Identification and functional analysis of serine protease inhibitor gene family of Eocanthecona furcellata (Wolff)

Abstract

The predatory natural enemy Eocanthecona furcellata plays a crucial role in agricultural ecosystems due to its effective pest control measures and defensive venom. Predator venom contains serine protease inhibitors (SPIs), which are the primary regulators of serine protease activity and play key roles in digestion, development, innate immunity, and other physiological regulatory processes. However, the regulation mechanism of SPIs in the salivary glands of predatory natural enemies is still unknown. In this study, we sequenced the transcriptome of E. furcellata salivary gland and identified 38 SPIs genes named EfSPI1∼EfSPI38. Through gene structure, multiple sequence alignment and phylogenetic tree analysis, real-time quantitative PCR (RT-PCR) expression profiles of different developmental stages and different tissues were analyzed. RNAi technology was used to explore the gene function of EFSPI20. The results showed that these 38 EfSPIs genes contained 8 SPI domains, which were serpin, TIL, Kunitz, Kazal, Antistasin, Pacifastin, WAP and A2M. The expression profile results showed that the expression of different types of EfSPIs genes was different at different developmental stages and different tissues. Most of the EfSPIs genes were highly expressed in the egg stage. The EfSPI20, EfSPI21, EfSPI22, and EfSPI24 genes of the Pacifastin subfamily and the EfSPI35 gene of the A2M subfamily were highly expressed in the nymphal and adult stages, which was consistent with the RT-qPCR verification results. These five genes are positively correlated with each other and have a synergistic effect on E. furcellata, and they were highly expressed in salivary glands. After interfering with the expression of the EfSPI20 gene, the survival rate and predatory amount of male and female adults were significantly decreased. Taken together, we speculated some EfSPIs may inhibit trypsin, chymotrypsin, and elastase, and some EfSPIs may be involved in autoimmune responses. EfSPI20 was essential for the predation and digestion of E. furcellata, and the functions of other EfSPIs were discussed. Our findings provide valuable insights into the diversity of EfSPIs in E. furcellata and the potential functions of regulating their predation, digestion and innate immunity, which may be of great significance for developing new pest control strategies.

Keywords: Eocanthecona furcellata (Wolff); RNA interference; natural enemy; salivary gland; serine protease inhibitor.

FIGURE 1

Gene ontology (GO) classification showed by the quantity of Eocanthecona furcellata transcripts.

FIGURE 2

Protein conserved domain of SPIs genes of Eocanthecona furcellata.

FIGURE 3

Multiple sequence alignment of the reactive center regions of the serpin subfamily and 6 subfamilies of canonical inhibitors of Eocanthecona furcellata. Predicted P1 residues are highlighted in red lines or red boxes. (A): Serpin domain; (B): Pacifastin domain; (C): KAZAL domain; (D): TIL domain; (E): Kunitz domain; (F): WAP domain; (G): Antistasin domain. Species and Gene Bank accession numbers are shown in Supplementary Table S4.

FIGURE 4

Phylogenetic relationship of serpins from Eocanthecona furcellata and four other insect species. The phylogenetic tree was constructed using the neighbor joining method for 1,000 times bootstrap replicates by MEGA 7.0.26 software. The first two letters in each of the serpins represent the acronym of the scientific name for a given species (Px: P. xylostella, Bm: B. mori, Ld: L. distinctifemur, Pp: P. plagipennis, Ef: Eocanthecona furcellata). The amino acid sequences used are shown in the Supplementary Material S1.

FIGURE 5

Phylogenetic analysis of thioester-containing proteins (TEPs) from Eocanthecona furcellata and other species. The phylogenetic tree was constructed using MEGA 7.0.26 with the neighbor joining method. The first two letters in each of the serpins represent the acronym of the scientific name for a given species. Species and Gene Bank accession numbers are shown in Supplementary Table S5.

FIGURE 6

Correlation network of EfSPIs gene family members. The red line indicates a positive correlation, and the blue line indicates a negative correlation.

FIGURE 7

Expression profiles of EfSPIs genes across eggs and salivary glands of different developmental stages (1-5 instar nymphs, female and male adults). Egg: egg stage; First: first instar nymphs; Second: second instar nymphs; Third: third instar nymphs; Fourth: fourth instar nymphs; Fifth: fifth instar nymphs; Male: male adults; Female: female adults; each developmental stage had 3 replicates, the mean value of 3 repeated FPKM values was used to make the expression calorimetric map. The log2 (FPKM +1) value in Heat Map represents the gene expression level. The deeper red and blue indicated higher and lower expression levels, respectively.

FIGURE 8

RT-qPCR of EfSPIs genes at different developmental stages. E: eggs, 1 L: first instar nymphs, 2 L: second instar nymphs, 3 L: third instar nymphs, 4 L: fourth instar nymphs, 5 L: fifth instar nymphs, M: male adults, F: female adults. EfRPL9 rRNA was used as a housekeeping gene. Error bars represent the mean ± SE deviations from three biological replicates. A one-way ANOVA was used to determine the significant difference with different lowercase letters (a–e) (p < 0.05).

FIGURE 9

RT-qPCR of EfSPIs genes at different tissues. Mt: Malpighian tube, Sg: salivary gland, Fb: fat body, Ov: ovary, Mg: midgut, He: head, Hem: hemolymph. EfRPL9 rRNA was used as a housekeeping gene. Error bars represent the mean ± SE deviations from three biological replicates. A one-way ANOVA was used to determine the significant difference with different lowercase letters (a–e) (p < 0.05).

FIGURE 10

The transcription level, survival rate and predation amount of EfSPI20 in salivary glands of Eocanthecona furcellata after RNAi 1, 2, 4, 6 days. In the figure, (A, B) are the interference efficiency of female and male adults, respectively. (C, D) are the survival rates of male and female adults, respectively. (E, F) are the changes in the feeding amount of male and female adults, respectively. The data are expressed as mean ± SE; different letters indicate significant differences at the 0.05 level.