Abstract
Eicosanoids are synthesized from phospholipids by the catalytic activity of phospholipase A2 (PLA2). Even though several PLA2s are encoded in the genome of different insect species, their physiological functions are not clearly discriminated. This study identified four PLA2 genes encoded in the western flower thrips, Frankliniella occidentalis. Two PLA2s (Fo-PLA2C and Fo-PLA2D) are predicted to be secretory while the other two PLA2s (Fo-PLA2A and Fo-PLA2B) are intracellular. All four PLA2 genes were expressed in all developmental stages, of which Fo-PLA2B and Fo-PLA2C were highly expressed in larvae while Fo-PLA2A and Fo-PLA2D were highly expressed in adults. Their expressions in different tissues were also detected by fluorescence in situ hybridization. All four PLA2s were detected in the larval and adult intestines and the ovary. Feeding double-stranded RNAs specific to the PLA2 genes specifically suppressed the target transcript levels. Individual RNA interference (RNAi) treatments led to significant developmental retardation, especially in the treatments specific to Fo-PLA2B and Fo-PLA2D. The RNAi treatments also showed that Fo-PLA2B and Fo-PLA2C expressions were required for the induction of immune-associated genes, while Fo-PLA2A and Fo-PLA2D expressions were required for ovary development. These results suggest that four PLA2s are associated with different physiological processes by their unique catalytic activities and expression patterns.
Similar content being viewed by others
Introduction
Phospholipase A2 (PLA2) catalyzes phospholipids (PLs) to release arachidonic acid (AA), which is usually used for the biosynthesis of eicosanoids1. Eicosanoids mediate various physiological processes such as immunity, metabolism, and reproduction in metazoans including insects2. However, most terrestrial insects possess little AA in their PLs and use linoleic acid, which is subsequently extended and desaturated into AA3. In mammals, AA released from PLs is oxygenated into prostaglandins (PGs) by cyclooxygenase (COX)4. Insects, which do not have COX orthologs, use a specific peroxidase called peroxinectin for the biosynthesis of PGs5. AA is also oxygenated by lipoxygenase (LOX)6. LOX or its equivalent enzyme has been not identified in insects. AA is also oxygenated by epoxygenase (EPX) into four different eicosatrienoic acids (EETs) in mammals and insects7.
All types of eicosanoids play a crucial role in mediating insect immunity8. Upon pathogen infection, the stimulation of sessile hemocytes by PGs induced their mobilization leading to an increase in the total number of circulatory hemocytes within 2 h9. In a beetle, Tribolium castaneum, RNA interference (RNAi) was systemically applied to suppress specific gene expression with high efficiency10. In this system, Toll/IMD signal pathways activated PLA2 to produce PGs and LTs, which led to the expression of specific antimicrobial peptides (AMPs) against different pathogens11. All four types of EETs were detected in a lepidopteran insect, Spodoptera exigua, and were found to mediate both cellular and humoral immune responses12. These findings indicate that PLA2 catalyzes the committed step for the biosynthesis of eicosanoids and finally mediates the immune responses.
After the discovery of the first PLA2 in snake venom, similar disulfide-rich PLA2s were also found in mammalian systems13,14,15,16. The subsequent recognition of non-disulfide bond-containing PLA2s from intracellular sources necessitated the classification of PLA2s into groups17. At least 16 PLA2 groups are now recognized, including five major types: secretory PLA2s (sPLA2s: groups I–III, V, IX, X, XI, XII, XIII, XIV, and XV), calcium-dependent intracellular PLA2 (cPLA2: group IV), calcium-independent intracellular PLA2 (iPLA2: group VI), lipoprotein-associated PLA2 (LpPLA2: groups VII and VIII), and adipose phospholipase A2 (AdPLA2: group XVI)18. sPLA2 and LpPLA2 are secretory proteins that act on extracellular membrane lipids, while cPLA2 and iPLA2 catalyze the hydrolysis of fatty acids from intracellular phospholipids. However, the localization of LpPLA2 and AdPLA2 is not clear.
The western flower thrips, Frankliniella occidentalis, is an invasive insect pest that infests various crops19. It is also known to transmit a plant virus, tomato spotted wilt virus (TSWV)20. The use of chemical insecticides to control this insect pest leads to the development of insecticide resistance21. An entomopathogenic fungus, Beauveria bassiana, was identified as an effective biological control agent22. However, the immune responses of the thrips, which are mediated by eicosanoids, play a crucial role in defending against the virulence of the fungi23. Furthermore, F. occidentalis also exhibits a potent antiviral response against TSWV involving apoptosis and AMPs via eicosanoid mediation24. However, the mechanism of eicosanoid biosynthesis in F. occidentalis remains unclear.
This study identified PLA2 genes from the F. occidentalis genome and analyzed their expressions. Based on their expression profile, the enzyme activities of PLA2 were analyzed in different stages of the development of F. occidentalis. Individual RNAi treatments were applied to assess their independent physiological roles in the development, immunity, and reproduction of F. occidentalis.
Results
Variation in PLA2 enzyme activities of F. occidentalis
All developmental stages of F. occidentalis from larva to adult exhibited PLA2 enzyme activities (Fig. 1). PLA2 enzymes extracted from different stages catalyzed two different phospholipid substrates (AA-PL and non-AA-PL) in a dose-dependent manner. However, the kinetic parameters of enzyme activities differed between the developmental stages (Table 1). Enzyme affinity to substrate measured by Michaelis–Menten constant (km) varied among different stages (F = 3.31; df = 6, 16; P = 0.0258) and between two substrate types (F = 13.93; df = 1, 16; P = 0.0018). Except for the larval stage, other developmental stages preferred AA-PL over non-AA-PL. AA-PL was the most preferred by male adults while non-AA-PL was the most preferred by larvae. The maximal catalytic capacities measured by Vmax varied among different stages (F = 9.58; df = 6, 16; P = 0.0001) and between two substrate types (F = 130.03; df = 1, 16; P < 0.0001). PLA2s of all developmental stages exhibited higher Vmax values in non-AA-PL (2.0–4.9 μmol/min/μg) than in AA-PL (0.13–0.38 μmol/min/μg). In both substrates, adult PLA2s showed higher Vmax values than immature stages.
The differential enzyme activities among developmental stages suggested multiple PLA2s in F. occidentalis. To test this hypothesis, PLA2 inhibitors specific to different PLA2 types were applied to the enzyme extracts (Fig. 2). Under AA-PL substrate, PLA2 activities were significantly suppressed by BEL (a specific inhibitor of iPLA2) treatment in most developmental stages except pupae (Fig. 2a). However, BPB (a specific inhibitor of sPLA2) or MAFP (a specific inhibitor of cPLA2) did not significantly inhibit the enzyme activities. In contrast, PLA2 activities at non-AA-PL substrate (Fig. 2b) were significantly suppressed by BPB treatment in all developmental stages but not by BEL and MAFP. These findings suggested that F. occidentalis possesses multiple PLA2s exhibiting different types of enzyme kinetics.
Four different PLA2 genes of F. occidentalis and their variation in molecular structure and expression profile
Four PLA2 genes (Fo-PLA2A, Fo-PLA2B, Fo-PLA2c, and Fo-PLA2D) were encoded in the F. occidentalis genome (Fig. 3). Functional domain analysis indicated that all four PLA2s have their own catalytic domains predicted to be ‘active site’, ‘patatin-like phospholipase’, or ‘lecithin-cholesterol acetyltransferase’ (Fig. 3a). In addition, two PLA2s (Fo-PLA2C and Fo-PLA2D) are secretory due to the presence of signal peptide domain while the other two (Fo-PLA2A and Fo-PLA2B) are not. In the secretory types, Fo-PLA2C has a calcium-binding site, but Fo-PLA2D does not. In the non-secretory types, Fo-PLA2A has an ankyrin-repeat domain but Fo-PLA2B does not.
A phylogenetic analysis of the four PLA2s along with already identified groups (‘I-XVI’) of PLA2s (Fig. 3b) showed that they were separately clustered with group III (Fo-PLA2C), group VI (Fo-PLA2A), group VIII (Fo-PLA2B), and group XV (Fo-PLA2D). Traditionally, these groups are classified into secretory PLA2 (sPLA2) for group III, calcium-independent and intracellular PLA2 (iPLA2) for group VI, lipoprotein PLA2 (LpPLA2) for group VIII, and lysosomal PLA2 (LPLA2) for group XV.
These four different PLA2 genes were expressed in all developmental stages of F. occidentalis (Fig. 3c). However, their expression patterns varied among different stages. Analysis of these expression variations indicated a distinct difference between the expression patterns of the immature stages and those of adults. Fo-PLA2A and Fo-PLA2D showed a higher expression in adults while Fo-PLA2B and Fo-PLA2C showed a higher expression in immature stages. This distinct pattern was also supported by the phylogenetic pattern analysis.
Fluorescence in situ hybridization (FISH) reveals tissue-specific PLA2s
Intestines and salivary glands of both larvae (Fig. 4a) and adults (Fig. 4b) of F. occidentalis were examined for the expressions of the four PLA2 genes by performing FISH. The four PLA2 transcripts were specifically detected with their antisense probes but not with the sense probes, supporting the specificity of the FISH analysis. Even though all four PLA2 genes were expressed in larvae and adults, the FISH signals against Fo-PLA2A and Fo-PLA2D mRNAs were stronger in the intestinal organs of adults (Fig. 4c). In contrast, Fo-PLA2B and Fo-PLA2C mRNAs were highly expressed in the intestinal organs of larvae. This difference was evident in the different colors of the merged images, in which Fo-PLA2A+Fo-PLA2B+Fo-PLA2D showed a red color in larvae due to a relatively strong expression of Fo-PLA2B but blue-green color in adults due to a relatively strong expression of Fo-PLA2D while Fo-PLA2A+Fo-PLA2B+FoPLA2C showed a white color in both stages.
In adult females, the four PLA2 mRNAs were examined in the ovary (Fig. 5). Four ovarioles were observed in each ovary, in which each ovariole was subdivided into previtellogenic (before formation of follicles), vitellogenic (growing oocytes in follicles), and choriogenic (terminal follicle in the ovariole undergoing chorion formation by follicular epithelium) regions (Fig. 5a). These ovarioles were tethered to the abdominal body wall through terminal filament. Some eggs were detected in the lateral oviduct by ovulation (see ‘egg’ in the oviduct). All four PLA2 genes were expressed in the ovary (Fig. 5b). Most PLA2s except Fo-PLA2D were expressed in the entire ovariole regions (Fig. 5c). Fo-PLA2D mRNA showed a low expression in the terminal ovariole area including the choriogenic follicle.
PLA2s associated with immature development
Multiple PLA2s and their differential expressions suggested that they mediate different physiological processes in F. occidentalis. To test this hypothesis, two different loss-of-function experiments were devised. One approach was to suppress specific gene expressions by using individual RNAi treatments specific to each of the four PLA2 genes. The other approach entailed the use of specific PLA2 inhibitors to suppress the enzyme activities of specific PLA2s.
Individual RNAi against each of the four PLA2 genes was performed using a feeding method with specific dsRNAs (Fig. 6). These RNAi treatments resulted in more than 50% reduction in their target genes while RNAi control specific to nontarget genes did not influence the target gene expressions. Under these RNAi conditions, the development of immature stages of F. occidentalis was monitored (Fig. 7). Two RNAi treatments specific to Fo-PLA2B and Fo-PLA2D expressions led to significant developmental retardation in the immature stages, which resulted in significant mortality (Fig. 7a). However, the other two RNAi treatments specific to Fo-PLA2A and Fo-PLA2C expressions showed no influence on the development of the immature stages.
The larvae were treated with two PLA2 inhibitors (BEL and BPB) via the feeding method (Fig. 7b). Treatment with both inhibitors led to significant developmental retardation of the immature stages, and BEL treatment resulted in significantly higher mortality.
PLA2s associated with immunity
An immune challenge with an entomopathogenic fungus, B. bassiana, significantly up-regulated the expressions of three phenoloxidase (PO) genes (PO1, PO2A, and PO2B) of F. occidentalis in both larvae and adults (Fig. 8). However, individual RNAi treatments specific to most PLA2 genes of F. occidentalis significantly prevented the induction of the PO genes except for RNAi treatment specific to Fo-PLA2D expression (Fig. 8a). Especially, RNAi treatment specific Fo-PLA2B expression significantly inhibited the gene induction in both larvae and adults except for PO2A of larvae. Among the two specific inhibitors, BEL treatment suppressed the induction of PO gene expressions while BPB treatment did not.
The fungal infection also significantly up-regulated the expressions of three PO-activating proteinase (PAP) genes (PAP2A, PAP2B, and PAP3) in both larvae and adults (Fig. 8b). This induction was significantly prevented by RNAi treatments against different Fo-PLA2 genes. In particular, RNAi treatments specific to Fo-PLA2B or Fo-PLA2C expression significantly inhibited the induction of three PAP genes except for PAP2A at the larval stage. Two specific PLA2 inhibitors also prevented the gene induction in response to the fungal infection. BEL treatment suppressed the induction of all three PAP gene expressions while BPB treatment inhibited PAP genes depending on the developmental stage.
The expressions of four different AMP genes were up-regulated in response to fungal infection (Fig. 8c). However, the gene inductions were significantly suppressed by at least one of RNAi treatments specific to four PLA2 genes except for transferrin gene expression at the larval stage. RNAi treatment specific to Fo-PLA2B expression prevented the up-regulation of two AMP genes in both developmental stages. RNAi treatment specific to Fo-PLA2C expression prevented the up-regulation of three AMP genes in adults. Both PLA2 inhibitors also prevented the gene induction of the AMPs in response to the fungal infection. BEL treatment suppressed the induction of most AMP gene expressions except defensin. However, BPB treatment inhibited the induction of defensin at the larval stage. All these loss-of-function assays against AMP expressions suggest that specific PLA2s mediate PO gene expression in response to the immune challenge.
To confirm the physiological roles of these PLA2s in immunity, the fungal virulence against larvae and adults of F. occidentalis was monitored after treatment with PLA2 inhibitors (Fig. 9). Fungal virulence was different in different developmental stages. Adults were more tolerant than larvae with > 50-fold higher median lethal concentration (LC50) though there was little difference in median lethal time (LT50). Both inhibitors of BPB and BEL significantly enhanced the fungal virulence against the thrips in larvae and adults, which led to significant decreases in lethal dose (= LC50) and speed-to-kill (= LT50).
PLA2s associated with reproduction
To determine specific PLA2 gene(s) that mediate ovary development, individual RNAi treatments specific to each of the four PLA2 genes were applied to F. occidentalis (Fig. 10). Two RNAi treatments specific to Fo-PLA2A or Fo-PLA2D expression led to a significant reduction in ovariole development while RNAi treatments specific to Fo-PLA2B or Fo-PLA2C expression did not (Fig. 10a). Especially, the RNAi treatment specific to Fo-PLA2A expression resulted in a significant decrease in fecundity (Fig. 10b). Reduced fecundity was also observed in the females treated with BEL, but not in those treated with BPB.
Discussion
Transcriptome analyses suggested that eicosanoids mediate the immune responses of F. occidentalis against viral or fungal infections23,24. However, the biosynthetic activity of the eicosanoids in the thrips against pathogen infections is not well characterized. To better understand the roles of eicosanoids in the thrips, this study focused on PLA2, which catalyzes the committed step in the biosynthesis of eicosanoids by assessing its biochemical characteristics and its catalytic modulation upon immune challenge. In particular, this study analyzed the independent roles of the four different PLA2s encoded in F. occidentalis by assessing the development and reproduction after individual RNAi treatments.
Four PLA2 genes are encoded and expressed in F. occidentalis. In addition to RT-qPCR, FISH was performed to determine their expressions in larval midgut and adult ovaries. They are classified into sPLA2 and iPLA2. All four PLA2s were expressed in larvae and adults of thrips. PLA2 have been found in all biological systems from bacteria to humans and are classified into at least 16 groups (I–XIV) based on their amino acid sequences8. These diverse PLA2s are divided into sPLA2, iPLA2 (Ca2+-independent cellular PLA2), and cPLA2 (Ca2+-dependent cellular PLA2). Groups III (Fo-sPLA2C) and XV (Fo-sPLA2D) are sPLA2s whereas groups VI (Fo-PLA2A) and VIII (Fo-PLA2B) are iPLA2s. No cPLA2 was encoded in the F. occidentalis genome like in other insects. Most insect sPLA2s are classified into group III, which are divided into venomous and non-venomous PLA2s8. This study identified a novel sPLA2 (= Fo-sPLA2D) in insects, which was classified into group XV. This type of PLA2 was first reported in a cnidarian invertebrate, Adamsia carciniopados, and has been regarded as an ancient PLA2 prototype25. Two different types of group VI PLA2s are found in insects and classified based on the presence of ankyrin repeat domains8. Fo-PLA2A has three ankyrin domains and is classified into ankyrin type of iPLA2. In contrast, Fo-PLA2B is a novel insect PLA2 classified into group VIII. The catalytic activity of group VIII PLA2s is known to be Ca++-independent and they display platelet-activating factor acetylhydrolase activity2. Thus, this study reports two novel insect PLA2s in F. occidentalis classified into groups VIII and XV.
All four Fo-PLA2 genes were expressed in different developmental stages. This explains the detection of PLA2 activities in all developmental stages. However, the PLA2 activities measured with two different substrates varied among developmental stages. Based on KM values indicating substrate affinity of the PLA2, adults preferred AA-linked phospholipid while larvae preferred non-AA phospholipid. This suggests that the different developmental stages of the thrips possess different compositions of the four PLA2s because cellular PLAs prefer AA type while sPLA2s do not have preference2. The different PLA2 compositions among the developmental stages were supported by the differential susceptibilities to different inhibitors. First, MAFP which is a specific inhibitor of cPLA2 activity26 did not inhibit the PLA2 activities in all the developmental stages. Second, BEL, a specific inhibitor of iPLA227 inhibited the enzyme activities only in AA type substrate, in which it significantly inhibited the enzyme activities of larvae and adults, but not that of pupae. Third, BPB a specific inhibitor of sPLA2 activity28 inhibited the enzyme activities in all developmental stages only in non-AA type substrate. These findings suggest that different developmental stages of F. occidentalis possess different PLA2 activities in terms of catalytic activity and substrate preference probably by modulating differential expression of the four PLA2 genes to mediate the specific physiological processes. This differential expression of the four PLA2 genes may explain the relative preference for AA-PL in male adults and non-AA-PL in larvae measured by KM values.
Inhibition of PLA2 activity led to developmental retardation of F. occidentalis. Both BPB and BEL, which are inhibitors of PLA2 activities of F. occidentalis, significantly interfered with the immature development of the thrips. Individual RNAi treatments specific to each of the PLA2 genes also resulted in developmental retardation, in which suppression of Fo-PLA2C expression was the most effective. sPLA2 is secreted from the midgut and plays a crucial role in digesting dietary lipids in Manduca sexta29,30. The mechanism of lipid digestion in insects is controversial due to the lack of bile salts to solubilize dietary lipids. According to a hypothesis, sPLA2 provides lysophospholipid (LPL) from dietary phospholipids to act like insect bile salts1. sPLA2 of S. exigua secreted from the midgut was specifically inhibited by benzylideneacetone (BZA), a specific PLA2 inhibitor, by feeding to larvae, which led to a significant decrease in gut content sPLA2 activity, body growth, and total hemolymph lipid contents31. However, the addition of a specific LPL, 1-palmitoyl-sn-glycero-3-phosphocholine, to BZA-treated larvae significantly rescued the digestibility and subsequent larval growth31. These findings explain the developmental retardation of the thrips by the suppression of PLA2 activities and suggest a digestive role of Fo-PLA2C in the thrips.
PLA2 activity of F. occidentalis was associated with immune responses and its suppression increased the susceptibility to a fungal pathogen, B. bassiana. Both BEL and BPB significantly enhanced the fungal virulence to the thrips in both larvae and adults. This supports a previous study23 which demonstrated a crucial role of eicosanoid-mediated immune responses in defending fungal pathogenicity. A pertinent question is which of the four PLA2(s) mediates the immune responses in F. occidentalis. Individual RNAi treatments showed that all four PLA2s mediated the gene expressions associated with AMPs, melanization, or production of reactive oxygen species. The regulation of the expression of immune-associated genes by the PLA2 activities may be performed by eicosanoids synthesized from the PLA2 catalytic activity32. A transcriptional factor called Repat33 is a downstream component of the eicosanoid immune signaling pathway in S. exigua, in which Repat33 mediates the immune-associated gene expression and cellular immune response33. In addition, prostaglandin E2 (PGE2) receptors have been identified in lepidopteran and dipteran insects and their downstream signals up-regulate cAMP and Ca2+ levels34,35,36. Thus, the elevated PLA2 activity upon immune challenge upregulates PLA2 activity, which produces eicosanoids to mediate the expressions of the immune-associated genes. In particular, Fo-PLA2B and Fo-PLA2C may play crucial roles in mediating various immune responses in F. occidentalis because their RNAi treatments mostly interfered with the immune-associated genes.
Oocyte development in F. occidentalis was found to be dependent on PLA2 activity. In particular, iPLA2 activity played a crucial role in oocyte development because treatment with BEL but not BPB inhibited oocyte development. Furthermore, in the individual RNAi treatments, suppression of Fo-PLA2A expression significantly inhibited oocyte development. In addition, RT-qPCR and FISH analysis showed high expression of Fo-PLA2A in adults. In Drosophila melanogaster and S. exigua, PGE2 was shown to mediate nurse cell dumping during early vitellogenesis in the polytrophic ovarioles37,38. In F. occidentalis, PGE2 was detected in growing ovarian follicles by an immunofluorescence assay, and aspirin (a specific cyclooxygenase inhibitor) treatment significantly suppressed the oocyte development in previtellogenesis and choriogenesis39. The present study suggests that Fo-PLA2A may play a crucial role in the production of PGE2 in the ovary to facilitate the oocyte development in F. occidentalis.
These results suggest that the four Fo-PLA2s are required for mediating development, immunity, and reproduction with their unique enzyme activities and differential expressions in different developmental stages and tissues of F. occidentalis. Specifically, our inhibitor assays coupled with individual RNAi treatments suggest that Fo-PLA2C mediates physiological processes especially in development, Fo-PLA2A in reproduction, and Fo-PLA2B/C in immunity though all four PLA2s are associated with the different physiological processes.
Experimental procedures
Thrips rearing and fungal culture
Adult F. occidentalis were collected from a hot pepper field in Andong, Korea, and reared on sprouted bean seed kernels under controlled laboratory conditions: a constant temperature of 27 ± 1 °C, photoperiod of 16:8 h (L:D), and relative humidity (RH) of 60 ± 5%. Under these conditions, the thrips underwent three larval stages (L1-L3), prepupa, and pupa before the emergence of adult. L2 stage and young adults (< 3 days after adult emergence) were used for pathogenicity tests. B. bassiana, an entomopathogenic fungus, was cultured for 14 days in a potato dextrose agar (PDA) plate at 25 ± 1 °C, 70 ± 5% relative humidity, and a 16:8 h (L:D) photoperiod.
Chemicals
PLA2 assay kits and methyl arachidonyl fluorophosphonate (MAFP) were purchased from Cayman Chemical (Ann Arbor, MI, USA). p-Bromophenacyl bromide (BPB), bromoenol lactone (BEL), bovine serum albumin (BSA), dimethylsulfoxide (DMSO), and t-octylphenoxy-polyethoxyethanol (Triton X-100) were purchased from Sigma Aldrich Korea (Seoul, Korea). Phosphate-buffered saline (PBS) was prepared with 100 mM phosphoric acid. Its pH was calibrated to 7.4 using 1 N NaOH.
PLA2 enzymatic activity
PLA2 activities in whole bodies of 100 individuals at each stage (L2 larva, pupa, and adult) were measured using sPLA2 and cPLA2 assay kits (Cayman Chemical) containing arachidonyl thio-phosphatidyl choline (PC) and diheptanoyl thio-PC substrates, respectively, based on the method described by Vatanparast et al.7. Whole body extracts were obtained after homogenizing in PBS. Moreover, to assess specific PLA2 inhibitors (BPB, BEL, and MAFP) were pre-incubated at 400 µM final concentration with each enzyme for 15 min, and residual enzyme activities were measured at 25 °C. All treatments were replicated three times. Protein concentration was determined by Bradford40 assay using BSA as standard.
Bioinformatics and phylogenetic analyses of PLA2s
Phospholipase A2 sequences (Fo-PLA2A, Fo-PLA2B, Fo-PLA2C, and Fo-PLA2D) of F. occidentalis were obtained from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) with accession numbers of XM_026421156.2, XM_026433123, XM_026415753, and XM_026437429, respectively. Phylogenetic analyses were performed using MEGA6.06 and ClustalW programs from EMBL-EBI (www.ebi.ac.uk). Bootstrapping values were obtained with 1000 iterations to support branching and clustering. Conserved domains of the four PLA2s were predicted using the NCBI Conserved Domain Database (www. ncbi.nlm.nih.gov/cdd).
FISH assay
To localize the four PLA2s in different tissues, the larval guts and adult ovaries were isolated onto a sterilized glass slide and fixed with 4% paraformaldehyde for 1 h at room temperature (RT). After washing with PBS, the tissues were permeabilized with 1% Triton X-100 in PBS for 2 h at RT. After washing with PBS, the tissues were rinsed in 2 × sodium saline citrate (SSC) and incubated at 42 °C with 25 μL of pre-hybridization buffer (2 μL yeast tRNA, 2 μL 20 × SSC, 4 μL dextran sulfate, 2.5 μL 10% SDS, and 14.5 μL deionized H2O) in dark and humid conditions for 1 h. Then, the pre-hybridization buffer was replaced with hybridization buffer (5 μL deionized formamide and 1 μL fluorescein-labeled oligonucleotide in 19 μL of the pre-hybridization buffer). DNA oligonucleotide probes were labeled at the 5′ ends with fluorescein amidite (FAM), rhodamine, or marina blue, which were purified using high-performance liquid chromatography (Bioneer, Daejeon, Korea). The probe sequences are listed in Table S1. The slides were covered with an RNAse-free cover slip and kept overnight (16–17 h) in a humid chamber at 42 °C. After hybridization, the tissues were washed twice with 4 × SSC for 10 min each and incubated with 4 × SSC containing 1% Triton X-100 in RT for 5 min. After washing three times with 4 × SSC, tissue samples were incubated at 37 °C with 1% anti-rabbit antibody (Thermo Fisher Scientific, Wilmington, DE, USA) in PBS under dark conditions for 30 min. After incubation, the tissues were washed twice with 4 × SSC for 10 min each, once with 2 × SSC, and then allowed to dry in air. After adding a drop of 50% glycerol and incubating at RT for 15 min, samples were covered by cover slip, and the slides were observed under a fluorescence microscope (DM2500, Leica, Wetzlar, Germany).
RNA extraction and RT-qPCR
Total RNA was extracted from different developmental stages of F. occidentalis using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. An experimental unit consisted of approximately 100 larvae, pupae, and adults. The extracted RNAs were quantified using a spectrophotometer (NanoDrop, Thermo Fisher Scientific). RNA extract (100 ng per reaction) was used for cDNA synthesis with an RT-premix (Intron Biotechnology, Seoul, Korea). 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 Biosystems, Singapore). The reaction mixture (20 μL) contained 10 pmol of gene-specific primers (Table S2) 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 (Table S2) for 30 s, and extension at 72 °C for 30 s. The target gene expression levels were normalized to those of EF1, a reference gene. Each treatment was replicated with three independently prepared biological samples. Quantitative analysis was performed using the comparative CT (2−ΔΔCT) method41.
RNA interference (RNAi)
Double-stranded RNAs (dsRNAs) were used for RNAi. To prepare dsRNAs specific to different genes, template DNAs were amplified with forward and reverse gene-specific primers containing the T7 promoter sequence at their 5ʹ ends. The resulting T7 promoter-tagged template DNAs were used to construct dsRNAs using the MEGAscript RNAi kit (Ambion, Austin, TX, USA). The newly-formed dsRNAs were mixed with Metafectene PRO (Biontex, Plannegg, Germany), a transfection reagent, at a 1:1 (v/v) ratio, and incubated at 25 °C for 30 min to form liposomes. These dsRNAs were treated by the feeding delivery method. Briefly, the beans were soaked in a dsRNA suspension at 500 μg/mL for 20 min. After removing the excess moisture, the treated beans were placed in a circular breeding container (100 mm × 40 mm) for 24 h, accessible to F. occidentalis individuals. RNAi efficiency was evaluated at different time intervals by RT-qPCR. Each treatment was replicated three times.
Effect of RNAi treatment on immature development
Specific dsRNAs were used to evaluate the different PLA2 functions in the immature developmental period. Within 6 h after the emergence of the first instar larvae, 10 newly emerged larvae were fed beans soaked in PLA2 dsRNAs. The treated diet was replaced every 24 h and the developmental stage period was measured every day till adult emergence. Three repetitions were performed for each treatment.
Effect of RNAi treatment on reproductive processes
Within six hours of adult emergence, a group consisting of twelve females and two males were provided with beans soaked in the dsRNA suspensions for a continuous 36-h period, with the beans being replaced every 12 h. Subsequently, fresh untreated bean cotyledons were provided for 48 h to facilitate egg laying by the test thrips. The count of eggs laid by the females over two days was determined by observing the newly hatched larvae on the beans. Furthermore, the same treatment method was employed to evaluate ovary size. After treatment, the ovaries were dissected and isolated, and their size was measured. Each treatment was replicated three times.
Effects of inhibitors (BPB and BEL) on immature development
To evaluate the impact of PLA2 inhibitors on the developmental period, 10 newly emerged first-instar larvae were provided with beans soaked in inhibitors (BEL and BPB) within 6 h of their emergence. The treated diet was refreshed every 24 h, and the developmental stages and mortality were monitored daily until adult emergence. This experiment was replicated three times for each treatment.
Effects of inhibitors (BPB and BEL) on reproductive processes
Six hours after adult emergence, adult thrips were exposed to beans soaked in PLA2 inhibitors (BPB and BEL) for a continuous 36-h period, renewing the treated beans every 12 h. Subsequently, untreated fresh bean cotyledons were provided for 48 h to facilitate egg deposition. The count of eggs laid by the females over two days was determined by monitoring the emergence of newly hatched larvae on the beans. Each treatment involved 10 females and 2 males, and the experiment was replicated three times.
Preparation of B. bassiana suspension and its virulence against F. occidentalis
Conidial suspension of B. bassiana was prepared by collecting the fungal colonies cultured on PDA medium in 1 mL of Triton X-100 (0.1%) (Duksan Pure Chemicals, Ansan, Korea) in PBS. Conidia of the suspension were counted using a Neuberger hemocytometer (Marienfeld-Superior, Lauda-Königshofen, Germany) under 40 × magnification.
To assess the virulence of B. bassiana, L2 larvae and adults were fed with different concentrations (1 × 104, 1 × 105, 1 × 106, 1 × 107, 1 × 108, 1 × 109 1 × 1010 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. After L2 larvae or adults were released into a petri dish (5 × 2 cm), the 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 Bates42. Dead insects were counted every 24 h up to 6 days by confirming mycosis development on insect cadavers. Three replicas of each treatment were used and each replicate used 20 insects.
Data analysis
Analysis of variance (ANOVA) followed by post hoc Tukey's test were conducted for statistical analysis using GraphPad Prism version 8.2.0 (La Jolla, CA, USA). Bioassay data were used to estimate the median lethal concentration (LC50) and time (LT50) using PoloPlus43. Significant differences between LC50 values were determined as described by Robertson et al.44.
Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information file. The genome sequence datasets generated and/or analyzed during the current study are available in the GenBank repository using accession numbers in Table S1.
References
Dennis, E., Cao, J., Hsu, Y. H., Magrioti, V. & Kokotos, G. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem. Rev. 111, 6130–6185 (2011).
Stanley, D. W. Eicosanoids in Invertebrate Signal Transduction Systems (Princeton University Press, 2000).
Hasan, M. A., Ahmed, S. & Kim, Y. Biosynthetic pathway of arachidonic acid in Spodoptera exigua in response to bacterial challenge. Insect Biochem. Mol. Biol. 111, 103179 (2019).
Tootle, T. L. Genetic insights into the in vivo functions of prostaglandin signaling. Int. J. Biochem. Cell Biol. 45, 1629–1632 (2013).
Park, Y., Kumar, S., Kanumuri, R., Stanley, D. & Kim, Y. A novel calcium-independent cellular PLA2 acts in insect immunity and larval growth. Insect Biochem. Mol. Biol. 66, 13–23 (2015).
Feussner, I. & Wasternack, C. The lipoxygenase pathway. Annu. Rev. Plant Biol. 53, 275–297 (2002).
Vatanparast, M., Ahmed, S., Sajjadian, S. M. & Kim, Y. A prophylactic role of a secretory PLA2 of Spodoptera exigua against entomopathogens. Dev. Comp. Immunol. 95, 108–117 (2019).
Kim, Y., Ahmed, S., Stanley, D. & An, C. Eicosanoid-mediated immunity in insects. Dev. Comp. Immunol. 83, 130–143 (2018).
Park, J. & Kim, Y. Change in hemocyte populations of the beet armyworm, Spodoptera exigua, in response to bacterial infection and eicosanoid mediation. Korean J. Appl. Entomol. 51, 349–356 (2012).
Mingels, L. et al. Extracellular vesicles spread the RNA interference signal of Tribolium castaneum TcA cells. Insect Biochem. Mol. Biol. 122, 103377 (2020).
Shrestha, S. & Kim, Y. Activation of immune-associated phospholipase A2 is functionally linked to Toll/Imd signal pathways in the red flour beetle, Tribolium castaneum. Dev. Comp. Immunol. 34, 530–537 (2010).
Vatanparast, M. et al. EpOMEs act as immune suppressors in a lepidopteran insect, Spodoptera exigua. Sci. Rep. 10, 20183 (2020).
Davidson, F. F. & Dennis, E. A. Amino acid sequence and circular dichroism of Indian cobra (Naja naja naja) venom acidic phospholipase A2. Biochim. Biophys. Acta 1037, 7–15 (1990).
Davidson, F. F. & Dennis, E. A. Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms. J. Mol. Evol. 31, 228–238 (1990).
Kramer, R. M. et al. Structure and properties of a human non-pancreatic phospholipase A2. J. Biol. Chem. 264, 5768–5775 (1989).
Seilhamer, J. J. et al. Novel gene exon homologous to pancreatic phospholipase A2: sequence and chromosomal mapping of both human genes. J. Cell. Biochem. 39, 327–337 (1989).
Dennis, E. A. Diversity of group types, regulation, and function of phospholipase A2. J. Biol. Chem. 269, 13057–13060 (1994).
Vasquez, A. M., Mouchlis, V. D. & Dennis, E. A. Review of four major distinct types of human phospholipase A2. Adv. Biol. Regul. 67, 212–218 (2018).
Reitz, S. R. et al. Invasion biology, ecology, and management of western flower thrips. Annu. Rev. Entomol. 65, 17–37 (2020).
Rotenberg, D., Jacobson, A. L., Schneweis, D. J. & Whitfield, A. E. Thrips transmission of tospoviruses. Curr. Opin. Virol. 15, 80–89 (2015).
Zhang, B., Qian, W., Qiao, X., Xi, Y. & Wan, F. Invasion biology, ecology, and management of Frankliniella occidentalis in China. Arch. Insect Biochem. Physiol. 102, e21613 (2019).
Zhang, X., Lei, Z., Reitz, S. R., Wu, S. & Gao, Y. Laboratory and greenhouse evaluation of a granular formulation of Beauveria bassiana for control of western flower thrips, Frankliniella occidentalis. Insects 10, 58 (2019).
Ahmed, S., Roy, M. C., Choi, D. & Kim, Y. HMG-like DSP1 mediates immune responses of the western flower thrips (Frankliniella occidentalis) against Beauveria bassiana, a fungal pathogen. Front. Immunol. 13, 875239 (2022).
Kim, C. Y., Ahmed, S., Stanley, D. & Kim, Y. HMG-like DSP1 is a damage signal to mediate the western flower thrips, Frankliniella occidentalis, immune responses to tomato spotted wilt virus infection. Dev. Comp. Immunol. 144, 104706 (2023).
Talvinen, K. A. & Nevalainen, T. J. Cloning of a novel phospholipase A2 from the cnidarian Adamsia carciniopados. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 132, 571–578 (2002).
Lio, Y. C., Reynolds, L. J., Balsinde, J. & Dennis, E. A. Irreversible inhibition of Ca2+-independent phospholipase A2 by methyl arachidonyl fluorophosphonate. Biochim. Biophys. Acta 1302, 55–60 (1996).
Ackermann, E. J., Conde-Frieboes, K. & Dennis, E. A. Inhibition of macrophage Ca2+-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J. Biol. Chem. 270, 445–450 (1995).
Park, Y. & Kim, Y. Xenorhabdus nematophilus inhibits p-bromophenacyl bromide (BPB)-sensitive PLA2 of Spodoptera exigua. Arch. Insect Biochem. Physiol. 54, 134–142 (2003).
Stanley, D. W., Sarath, G. & Rana, R. L. A digestive phospholipase A2 in midguts of tobacco hornworms, Manduca sexta L.. J. Insect Physiol. 44, 297–303 (1998).
Rana, R. L. & Stanley, D. W. In vitro secretion of digestive phospholipase A2 by midguts isolated from tobacco hornworms, Manduca sexta. Arch. Insect Biochem. Physiol. 42, 179–187 (1999).
Sajjadian, S. M., Vatanparast, M., Stanley, D. & Kim, Y. Secretion of secretory phospholipase A2 into Spodoptera exigua larval midgut lumen and its role in lipid digestion. Insect Mol. Biol. 28, 773–784 (2019).
Kim, Y. & Stanley, D. Eicosanoid signaling in insect immunology: New genes and unresolved issues. Genes 12, 211 (2021).
Hrithik, M. T. H., Vatanparast, M., Ahmed, S. & Kim, Y. Repat33 acts as a downstream component of eicosanoid signaling pathway mediating immune responses of Spodoptera exigua, a lepidopteran insect. Insects 12, 449 (2021).
Kwon, H. et al. Characterization of the first insect prostaglandin (PGE2) receptor: MansePGE2R is expressed in oenocytoids and lipoteichoic acid (LTA) increases transcript expression. Insect Biochem. Mol. Biol. 117, 103290 (2020).
Kim, Y. et al. Deletion mutant of PGE2 receptor using CRISPR-Cas9 exhibits larval immunosuppression and adult infertility in a lepidopteran insect. Spodoptera exigua. Dev. Comp. Immunol. 111, 103743 (2020).
Ahmed, S. & Kim, Y. Prostaglandin catabolism in Spodoptera exigua, a lepidopteran insect. J. Exp. Biol. 223, jeb233221 (2020).
Tootle, T. L. & Spradling, A. C. Drosophila Pxt: a cyclooxygenase-like facilitator of follicle maturation. Development 135, 839–847 (2008).
Al Baki, M. A. & Kim, Y. Inhibition of prostaglandin biosynthesis leads to suppressed ovarian development in Spodoptera exigua. J. Insect Physiol. 114, 83–91 (2019).
Choi, D. Y. & Kim, Y. Transcriptome analysis of female western flower thrips, Frankliniella occidentalis, exhibiting neo-panoistic ovarian development. PLoS ONE 17, e0272399 (2022).
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254 (1976).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data analysis using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25, 402–408 (2001).
Winston, P. W. & Bates, D. H. Saturated solutions for the control of humidity in biological research. Ecology 41, 232–237 (1960).
Software, L. O. Version 2.0. Polo plus: A user’s guide to probit or logit analysis. Petaluma, CA: LeOra Software Company (2007).
Robertson, J. L., Jones, M. M., Olguin, E. & Alberts, B. Bioassays with Arthropods (CRC Press, 2017).
Acknowledgements
This work was supported by a grant (2022R1A2B5B03001792) to YK from the National Research Foundation (NRF) funded by the Ministry of Science, ICT and Future Planning, Republic of Korea.
Funding
Funding was provided by National Research Foundation of Korea (Grant number: 2022R1A2B5B03001792, 2022R1A2B5B03001792).
Author information
Authors and Affiliations
Contributions
M.E.: methodology, validation, formal analysis, investigation, writing. Y.K.: conceptualization, writing, writing—review and editing, supervision, project administration.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Esmaeily, M., Kim, Y. Four phospholipase A2 genes encoded in the western flower thrips genome and their functional differentiation in mediating development and immunity. Sci Rep 14, 9766 (2024). https://doi.org/10.1038/s41598-024-60522-8
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-024-60522-8
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.