI content and recording the spore suspension with the LC50 value of 4.75 × 108 spores ml−1 treated with B. bassiana spores at 12, 24, 48 and 96 h. Corrected mortality (%) = [(pest mortality of the treatment group–pest mortality of the control group)/(1–pest mortality of the control group)] × 100. Meanwhile, the RNAi-treated larvae were also used to observe the developmental duration of larvae and pupae, as well as the preoviposition duration and longevity of male and female moth. The weight of larvae (first day of fourth instar) and pupae (second day of pupation), pupation rate, emergence rate and number of eggs per female were also calculated.
Determination of MsKaSPI protein content
The above-mentioned third-day third-instar larvae of M. separata treated with B. bassiana and RNAi at different time points were grounded in a glass homogeniser for 5 min, then centrifuged at 4°C for 15 min at 4 500 × g. The supernatant was and reserved. We used the insect KaSPI enzyme-linked immunosorbent assay (ELISA) kit (Meibiao, Jiangsu) to determine the change of MsKaSPI content in M. separata treated with B. bassiana and RNAi by double-antibody sandwich method. There were three biological replicates for each treatment, and three technical replicates for each sample.
Determination of serine protease activity
The insect SP, trypsin, and chymotrypsin ELISA kits (Meibiao) were used to determine the corresponding enzymatic activity by double-antibody sandwich method. There were three biological replicates for each treatment, and three technical replicates for each sample. The total protein content of the samples from the whole body of third-instar larvae were determined with reference to the Coomassie Brilliant Blue G-250 staining method described by Bradford (Reference Bradford1976). Sample converted enzyme activity = the actual enzyme activity of the sample/the total protein content of the sample, and the converted enzyme activity is used as the sample enzyme activity in results.
Statistical analysis
The relative expression levels were measured using the 2−ΔΔCT method (Livak and Schmittgen, Reference Livak and Schmittgen2001) and mean expression ratio (±SE) of three biological replicates were calculated. Statistical analyses were performed using SAS 9.0 software (IBM Corp., Armonk, New York, USA) single-factor variance Duncan's multiple comparison method to analyse the relative expression of genes at different time points, as well as the significance of enzyme content and the significance of the differences in enzyme activities. The significance of the difference in mortality and RNAi on the growth and development-related parameters of M. separata was analysed by the t-test (P < 0.05). The numerical calculation was carried out in Excel 2010 and chart production was by GraphPad Prism 8.
Results
Identification and analysis of MsKaSPI
We cloned and identified a novel KaSPI cDNA sequence from M. separata, referred to as MsKaSPI (GenBank accession number MN931651). The full-length cDNA sequence is 869 nucleotides long and contains a 540-nucleotide open reading frame and 3′, 5′ untranslated regions. The cDNA encodes an amino acid sequence with 179 residues and contains conserved GXDXXTYXNXC motif and six non-conserved cysteine sequences (fig. 1), which forms the disulphide bonds and is a characteristic of the KaSPI family. A 20 amino acid putative signal peptide was detected using the Signal P 4.1Server (http://www.cbs.dtu.dk/services/SignalP/) and the molecular weight of the gene is about 19.30 kDa and the isoelectric point is 4.95 by the compute pI/Mw tool software of ExPASy (http://ca.expasy.org/tools/). The phylogenetic tree showed that the MsKaSPI was closely related to Spodoptera litura (XP_022823076), Spodoptera frugiperda (XP_035444185) and Helicoverpa armigera (XP_021186066), but far from Nasonia vitripennis (NM001170879) and A. aegypti (XP_001658905) (fig. 2). There is a certain correlation between the phylogenetic relationship of KaSPIs.
Nucleotide and deduced amino acid sequences of MsKaSPI cDNA. The start codon (ATG) and stop codon (TAA) are boxed. The signal peptides are underlined with double underline. Conserved cysteines are underlined with a single line. Kazal structure domain residues are indicated by grey shade.
Phylogenetic tree of KaSPI proteins from Mythimna separata and other insects by neighbour-joining method based on amino acid sequence (1 000 replicates). Origin species of KaSPI proteins: SfKaSPI: Spodoptera frugiperda; SlKaSPI: Spodoptera litura; AcKaSPI: MsKaSPI-1, MsKaSPI-2: M. separata; HaKaSPI: Helicoverpa armigera; TnKaSPI: Trichoplusia ni; MsKaSPI: Manduca sexta; GmKaSPI: Galleria mellonella; CmKaSPI: Callosobruchus maculatus; PxKaSPI: Papilio xuthus; VtKaSPI: Vanessa tameamea; NvKaSPI: Nicrophorus vespilloides; RpKaSPI: Rhodnius prolixus; TiKaSPI: Triatoma infestans; PmKaSPI: Panstrongylus megistus; AmKaSPI: Antheraea mylitta; BmKaSPI: Bombyx mori; NvKaSPI-1, NvKaSPI-2: Nasonia vitripennis; AaKaSPI: Aedes aegypti; DnKaSPI: Diuraphis noxia; SflKaSPI: Sipha flava.
Stage and tissue expression patterns of MsKaSPI
The MsKaSPI was expressed at different developmental stages of M. separata, and the expression level was the highest in fifth-instar larvae, which was 13.77, 2.39, 1.99, 1.76, 1.14, 2.34 and 26.79-fold higher than that in egg, first to sixth-instar larvae and adult, respectively (P < 0.05), but there was no significant difference from the pupal stage (fig. 3).
Expression levels of MsKaSPI at different developmental stages of M. separata. Three biological replicates were conducted. The relative expression levels were measured using the 2−ΔΔCT method and mean ± SE were calculated. Different lowercase letters above bars indicate significant difference (P < 0.05, Duncan's multiple range test).
Expression pattern analysis of MsKaSPI in third-instar larvae showed that the expression level in the midguts was significantly higher than in the other tested tissues and was 2.30, 2.38, 2.04, 8.40, 9.19 and 1.74-fold higher than in foreguts, hindguts, Malpighian tubules, fat bodies and integuments, respectively (P < 0.05). As shown in fig. 4.
Expression levels of MsKaSPI in different tissues of M. separata. Fg: foreguts; Mg: midguts; Hg: hindguts; Sg: salivary glands; Mt: malpighian tubules; Fb: fat bodies; Ig: integuments. The relative transcript levels were measured using the 2−ΔΔCT method and mean ± SE were calculated. Different lowercase letters above bars indicate significant difference in the gene expression level at different developmental stages (P < 0.05, Duncan's multiple range test).
Effect of ecdysone on MsKaSPI
Different concentrations of 20E were used to induce the expression of MsKaSPI. We found that the trend in MsKaSPI expression variations was consistent among different concentrations of 20E treatment groups at the different time points. As the 20E concentration increased from 5 to 10 μg μl−1, the MsKaSPI expression level showed an upward trend. MsKaSPI expression increased from 3 to 24 h reaching its highest level at 24 h, decreased at 48 h.The expression level of MsKaSPI was highest 24 h after injection of 10μg μl−1 20E, which was 3.24-fold than control. The 5 and 10 μg μl−1 20E significantly up-regulated the expression of MsKaSPI gene after 12 and 24 h, while the high concentration 20E (20 μg μl−1) did not show any effect on up-regulation of the expression of MsKaSPI (fig. 5).
Effect of different doses of 20-hydroxyecdysone (20E) on the relative MsKaSPI expression. Total RNA was extracted from third-day third-instar larvae injected with 20E concentrations of 5, 10 and 20 μg μl−1. The time points on the x-axis indicate hours after the injection. DMSO is the control group. The relative transcript levels were measured using the 2−ΔΔCT method and mean ± SE were calculated. Different lowercase letters above bars indicate significant difference in the gene expression level among different treatment time points (P < 0.05, Duncan's multiple range test).
Effects of B. bassiana infection on the MsKaSPI in M. separata
The expression level of MsKaSPI within 72 h in M. separata after being infected with LC50 B. bassiana peaked at 24 h compared with the control group (P < 0.05), which is 4.18-fold of the control group (fig. 6a). As shown in fig. 6b, the protein content of MsKaSPI was significantly different from that of the control group after 12, 24 and 48 h after infection with B. bassiana (P < 0.05), which were 1.94, 3.11 and 2.53-fold, respectively. However, there was no significant difference in protein content at 72 h after infection. It can be seen that B. bassiana induced the expression of MsKaSPI.
Effect of MsKaSPI expression pattern in M. separata after infection by B. bassiana. The expression of MsKaSPI at the mRNA level (a) and the MsKaSPI content (b) at different times after the larvae were treated with B. bassiana or blank control (CK). The spore suspension with the LC50 value of 4.75 × 108 spores ml−1 was used for infection, and sterile water containing 0.1% Tween 80 was used as the CK. The relative transcript levels were measured using the 2−ΔΔCT method and mean ± SE were calculated. Different lowercase letters above bars indicate significant difference among different treatment time points (P < 0.05, Duncan's multiple range test).
Effect of B. bassiana infection on serine protease activity in M. separata
The activity of SP (fig. 7a) in M. separata infected by B. bassiana compared with the control significantly decreased by 36.39 and 34.31% after treatment for 12 and 48 h. While the trypsin activity (fig. 7b) was only significantly decreased from the control group at 48 h by 41.18% (P < 0.05), the chymotrypsin (fig. 7c) activity was significantly different from that of the control group at 12 and 72 h (P < 0.05), and the enzyme activity decreased by 30.95 and 28.21%, respectively.
Effects of B. bassiana infection on the activities of serine proteinase (a), trypsin (b) and chymotrypsin (c) in M. separata.
Influence of RNA interference on MsKaSPI
The efficiency of MsKaSPI knowdown was calculated using qRT-PCR. At 6, 12 and 24 h point of injection of siMsKaSPI, the expression levels in third-day third-instar larvae were repressed by 73.44, 49.06 and 27.23% compared to the expression levels after injection of siNC, respectively (fig. 8a).
Effects of RNAi on MsKaSPI expression levels. The expression of MsKaSPI at the mRNA level (a) and the protein content of MsKaSPI (b) at different times after the larvae were injected with siRNA(siMsKaSPI) or negative control (siNC). Total RNA was extracted at 3, 6, 12, 24 and 48 h after injection with siRNA. The relative transcript levels were measured using the 2−ΔΔCT method and mean ± SE were calculated. Different lowercase letters above bars indicate significant difference among different treatment time points (P < 0.05, Duncan's multiple range test).
In order to further study the function of MsKaSPI in the third-day third-instar larvae in response to B. bassiana infection, the changes of MsKaSPI protein content during 3, 6, 12, 24 and 48 h were detected by the double-antibody sandwich method after being treated with siRNA. As shown in fig. 8b, at different time points of RNAi, the content of MsKaSPI decreased in varying degrees. At 6 and 12 h, the MsKaSPI content was significantly different from that of the control (P < 0.05), and decreased by 40.02 and 55.66%, respectively.
In addition, the MsKaSPI gene-silenced larvae showed slow growth, and the larval and pupal stages were prolonged (P < 0.05), but it had no effect on the pre-oviposition and adults lifespan. Compared with the control group, the oviposition of females was reduced by 181 (P < 0.05) (table 1), but there was no significant difference in pupation rate and emergence rate. The silence of MsKaSPI slightly increased the mortality of M. separata after 48 and 96 h, but the differences were not significant (fig. 9). While the insect mortality rate has significantly increased in MsKaSPI-silenced larvae after 24, 48 and 96 h infection with B. bassiana (P < 0.05), which increased by 8.33, 13.56 and 18.96%, respectively (fig. 9). It indicated that the silence of MsKaSPI could help to increase the infection rate of B. bassiana to M. separata.
Growth and development parameters of M. separata under different treatments
Data in the table are means ± SEs. Asterisk following the data in a column indicates significant difference from the control (P < 0.05, t-test). 30 worms were observed (including 14 females and 16 males) and conducted growth parameter data statistics every 24 h. The number of eggs (per female) were counted for 14 female adults.
Corrected mortality of MsKaSPI RNAi treated M. separata and infection by B. bassiana. Corrected mortality (%) = [(mortality of the treatment group–mortality of the control group)/(1–mortality of the control group)] × 100. Data in the figure are means ± SEs. Asterisk following the data in a column indicates significant difference from the control (**P < 0.05, ***P < 0.01, Duncan's multiple range test).
Effects of RNAi of MsKaSPI on enzyme activities in M. separata
After RNAi treatment, the SP (fig. 10a) and trypsin (fig. 10b) activities showed an overall trend of initial increase and then decrease. At 6 h after treatment, the SP activity began to increase significantly (P < 0.05). The trypsin activity showed a significant increase from 3 to 24 h after treatment (P < 0.05); chymotrypsin (fig. 10c) activity increased slightly after treatment, but not significantly different from the control group. By silencing of MsKaSPI, the activity of SP and trypsin in M. separata was enhanced, indicating that MsKaSPI may effectively regulate related SPs by adjusting the content of SPIs.
Effects of RNAi of MsKaSPI on the activities of serine proteinase (a), trypsin (b) and chymotrypsin (c) in M. separa.
Discussion
SPIs play an important role in insect growth and development, metabolism and immune defence. In order to further study the mechanism of SPIs in insects, a cDNA sequence MsKaSPI of SPI gene with Kazal domain was obtained by transcriptome sequencing. The sequence deduced by MsKaSPI contains three Kazal-type domains formed by three disulphide bonds and six cysteine residues in a specific pattern (CysI-CysV, CysII-CysIV and CysIII-CysVI). Sequence analysis shows that this MsKaSPI protein belongs to group II of the non-classical KaSPI (Cabrera-Muñoz et al., Reference Cabrera-Muñoz, Valiente, Rojas, Alonso-Del-Rivero Antigua and Pires2019).
The MsKaSPI had the highest expression in fifth-instar larvae and the midgut of M. separata. This expression pattern indicates it may be related to food digestion and absorption in M. separata. The expression of BmSPI3 was the highest both in the fifth-instar larvae and midguts of the fifth-instar larvae (Zheng et al., Reference Zheng, Xu, Zhou, Lv and Zhang2010). ApKTSPI was specifically highly expressed in the fat body of fifth-instar larvae in A. pernyi (Wang et al., Reference Wang, Qiu, Qian, Zhu and Liu2014). In addition, it was also found that the expression of insect KaSPI was positively regulated by ecdysone 20E which participated in the regulation of insect innate immunity (Tian et al., Reference Tian, Guo, Diao, Zhou, Peng, Cao, Ling and Li2010; Rus et al., Reference Rus, Flatt, Tong, Aggarwal, Okuda, Kleino, Yates, Tatar and Silverman2013; Sun et al., Reference Sun, Shen, Zhou and Zhang2016). The CG7906 and CG7924 genes in D. melanogaster were successfully induced after ecdysone 20E treatment, the expression levels were 3.49 and 2.41 times higher than control (Beckstead et al., Reference Beckstead, Lam and Thummel2007), respectively. In this experiment, the expression of MsKaSPI in the third-instar larvae was the highest 24 h after injection of 10 μg μl−1 20E. It was speculated that 20E could induce MsKaSPI expression. When the concentration of 20E increased from 5 to 10 μg μl−1, the expression of MsKaSPI increased gradually, but it decreased when injected with the concentration of 20 μg μl 20E, which may be because although the growth and development of larvae were regulated by ecdysone and juvenile hormone, excessive concentrations would affect the normal physiological process of insects. There was a similar situation in B. mori, the expression of BmKaSPI was significantly up-regulated in silk glands of both female and male silkworms 12 and 24 h after treatment with low concentration of 20E, while there was no up-regulation of BmKaSPI after treatment with high concentration of 20E (Gan et al., Reference Gan, Liu and Li2016). However, 20E regulates insect innate immunity in a variety of ways, and the specific mechanism is not clear (Wu et al., Reference Wu, Chang and Nan2016).
After treatment with B. bassiana, the expression of MsKaSPI was significantly up-regulated, and the activities of SP, trypsin and chymotrypsin were significantly inhibited, indicating that MsKaSPI may participate in the resistance of armyworm to the invasion of pathogens. ApKaSPI was up-regulated after nuclear polyhedrosis virus, E. coli and B. bassiana (Wang et al., Reference Wang, Qiu, Qian, Zhu and Liu2014). AaKaSPI was up-regulated after immune stimulation by virus DENV (Soares et al., Reference Soares, Gonzalez, Torquato, Lemos and Tanaka2018). After leafhoppers were stimulated by the bacteria and E. coli, the expression of KaSPI in the leafhoppers midgut, blood cells and whole insects were collectively up-regulated (Gonella et al., Reference Gonella, Mandrioli, Tedeschi, Crotti, Pontini and Alma2019). The results of these studies showed that insect protease inhibitors are involved in the immune response, but the gene expression patterns are different under the immune stimulation of different pathogens. In addition, it was also found that after treatment with B. bassiana, the content of MsKaSPI protein increased significantly (fig. 6b). However, it has decreased from 24 h, which was not consistent with the expected results. According to the trend that ecdysone first increased and then decreased before and after moulting in insects and the results of MsKaSPI expression induced by 20E in vitro (fig. 5), it was speculated that the increase of MsKaSPI protein content might be caused by keeping a certain concentration of ecdysone for a short time after 24 h treatment of third-instar larvae.
RNAi technology was taken to further study the function of MsKaSPI in the induced metabolism of M. separata by B. bassiana. The results showed that the expression of MsKaSPI and the protein content of KaSPI were significantly decreased, and the SP and trypsin activities were significantly enhanced, indicating that MsKaSPI silencing would cause changes in related proteins and enzyme activities. After silence of MsKaSPI, the mortality of M. separata increased after B. bassiana infection. It is inferred that MsKaSPI functions in the resistance to B. bassiana. The bacteriostatic effect of KaSPI has also been reported in other insects. For example, it was found that over-expressed BmKaSPI in silkworms can effectively inhibit subtilisin which plays an important role in resisting pathogen invasion in silkworms (Guo et al., Reference Guo, Dong, Xiao, Li, Zhang, He, Xia and Zhao2015; Chen and Lu, Reference Chen and Lu2017); AmKaSPI can bind to the surface of bacteria and fungi, and exhibits antimicrobial activity against fungi and Gram-positive and -negative bacteria (Qian et al., Reference Qian, Fang, Wang and Ye2015; Yang et al., Reference Yang, Lee, Kim, Choi, Yoon, Jia and Jin2017), it can be seen that KaSPI plays an important role in resisting pathogen invasion in insects.
Meanwhile, it was also found that the number of eggs per female was significantly reduced after the RNAi expression of MsKaSPI. A KaSPI gene named Greglin related to reproduction was also found in migratory locusts. Knockdown of Greglin in adult female locusts results in a significant reduction in Greglin content, oocyte maturation was blocked, ovarian growth stagnated and follicular epithelial cells atrophied in L. migratoria, and the number of eggs laid and hatching rate decreased, indicating that the gene was involved in the reproductive process of locusts (Guo et al., Reference Guo, Dong, Xiao, Li, Zhang, He, Xia and Zhao2015). It can be seen that MsKaSPI not only plays an important role in resisting the invasion of pathogens, but also has a great impact on the reproduction of M. separata.
Conclusions
In summary, we described the molecular characteristics of MsKaSPI as well as its structure properties and the spatio temporal expression profiles. The MsKaSPI gene expression was induced by 20E treatment and the infection of B. bassiana. MsKaSPI gene knockdown could significantly increase the activities of SP, trypsin and the mortality by the infection of B. bassiana, and reduced the number of eggs in M. separata. Our results provide further insights into the role of MsKaSPI on reproduction and immune defence, and potential target for pest control.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S000748532300041X
Acknowledgements
This work was supported financially by the National Key R&D Program of China (2018YFD0201000) and Special Fund for the Construction of Modern Agricultural Industrial Technology System (CARS-04).
Competing interests
None.
