Fig 1.
Variation in gene (Se-sPLA2) expression and enzyme activity of sPLA2 in different developmental stages of S. exigua.
(A) Expression profile of Se-sPLA2 in different stages using RT-PCR (upper panel) and RT-qPCR (lower panel). A ribosomal protein, RL32, expression was used for control for RT-PCR and normalization for RT-qPCR. (B) Its enzyme activities from the whole bodies of different stages, in which the upper panel shows a western blot. Monoclonal antibody specific to α-tubulin was used for control to confirm the same amount protein loading. (C) Up-regulation in sPLA2 activity in the hemolymph upon an immune challenge with 5 × 104 X. nematophila killed at 95°C for 5 min. Left panel shows the Se-sPLA2 protein level in the plasma by a western blot. All treatments were replicated three times. Different letters above the standard deviation bars indicate the significant difference among means at Type I error = 0.05 (LSD test).
Fig 2.
Mutagenesis using CRISPR/Cas9 against Se-sPLA2.
(A) Design of single stranded guide RNAs (sgRNA1 and sgRNA2) from exon 2 (E2) on the genomic DNA of Se-sPLA2. The genome map (scaffold 52640) was obtained from GenBank accession number of GCA_022117675.1. Two sgRNAs are 85 bp apart based on their cleavage sites. (B) Demonstration of CRISPR mutagenesis using two sgRNAs and Cas9. Two arrows indicate diagnostic primers (FP and RP), which produce 230 bp PCR product including 58 bp and 67 bp neighboring the cleavage sites. In contrast to wild type (WT), a mutant (M1) produced 156 bp by 85 bp loss and some indels.
Fig 3.
Construction of the deletion mutants in Se-sPLA2 of S. exigua using CRISPR/Cas9.
(A) Preparation of the newly laid eggs for microinjection of sgRNAs and Cas9. (B) Outcomes of the mutagenesis. Mutagenic treatment (ΔsPLA2) used both sgRNA and Cas9 for the injection while the control (CON) used only sgRNA. After CRISPR mutagenesis, the final live larvae were counted at fifth instar (L5). (C) Confirmation of the mutagenesis by sequencing at the target sites (230 bp) produced by diagnostic primers. All 18 mutants (M1-M18) were obtained based on the sequence comparison with the wild type (WT) sequence and reference (REF) sequence reported in GenBank (MH061374). (D) Selection of homozygote (-/-) mutants. At the first generation (G0), all the mutants were heterozygote (-/+). These inbred line (5 × 6 male and female pair) gave the homozygote mutants at the next generation (G1). The homozygotes (6 × 7 male and female pair) were inbred to get all homozygote mutants at G2 generation.
Fig 4.
RNASeq analysis of CRISPR mutants (ΔsPLA2) of S. exigua in Se-sPLA2.
Both mutant wild type (WT) larvae were immune challenged with 5 × 104 X. nematophila killed at 95°C for 5 min. These four treatments are WT-naïve, WT-immune, ΔsPLA2-naïve, and ΔsPLA2-immune. RNASeq was performed three replications in each treatment. (A) Hierarchical clustering analysis of 12,878 unigenes among 12 RNASeq samples. (B) Differentially expressed genes (DEGs) selected from two comparison groups (G1 and G2). DEGs of G1 represent immune-associated genes by comparing transcripts of naïve and immune challenge in WT. DEGs of G2 represent the target genes associated with sPLA2 expression by comparing wild type and mutant transcripts. (C) Venn diagram to indicate the immune-associated genes controlled by sPLA2 expression. (D) Heatmap analysis depicting the expression profiles of 55 canonical immune-associated genes among four treatments. A phylogeny analysis was obtained using ClustVis online tool (https://biit.cs.us.ee/clustvis/). It indicates little responsiveness of the CRISPR mutant to the immune challenge in the canonical immune genes compared to wild type larvae.
Table 1.
KEGG analysis of 8,944 DEGs (G2 in Fig 4B) associated with sPLA2 expression in S. exigua.
Fig 5.
Suppression in immune responses of the CRISPR mutants (ΔsPLA2) in Se-sPLA2.
(A) Suppression in a cellular immune response assessed by nodule formation. L5 larvae were injected with 5 × 104 cells of Heat-killed X. nematophila (HK-Xn). After 8 h, the larvae were dissected for counting nodules. Each treatment was replicated with 10 larvae. Arachidonic acid (AA) was injected to the larvae 1 μg per larvae. (B) Suppression in humoral immune response. In two immune-associated tissues, eight AMP genes were assessed in RT-qPCR after immune challenge: Apo (apolipophorin III), Att1 (attacin 1), Att2 (attacin 2), Def (defensing), Gal (gallerimycin), Tf1 (transferrin 1), Tf2 (transferrin 2), and Hem (hemolin). Different letters above the standard deviation bars indicate significant difference among means in each AMP at Type I error = 0.05 (LSD test).
Fig 6.
Developmental and reproductive alterations of the CRISPR mutants (ΔsPLA2) in Se-sPLA2.
(A) Expression analysis of Se-sPLA2 in mutant and wild type (WT) during entire developmental stages of S. exigua. (B) Alteration in immature development. Reduced pupal size (n = 10) and pupation percentage. Pupal weight was measured within 10 h after pupation. (C) Alteration in reproduction assessed by fecundity (egg laying number) and fertility (egg hatch). Different letters or asterisk above the standard deviation bars indicate significant difference among means at Type I error = 0.05 (LSD test).