Adults of western flower thrips (F. occidentalis) were obtained from Bio Utility (Andong, Korea) and reared under laboratory conditions (temperature of 27 ± 1°C; photoperiod of 16:8 h (L: D), and relative humidity (RH) of 60 ± 5%). Two larval instars (L1 and L2) and adults were reared on sprouted bean seed kernels. L2 stage and adults < 3 days after adult emergence were used for immune and pathogenicity tests. An entomopathogenic fungus, B. bassiana, was cultured in a potato dextrose agar (PDA) plate at 25 ± 1°C and RH of 70 ± 5% with a 16:8 h (L:D) photoperiod for 14 days.

3-Ethoxy-4-methoxyphenol (EMP) was purchased from Sigma-Aldrich Korea (Seoul, Korea) and dissolved in dimethyl sulfoxide (DMSO). Triton X-100 (t-octylphenoxy-polyethoxyethanol) was purchased from Sigma-Aldrich Korea and used as an adjuvant in fungal bioassay analysis. p-Bromophenacyl bromide (BPB) and methylarachidonyl fluorophosphate (MAFP) were purchased from Sigma-Aldrich Korea. DAPI (4,6-diamidino-2-phenylindole) was purchased from Thermo Scientific (Steingrung, Dreieich, Germany) and dissolved in DMSO. BZA (benzylideneacetone) was purchased from Sigma-Aldrich Korea and dissolved in DMSO. Phosphate-buffered saline (PBS) was prepared with 100 mM phosphoric acid. Its pH was adjusted to 7.4 using NaOH.

DSP1 sequence of F. occidentalis (Fo-DSP1) was obtained from GenBank with an accession number of XP_026278027.1. Sequences of F. occidentalis were obtained for secretory phospholipase A₂ (Fo-sPLA₂A and Fo-sPLA₂B), calcium-independent phospholipase A₂ (Fo-iPLA₂A and Fo-iPLA₂B), microsomal prostaglandin E synthase type 2 (Fo-PGES2) and dual oxidase (Fo-Duox), phenoloxidase-activating protease (Fo-PAP2A, Fo-PAP2B and Fo-PAP3), phenoloxidase (Fo-PO1, Fo-PO2A and Fo-PO2B), antimicrobial peptide genes (Fo-Apol, Fo-Def, Fo-Lyz, and Fo-Tra1) from National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). GenBank accession numbers of these genes and related orthologs are described in Supplementary Tables S1 – S7 . Phylogenetic analyses were performed using MEGA6.06 and ClustalW programs from EMBL-EBI (www.ebi.ac.uk). Bootstrapping values were obtained with 1,000 repetitions to support branching and clustering. Protein domains were predicted using Prosite (https://prosite.expasy.org/) and SMART search program (http://smart.embl-heidelberg.de/).

Conidial suspension of B. bassiana was prepared by scraping the fungal culture from 14 days old PDA medium into an Eppendorf tube containing 1 mL of autoclaved Triton X-100 (0.1%) solution (Duksan Pure Chemicals, Ansan, Korea). Conidia of the suspension were counted using a Neubauer hemocytometer (Marienfeld-Superior, Lauda-Königshofen, Germany) under 40× magnification.

To assess the pathogenic activity of B. bassiana, L2 larvae and adults were fed with different concentrations (1×10⁸, 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴ conidia/mL) of conidial suspension. Briefly, a piece of sprouted bean seed kernel was dipped in 1 mL of conidial suspension from each concentration for 5 min and kept for 10 min to dry under a clean bench. The aseptic condition was strictly followed throughout the process. After L2 larvae or adults were released into a Petri dish (5 × 2 cm), the Petri dish was sealed with parafilm (Bemis Company, Zurich, Switzerland). These Petri dishes were kept in a desiccator (4202-0000, Bel-Art Products, Pequannock, NJ, USA) with a constant temperature of 25 ± 1°C and 75 ± 5% RH which was maintained using a saturated solution of NaCl according to Winston and Bates (21). Dead insects were counted every 12 h up to 5 days by confirming mycosis development on insect cadaver. Each treatment had three replicates. Each replication used 10 larvae or adults.

Sprouted bean seed kernels were dipped into 1 mL of conidial suspension (1×10⁶ conidia/mL) for 5 min and dried for 10 min under aseptic conditions. They were then placed in Petri dishes (5 × 2 cm), in which test insects were fed. After 3 h, the diet was replaced with the fresh kernel. For RNA extraction, both larvae and adult insects were collected at 6, 12, and 24 h after fresh kernel feeding. Each time point was replicated three times with 100 larvae and 80 adults.

RNA was extracted from different developmental stages (~100 L2, ~100 pupae, or ~100 adults per sample) of F. occidentalis using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Extracted RNA was quantified using a spectrophotometer (NanoDrop, Thermo Scientific, Wilmington, DE, USA). RNA extract (100 ng per reaction) was used for cDNA synthesis with an RT-premix (Intron Biotechnology, Seoul, Korea). Synthesized cDNA was used as a template for PCR amplification with gene-specific primers ( Supplementary Table S8 ). To determine cDNA integrity, the elongation factor 1 (EF1) gene was used. RT-PCR began with initial heat treatment at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 1 min, annealing at a specific temperature depending on primers ( Supplementary Table S8 ) for 30 s, and extension at 72°C for 1 min. PCR reaction was terminated with a final chain extension step at 72°C for 10 min.

Quantitative PCR (qPCR) was performed using SYBR Green Real-Time PCR master mixture (Toyobo, Osaka, Japan) on a Real-Time PCR System (Step One Plus Real-Time PCR System, Applied Biosystem, Singapore). The reaction mixture (20 μL) contained 10 pmol of primers used in RT-PCR and 80 ng of cDNA template. After activating Hotstart Taq DNA polymerase at 94°C for 5 min, the reaction was amplified with 40 cycles of denaturation at 94°C for 30 s, annealing at a specific temperature depending on primers ( Supplementary Table S8 ) for 30 s, and extension at 72°C for 30 s. The expression level of EF1, a reference gene, was used to normalize target gene expression levels. Each treatment was replicated with three independent biological sample preparations. Quantitative analysis was performed using the comparative CT (2^-ΔΔCT) method (22).

A commercial assay kit (sPLA₂ Assay Kit, Cayman Chemical) was used with diheptanoyl thio-phosphatidylcholine as enzyme-substrate to measure PLA₂ activities in whole bodies of 100 individuals at each stage (L2 larva, pupa, and adults). Whole body extracts were obtained after homogenizing in 100 mM phosphate-buffered saline (PBS, pH 7.4). For inhibitor assay, protein samples were incubated at 25°C with inhibitors (BPB or MAFP) for 30 min. For the immune challenge, larva and adult insects were challenged with B. bassiana (1×10⁶ conidia/mL) as mentioned earlier. Samples were collected at 6 h, 12 h, and 24 h post-treatment. sPLA₂ enzyme activities were measured using Ellman’s reagent [5,5′-dithio-bis-(2-nitrobenzoic acid), DTNB] to produce 5-thio-2-nitrobenzoic acid. DTNB was prepared at 10 mM in 0.4 M Tris buffer (pH 8.0). Assay buffer used 25 mM Tris (pH 7.5) containing 10 mM CaCl₂, 100 mM KCl, and 0.3 mM Triton X-100. The reaction mixture (175 μL) contained 10 μL of plasma sample, 10 μL of DTNB, 5 μL of assay buffer (or inhibitor for inhibition assay), and 150 μL of the substrate. All inhibitors were used at 100 µM. For negative controls, the same volume of reaction mixture consisted of 10 μL of DTNB, 15 μL of assay buffer, and 150 μL of the substrate. Absorbance was measured at 405 nm. Absorbance for non-enzymatic blank controls was calculated and subtracted from sample wells. The actual extinction coefficient for DTNB was 10.66 mM⁻¹. Specific enzyme activity (pmol/min/μg) was calculated by dividing absorbance change by protein amount used as enzyme source for the reaction. Each treatment was replicated with three biologically independent enzyme preparations using different larval or adult samples. Protein concentration was determined by Bradford (23) assay using bovine serum albumin (BSA) as standard.

B. bassiana (1×10⁵ conidia/mL) was treated along with EMP (1,000 ppm) or BZA (1,000 ppm). Briefly, sprouted bean seed kernels were dipped into 1 mL of the conidial suspension (1×10⁵ conidia/mL) containing EMP or BZA for 5 min and dried at 25°C for 10 min under sterile condition. These treated kernels were provided for feeding for 3 h. After 3 h of feeding, treated kernels were replaced with fresh kernels. Samples for RNA extraction were collected at 24 h post-treatment. Each treatment was replicated three times with each replicate having 100 insects.

Midgut from each adult was collected onto slide glass containing 10 µL of TC100 insect tissue culture medium (Welgene, Gyeongsan, Korea) and incubated at 25°C in a wet chamber for 10 min. After removing TC100, the midgut was then fixed with 4% formaldehyde for 20 min at room temperature (RT). Fixative was replaced with PBS followed by incubation at 25°C for 10 min. After washing with PBS twice, the midgut was permeabilized with 0.2% Triton X-100 in PBS for 10 min at RT. After washing with PBS thrice, the midgut was blocked with 5% skimmed milk in PBS at RT for 20 min. After washing with PBS once, the midgut was incubated with 10 µL of primary antibody (16) raised against Se-DSP1 in rabbit which was diluted in 3% BSA in PBS (1:100) at RT for 1 h 20 min. After washing with PBS thrice, the midgut was incubated with 10 µL of FITC-tagged anti-rabbit secondary antibody (Sigma-Aldrich Korea, diluted with 3% BSA in PBS at 1:5,000) at RT for 1 h. After washing with PBS thrice, the midgut was incubated with 10 µL of 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL) (Thermo Scientific, Rockford, IL, USA) in PBS at RT for 5 min for nucleus staining. Finally, after washing with PBS thrice, 5 µL of glycerol and PBS (1:1) mixture was added and cover glass was placed on it. It was then observed under a fluorescence microscope (DM2500, Leica, Wetzlar, Germany) at 400 × magnification.

Data of continuous variables were subjected to one-way analysis of variance (ANOVA) using PROC GLM in the SAS program (24). Means were compared with the least significant difference (LSD) test at Type I error = 0.05. Median lethal concentration (LC₅₀) and time (LT₅₀) were subjected to Probit analysis using EPA Probit Analysis Program, ver. 1.5 (U.S. Environmental Protection Agency, USA).

Susceptibility of F. occidentalis to B. bassiana was assessed by oral feeding bioassays using larvae or adults ( Figure 1 ). Both developmental stages were susceptible to B. bassiana. Dead insects showed sporulation symptoms on body surfaces ( Figure 1A ). The fungal virulence increased in a dose-dependent manner after treatment with fungal spores. However, larvae and adults showed different susceptibilities to B. bassiana. Most (> 95%) larvae were dead after treatment with fungal spores at 10⁸ spores/mL. However, only 60% of adults were dead after the same treatment. Median lethal concentrations were different by more than 30 folds between these two developmental stages ( Figure 1B ). In addition, the speed-to-kill was apparently slower for adults by almost two folds than for larvae at the same fungal treatment.

The apparent difference between larvae and adults in fungal pathogenicity suggests a differential defense in immune responses of F. occidentalis. DSP1, a DAMP molecule in other insects, was suspected to be activated in the gut of F. occidentalis in response to an oral infection of B. bassiana. Domains of Fo-DSP1 possess HMG Box A and Box B with additional N- and C-terminal extensions ( Figure 2A ). Its predicted amino acid sequence was clustered with other insect DSP1s ( Figure 2B ). Midguts were isolated from adult insects and used for the analysis of Fo-DSP1 localization with a polyclonal antibody raised against Se-DSP1 of Spodoptera exigua ( Figure 2C ). The cross-reactivity of the antibody was supported by high sequence homology (69.6%) between Fo-DSP1 and Se-DSP1. Immunofluorescence assay showed that Fo-DSP1 was localized in the nucleus stained with DAPI. Fo-DSP1 was expressed in larval to adult stages, with adult males showing the highest expression level ( Figure 2D ). Fungal challenge used diet contaminated with B. bassiana significantly increased the expression level of Fo-DSP1 in both larval and adult stages ( Figure 2E ). However, there was a difference in their induction patterns. Fo-DSP1 was induced only at 12 h after the fungal treatment at the larval stage while it was induced expression constantly from 6 to 24 h at the adult stage.

DSP1 can mediate immune responses by activating PLA₂ activity in insects (16, 18). Four PLA₂ genes were obtained from GenBank ( Figure 3A ). Among 16 gene Groups (‘I-XVI’) of PLA₂, four PLA₂s of F. occidentalis were separately clustered with Group III (Fo-sPLA₂A), Group XV (Fo-sPLA₂B), Group VI (Fo-iPLA₂A), and Group VIII (Fo-iPLA₂B). Two secretory PLA₂s (Fo-sPLA₂A and Fo-sPLA₂B) were found to have a signal peptide or Ca²⁺-binding domain while two Ca²⁺-independent cellular PLA₂s (Fo-iPLA₂A and Fo-iPLA₂B) were not. These two iPLA₂s were different in the ankyrin repeat domain, which was predicted only in Fo-iPLA₂B.

Four PLA₂ s of F. occidentalis were expressed in all developmental stages. They were highly expressed in adult stages ( Figure 3B ). The fungal challenge with B. bassiana significantly increased expression levels of four PLA₂ s in both larval and adult stages ( Figure 3C ). Especially, Fo-sPLA₂B was highly induced by several hundred folds in its expression levels in both larval and adult stages in response to the fungal challenge.

PLA₂ enzyme activity was analyzed using different PLA₂ inhibitors: BPB as a specific inhibitor for sPLA₂ and MAFP as a specific inhibitor for cPLA₂ ( Figure 3D ). Protein extracts of larvae had PLA₂ enzyme activities, which were significantly inhibited by both inhibitors. PLA₂ activities were detected in different developmental stages of F. occidentalis. They were increased in both larval and adult stages after the fungal infection ( Figure 3E ). The susceptibility of PLA₂ enzyme activity to MAFP suggests a presence of cPLA₂ in F. occidentalis, but its genome does not have a typical cPLA₂ ortholog.

PGE₂ is one of the PGs commonly detected in different insects (15). F. occidentalis ortholog of PGE₂ synthase 2 (Fo-PGES2) was obtained from GenBank ( Table S3 ). Its amino acid sequence indicated an enzyme catalytic domain ( Figure S1A ). Phylogenetic analysis indicated that Fo-PGES2 was clustered with other membrane-bound PGES2 genes ( Figure S1B ). Fo-PGES2 was expressed in different developmental stages ( Figure 4A ). Its expression was highly inducible in larval and adult stages by the fungal infection ( Figure 4B ).

EMP is known to bind to and inactivate DSP1 in S. exigua and Tenebrio molitor (16, 18). BZA is a specific inhibitor for PLA₂ of insects (25). These two inhibitors were used to treat larvae and adults of F. occidentalis along with B. bassiana ( Figure 5 ). Both inhibitors increased the insecticidal activity of B. bassiana to F. occidentalis in a dose-dependent manner ( Figure 5A ).

To clarify the effects of inhibitors on the enhancement of fungal pathogenicity, expression levels of Fo-DSP1, Fo-PLA₂ s, and Fo-PGES2 were examined after inhibitor treatments. The induction of Fo-DSP1 after fungal treatment was significantly suppressed by EMP treatment in both developmental stages ( Figure 5B ). Fo-DSP1 expression after treatment with EMP was also observed in the negative control when EMP alone was used for treatment. Results showed that EMP suppressed the basal level of Fo-DSP1 in naïve F. occidentalis. EMP treatment also suppressed expression levels of all four PLA₂s ( Figure 5C ). The negative control of PLA₂ gene expressions by EMP was supported by the suppression of PLA₂ activities ( Figure 5D ). The increase of PLA₂ activity after fungal infection was significantly suppressed by the addition of EMP or BZA, a specific PLA₂ inhibitor. Similarly, BZA or EMP treatment significantly suppressed Fo-PGES2 expression in both larvae and adults, although the suppression was more prominent in adults than in larvae ( Figure 5E ).

Fungal infection used a feeding method to determine whether F. occidentalis might defend the fungal conidia through its gut immunity. Dual oxidase (Duox) plays a crucial role in gut immunity in insects (26). A Duox gene (Fo-Duox) was obtained from GenBank ( Table S4 ). The additional peroxidase domain in N-terminus supports its identity from NADPH-dependent oxidase ( Figure S2A ). Its predicted amino acid sequences shared homologies with other insect Duox genes ( Figure S2B ). Fo-Duox was expressed in different developmental stages ( Figure 6A ). Its expression was highly up-regulated in larvae and adults upon the fungal challenge ( Figure 6B ). However, the induction of this gene was prevented by the addition of an inhibitor specific to DSP1 or PLA₂ ( Figure 6C ).

In hemocoel, acute cellular immune responses are likely to be activated by hemocytes with help of the catalytic activity of phenoloxidase (PO), leading to melanization against pathogens (27). PO is produced in an inactive prophenoloxidase (PPO) form, which is cleaved by a specific serine protease called PO-activating protease (PAP) (28). Three PAP genes (Fo-PAP2A, Fo-PAP2B, and Fo-PAP3) were obtained from GenBank ( Table S5 ). Domain analyss of these three PAPs possess typical clip domain along with catalytic domain ( Figure S3A ). Different subtypes of PAPs were clustered with their clade members ( Figure S3B ). All three PAPs were expressed in different developmental stages, with adult females having the highest expression levels ( Figure 7A ). Fungal infection induced their expressions, with larvae responding to the fungal infection earlier than adults ( Figure 7B ). The addition of inhibitors specific to DSP1 or PLA₂ significantly suppressed the up-regulation of the three PAPs ( Figure 7C ).

Three PO genes (Fo-PO1, Fo-PO2A, and Fo-PO2B) were obtained from GenBank ( Table S6 ). Two POs (Fo-PO1 and Fo-PO2B) are likely to be secretory proteins due to their signal peptides in their N termini ( Figure S4A ). However, Fo-PO2A appeared to be a transmembrane protein based on domain analysis. A phylogeny tree analysis showed that Fo-PO2A and Fo-PO2B were closely clustered away from Fo-PO1 ( Figure S4B ). All three POs were expressed in different developmental stages, with the adult stage having the highest expression levels ( Figure 8A ). Fungal infection induced their expression, with larvae responding to the fungal infection earlier than adults ( Figure 8B ). The addition of an inhibitor specific to DSP1 or PLA₂ significantly suppressed the up-regulation of these three POs in response to the fungal infection ( Figure 8C ).

Four AMPs were obtained from GenBank. All AMPs were expressed in different developmental stages, in which most of them except defensin (Fo-Def) showed the higher expressions in adults compared to immature stages ( Figure 9A ). All four AMPs were inducible to the fungal infection, in which larvae up-regulated the AMP expressions earlier than adults except lysozyme ( Figure 9B ). The addition of inhibitors specific to DSP1 or PLA₂ significantly suppressed the up-regulation of these AMPs in response to the fungal infection ( Figure 9C ).