Lipidomics Reveals How the Endoparasitoid Wasp Pteromalus Puparum Manipulates Host Energy Stores for Its Young

Lipidomics reveals how the endoparasitoid wasp Pteromalus puparum manipulates host energy stores for its young

Item Type Article

Authors Wang, Jiale; Jin, Hongxia; Schlenke, Todd; Yang, Yi; Wang, Fang; Yao, Hongwei; Fang, Qi; Ye, Gongyin

Citation Wang, J., Jin, H., Schlenke, T., Yang, Y., Wang, F., Yao, H., ... & Ye, G. (2020). Lipidomics reveals how the endoparasitoid wasp Pteromalus puparum manipulates host energy stores for its young. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 158736.

DOI 10.1016/j.bbalip.2020.158736

Publisher ELSEVIER

Journal BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS

Download date 08/10/2021 08:45:24

Item License http://rightsstatements.org/vocab/InC/1.0/

Version Final accepted manuscript

Link to Item http://hdl.handle.net/10150/648043 Lipidomics reveals how the endoparasitoid wasp Pteromalus puparum manipulates host energy stores for its young

Jiale Wang 1,2, Hongxia Jin 1, Todd Schlenke 2, Yi Yang 1, Fang Wang1, Hongwei Yao1, Qi Fang1 and Gongyin Ye 1*

1State Key Laboratory of Rice Biology & Agricultural and Rural Affairs Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China 2Department of Entomology, University of Arizona, Tucson AZ, USA

*Corresponding author: Gongyin Ye, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China (e-mail: [email protected]).

Highlights:  After the infection by an endoparasitoid Pteromalus puparum, levels of 87 and 117 lipids significantly change in the fat body and hemolymph of the cabbage butterfly Pieris rapae, respectively.  In host P. rapae fat body, levels of highly unsaturated triacylglycerides increased post-infection while more saturated forms decreased.  Membrane phospholipid levels mostly decreased in host fat body but increased in hemolymph post-infection.  Levels of the necessary dietary lipid for insects - cholesteryl esters - dramatically increased in host hemolymph following infection.

Abstract: Endoparasitoid wasps inject venom along with their eggs to adjust the physiological and nutritional environment inside their hosts to benefit the development of their offspring. In particular, wasp venoms are known to modify host lipid metabolism, lipid storage in the fat body, and release of lipids into the hemolymph, but how venoms accomplish these functions remains unclear. Here, we use an UPLC-MS-based lipidomics approach to analyze the identities and concentrations of lipids in both fat body and hemolymph of host cabbage butterfly (Pieris rapae) infected by the pupal endoparasitoid Pteromalus puparum. During infection, host fat body levels of highly unsaturated, soluble triacylglycerides (TAGs) increased while less unsaturated, less soluble forms decreased. Furthermore, in infected host hemolymph, overall levels of TAG and phospholipids (the major component of cell membranes) increased, suggesting that fat body cells are destroyed and their contents are dispersed. Altogether, these data suggest that wasp venom induces host fat body TAGs to be transformed into lower melting point (more liquid) forms and released into the host hemolymph following infection, allowing simple absorption and nutritional acquisition by wasp larvae. Finally, cholesteryl esters (CEs, a dietary lipid derived from cholesterol) increased in host hemolymph following

infection with no concomitant decrease in host cholesterol, implying that the wasp may provide this necessary food resource to its offspring via its venom. This study provides novel insight into how parasitoid infection alters lipid metabolism in insect hosts, and begins to uncover the wasp venom proteins responsible for host physiological changes and offspring development.

Keywords: Lipidomics; Hemolymph; Fat body; Pteromalus puparum; Pieris rapae; Lipid.

1 Introduction

Parasitoid wasps are used in the biocontrol of insect pest populations around the world [1]. These wasps lay their eggs inside (endoparasitoids) or outside (ectoparasitoids) of their hosts’ bodies, and once hatched the wasp larvae consume their hosts as they proceed through development. Juvenile wasps are thus reliant on their hosts for most of their developmental nutrition needs.

Many parasitoid wasp lineages have lost the ability to synthesize lipids themselves, instead evolving strategies for efficiently obtaining lipids from hosts [2-4]. For example, adult female wasps inject venom (virulence) proteins, which function to regulate host physiology, into their hosts while laying eggs [5-12]. Venom proteins can play important roles in modifying host lipid metabolism, like altering host lipid synthesis and accumulation, and causing hosts to release lipids from their main storage tissue, the fat body [13]. All of these effects are thought to help provision the developing wasp larvae. Although wasp venoms are essential for host manipulation and offspring development, we still lack a detailed understanding of how venoms actually regulate host lipid availability.

Parasitoids have evolved multiple times in the wasps [14], as have the necessary virulence strategies and the parasitoid-associated factors that accomplish them. In some parasitoid wasp lineages (e.g. Braconidae, Ichneumonidae ), virulence factors are made up of individual venom proteins as well as supramolecular structures like polydnaviruses (PDVs) and virus-like particles (VLPs) [15, 16]. The proteins that make up these structures can be difficult to functionally characterize individually, and thus we have focused on a wasp species representing a lineage that mainly depends on venom but lacks those supramolecular factors PDVs or VLPs: Pteromalus puparum (Hymenoptera: Pteromalidae). P. puparum is a pupal endoparasitoid that regularly infects Pieris rapae (Lepidoptera: Pieridae), the “small white” butterfly or “cabbage

worm”, a worldwide pest of cabbage and other mustard family crops. After parasitization, infected P. rapae pupa stop developing and change color from green- gray to brown as the wasp larvae develop inside of them. The numerous single-protein venoms that P. puparum produces to enable successful infection have been identified, including lipases and phospholipases, but so far the venom constituents have only been functionally characterized in relation to their ability to suppress the host immune response against wasp eggs [6, 8]. Although P. puparum is one of the small proportion of parasitoids that have re-evolved the ability to synthesize some lipids [3], we assume that lipid acquisition from hosts remains a requisite of juvenile development. Thus, we believe that the P. puparum - P. rapae system is ripe for exploiting as a model system for how and why parasitoid infection alters lipid metabolism in insect hosts.

Lipids are a class of organic compounds made up of fatty acids or their derivatives that have multiple roles in living organisms, including energy storage, signal transduction, and cell membrane building blocks. The past 25 years have seen a remarkable increase in our ability to differentiate and identify lipids from biological samples, leading to the new field of lipidomics [17-20]. The typical approach uses liquid chromatography and mass spectrometry to identify and quantify lipid species. Lipidomics data representing different biological treatments help illuminate the molecular biology of lipid metabolism as well as the functional roles that various lipids play in host physiology.

Here, we used an ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-TOF-MS)-based lipidomics approach to analyze the identities and concentrations of lipids in P. rapae hosts parasitized by P. puparum wasps. We found that concentrations of 30 lipid components increased but 57 decreased in the host fat body post-infection, while 89 increased but 28 decreased in

the host hemolymph post-infection. Analysis of these data provides novel insight into how parasitoid wasp venom alters host lipid metabolism for the benefit of the developing wasp larvae.

2 Materials and Methods 2.1 Insect rearing Young P. rapae larvae were collected from greenhouse-grown cabbage in the suburbs of Hangzhou, China. Subsequently, larvae were maintained in the lab under the condition of 10 h: 14 h (light: darkness) photoperiod at 25 ± 1℃ and fed fresh cabbage until pupation. Adult P. puparum were reared as described by Cai et al. [21] under the same condition above and fed with 20% (v/v) honey solution to lengthen their life spans.

2.2 Samples collection For experimental infections, we selected P. rapae pupae of similar sizes that had pupated within the previous 6 h, and then independently exposed them to a two-day- old mated endoparasitoid P. puparum female. They were placed together in a plastic tube (18 × 82 mm). To minimize wasp superparasitism in hosts, we used a light parasitization method [22] where we ensured that only a single oviposition occurred within the period of 1 h. When this occurred, the parasitoid wasp was removed and the P. rapae pupae were left alone for 24 h (the parasitized groups). Uninfected control P. rapae were treated the same except without parasitization.

Parasitoid larvae consume host hemolymph early on in development before feeding directly on the host fat body and other hard tissues later in development [23, 24]. Thus, we made lipid extractions from the host fat body and hemolymph, separately. Furthermore, P. puparum embryos hatch in 1.5 d under our experimental conditions, so assaying host lipids 25 h post-infection is not influenced by the presence of any parasitoid larvae.

2.3 Pieris rapae lipid extraction

The collected P. rapae pupae were surface-sterilized with 75% ethanol. A dissecting pin was used to poke a hole in the cuticle near the pupa wing bud, and the hemolymph was collected based on the method described by Fang et al [25]. Hemolymph was transferred into a sterilized 1.5 ml Eppendorf tube containing 5 μl of supersaturated 1- phenyl-2-thiourea (PTU, a chelator of copper ions acting as an inhibitor of phenoloxidase) (Sigma, Taufkirchen, Germany) solution in 10 mM phosphate- buffered saline (PBS) using a micropipette (hemolymph from three pupae per tube). After the gentle centrifugation at 200 g for 10 min at 8℃, the supernatant was transferred to a new clean tube. The same pupae were used for fat body dissections.

Dissected fat bodies were cleaned with ddH2O several times, and then moved into 1.5 ml tubes (3 pupae per tube). All samples were stored at -80℃ until used in the UHPLC-QTOF-MS.

2.4 Lipid sample preparation The frozen fat body samples (15 ± 0.5 mg) were homogenized in 350 μl ice-cold methanol (Honeywell, USA)/water (2: 5, v/v) and then processed by ultra-sonication (100 Hz) for 2 min (5 cycles with 3-minute intervals) at - 80°C. 500 μl MTBE (methyl tert-butyl ether) was then added and the mixture was centrifuged at 3000 rpm for 15 min. 400 μl of supernatant was pipetted out nitrogen-drying, and re-dissolved in 200 μl dichloromethane/methanol (1: 1, v/v) for UHPLC-QTOF-MS analysis. 5 μl of this solution was injected for ESI- mode, the remaining solution was diluted 1: 10 (10%, v/v) and 2 μl was injected for ESI+ mode. Similarly, each 20 μl hemolymph sample was mixed with 280 μl of ice-cold methanol/water (2: 5, v/v) and 400 μl MTBE. The mixture was vortexed for 1 min and maintained at 4°C for 2 h, followed by centrifugation at 3000 rpm for 15 min. 280 μl of supernatant was removed for evaporation drying using nitrogen, and re-dissolved in 100 μl of dichloromethane/methanol (1: 1, v/v) for UHPLC-QTOF-MS analysis. 0.5 μl of this solution was injected for ESI+ mode or 4 μl for ESI- mode. This work, as well as the

following UHPLC-QTOF-MS and statistical analysis steps were performed at

Shanghai ProfLeader Biotech Co. (China).

2.5 UHPLC-QTOF-MS The chromatographic separation was performed on an Agilent UHPLC system (1290) with a Phenomenex Kinetex C18 column (2.1mm × 100 mm, 1.7 μm) at a flow speed of 0.3 ml/min and 55°C column temperature. The eluent was analyzed in both positive and negative ion modes on a hybrid quadrupole time-of-flight mass spectrometer (Triple TOF 5600 system, AB Sciex, Concord, ON, Canada) equipped with a DuoSpray ion source. The pressures of nebulizer gas (GS1), heater gas (GS2) and curtain gas (CUR) were set to 60 psi, 60 psi, and 30 psi, respectively. The ion source temperature was 600°C and ionization voltage (ISVF) was set to 5000 V. The software Analyst TF 1.7 (AB Sciex, Concord, ON, Canada) was used to control the instrument and collected data.

2.6 Statistical analysis and identification of metabolites The raw UPLC-QTOF-MS data were first transformed to mzXML format by ProteoWizard and then processed by XCMS and CAMERA packages in the R software platform. In the XCMS package, peak picking (method = centWave, ppm = 15, peakwidth = c (5, 20), snthresh = 10), alignment, and retention time correction (method = obiwarp) were used. In the CAMERA package, annotations of isotope peak, adducts, and fragments were performed. The final data were exported as a peak table file, including observations, variables, and peak areas. They were also normalized against total peak areas before performing univariate and multivariate statistics.

For multivariate statistical analysis, the normalized data were imported into SIMCA software (version 14.1, Umetrics, Umeå, Sweden), where the data were preprocessed by Pareto scaling and mean centering before performing PCA and OPLS-DA models. Additionally, they were analyzed by Welch’s t-test. The variables with both VIP (variable importance in projection) values from the OPLS-DA model larger than 1

and p values from univariate statistical analysis lower than 0.05 were identified as potential differentially represented metabolites. The mass-to-charge ratio (m/z) of precursors and productions were used to identify the metabolites. They were searched against LipidBlast database [26] and an in-house database constructed by Shanghai ProfLeader Biotech Co. (China).

2.7 Identification of desaturases

Recognized desaturase sequences of Lepidoptera butterflies and Hymenoptera bees were downloaded from NCBI RefSeq database (Supplementary file 2). They were

respectively used as queries against P. rapae transcriptome database [27] and P. puparum genome database (unreleased) using local BLASTP program (E-value 1e-5), and manually validated using a series BLAST programs on NCBI.

2.8 Analysis of RNA-seq data

For the host P. rapae, the relative expression levels of infected and control pupal transcripts have been measured in our previous study [27]. The KEGG pathways enrichment analysis of DEGs has been displayed as well. For the transcriptome analysis of P. puparum in different development stages or tissues, a similar process was applied as described by Yan et al. previously [28]. Transcriptomic raw data from each library was previously generated and assembled in our lab (unreleased). The expression levels of identified desaturases were estimated by the expected number of Fragments Per Kilobase of exon per Million fragments mapped (FPKM) by software TopHat and Cufflinks [29].

3 Results and Analyses

3.1 Overview of differentially expressed lipids following infection Newly pupated P. rapae were separated into P. puparum infection and control treatments. Twenty-four hours post-infection, with wasps still at the egg stage, P.

rapae hosts were dissected and fat body and hemolymph samples were collected from each. There were a total of 24 lipidomics samples: 6 replicates each of fat body or hemolymph tissues from infected or uninfected treatments. Each sample was derived from three individual P. rapae pupae, for a total of 72 P. rapae individuals in the experiment.

All samples of fat body and hemolymph were run in both positive and negative-ion electrospray modes to identify the fullest set of lipids possible [30]. One parasitized/fat body sample was discarded as a statistical outlier as it showed an abundance of high molecular weight lipids not found in other samples (Fig. S1). For the P. rapae fat body lipid extracts, 1,706 positive peaks (representing specific lipid structures) and 1,640 negative peaks were detected; for the hemolymph lipid extracts 1,800 positive peaks and 2,000 negative peaks were detected. We performed principal component analysis (PCA) using the software SIMCA (version 14.1) to visualize inherent clustering among our lipid samples, after combining the positive and negative-ion electrospray data for each sample. As shown in Fig. 1 (A-D), for both the fat body and hemolymph tissues, parasitized and unparasitized samples formed discrete clusters (statistical test p-values here). PCA is an unbiased method for identifying clusters, but given clustered samples, a “supervised” approach such as orthogonal partial least squares discriminant analysis (OPLS-DA) can distinguish which variables (i.e. which kinds of lipids) yield the largest power in discriminating the clusters [31]. OPLS-DA analyses also revealed discrete clustering of parasitized and unparasitized samples (Fig. 1, E-H) (statistical test p-values here). For all PCA and OPLS-DA models, quality parameters were good and permutation tests revealed no overfitting (Table S1).

In order to identify the differentially expressed lipids in our samples, the “variable importance in projection” (VIP) values from the OPLS-DA model were combined

with p-values based on Welch’s t-test comparisons of lipid concentrations across treatments [32]. Only those lipid components with both VIP values greater than 1 and t-test p-values less than 0.05 (VIP > 1 and p-values < 0.05) were scored as differentially expressed, although we also considered a less strict significance threshold of (p-values < 0.05) only. Using the strict significance criteria, we identified 87 differentially expressed lipids in host fat body (30 increased and 57 decreased after infection), and 117 differentially expressed lipids in host hemolymph (89 increased and 28 decreased after infection) (Fig. 2) [33]. Overall, many of the differentially expressed lipids in the fat body had decreased levels post-infection (Fig. 2, blue bars), while many of the differentially expressed lipids in the hemolymph had increased levels post-infection (Fig.2, red bars). Furthermore, of the 21 lipid groups considered (Fig.2), five groups showed both decreased expression in the fat body and increased expression in the hemolymph. These included triacylglycerides (TAGs) and various phospholipids like sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI).

The full list of lipids differentially expressed in parasitized versus unparasitized samples is shown in Table S2. As described above, some lipid groups showed a decrease in abundance in the fat body and an increase in abundance in the hemolymph post-infection (e.g. TAGs), but only a subset of the specific lipids in these groups were differentially expressed in both tissues. We denote individual lipids using a standard convention. For example, PC 16 : 0-18 : 2 and PC 18 : 1-14 : 0 are distinct species of the compound phosphocholine (PC). PC 16 : 0-18 : 2 consists of two carbon chains: the first chain has 16 carbons without any double bonds while the second chain has 18 carbons with 2 double bonds. Note that lipid species with the same number of carbons and double bonds can be distinct and represented by two peaks in the lipidomics analysis (and considered separately in our analyses) if the double bonds are found in different positions. A total of 23 specific lipids were

differentially expressed (using the strict criteria of VIP > 1 and p-value < 0.05) in both the fat body and the hemolymph following infection (Fig.3). In this subset of lipids, only three showed decreased levels in the hemolymph (two diacylglycerides (DAGs) and a lysophosphatidylcholine (LPC)). The other twenty such lipids (five TAGs and fifteen phospholipids) showed decreased levels in the fat body and increased levels in the hemolymph. More information about these individual lipids is shown in Table S3.

3.2 Host triacylglyceride levels increased post-infection while hemolymph diacylglyceride levels decreased TAGs are the predominant form of lipids in insects, functioning as energy storage molecules and as precursors for other molecules such as semiochemicals [34]. TAGs are mobilized from the fat body for energetically costly physiological functions such as flight and provisioning of egg yolks for developing embryos [35, 36]. However, many endoparasitoid wasp groups produce hydropic (or at least partially hydropic) eggs, meaning that the eggs are small and incompletely provisioned (making it easier to send them through hypodermic ovipositors) and need to absorb nutrients from the host for development [1]. Thus, wasp venom proteins are thought to manipulate host physiology to increase access of their eggs/larvae to host lipids in the host hemolymph.

We observed a dramatic increase in the levels of TAGs in host hemolymph post- infection. At this same time, we observed a dramatic decrease in host hemolymph levels of diacylglycerides (DAGs) (Fig. 2), the main form in which lipids are transported between host tissues [37]. These data suggest that host hemolymph DAGs are converted to TAGs post-infection. Transcriptomics/proteomics analyses from P. rapae hosts and P. puparum venoms provide insight into potentially how and why this occurs. P. rapae differentially express a number of enzymes involved in

glycolysis and gluconeogenesis following infection (Fig. S2). Specifically, the transcript for the last key enzyme in TAG synthesis, diacylglycerol acyltransferase (DGAT2), which converts DAGs to TAGs, is nearly 5 -fold upregulated. P. puparum venoms do not contain DGAT homologs, but do contain a number of predicted lipases [28] -venom lipases could catalyze the hydrolysis of TAGs but versions missing their catalytic triads are also known to function in the binding and transport of these lipids to the developing embryo [38, 39]. Thus, it appears that a host enzyme is responsible for DAG to TAG conversion. The host could be attempting to sequester hemolymph lipids from the parasite, or the parasite may be manipulating the host into increasing DGAT2 expression as a means of increasing hemolymph TAG levels. As discussed further below, the parasite appears to induce increased TAG levels in host hemolymph in other ways as well.

3.3 Host fat body levels of highly unsaturated triacylglycerides increased post- infection while more saturated forms decreased After being parasitized, P. rapae fat body concentrations of 20 TAGs increased while 14 of them decreased (Fig. 4A). Nearly all TAGs with increasing concentrations had high numbers of double bonds (average = 6.9) and were thus highly unsaturated, while those with decreasing concentrations had many fewer double bonds (average = 3.0) and were more saturated. At the same temperature, increasing the number of TAG double bonds (increased unsaturation) increases TAG solubility [40], which might allow wasp offspring to acquire and utilize TAGs more easily. However, no such change in TAG saturation was found in host hemolymph TAGs post-infection, so it is not yet clear why this effect would benefit the wasp offspring.

The main type of enzyme responsible for creating double (unsaturated) bonds in fatty acyl chains is fatty acid desaturase [41], so we sought to identify desaturases potentially involved in this TAG transformation from both the hosts and the wasps.

Transcriptomic analyses of P. rapae hosts identified four fatty acid desaturases that were significantly upregulated following infection, including one that was more than 218 -fold upregulated (Fig. 4B). Furthermore, although proteomic analyses of P. puparum venom glands did not identify a venom desaturase [42], RNA-seq data comparing gene expression in different P. puparum tissues identified a single desaturase (acyl-CoA desaturase-like gene PPU08555) that is highly expressed in wasp larvae and adult females, especially in female venom glands (Fig. 4C). Further experiments will be required to determine whether any of these host and/or wasp desaturases is responsible for the desaturation of TAGs in the infected host fat body.

3.4 Levels of membrane phospholipids decreased in the host fat body but increased in hemolymph post-infection Phospholipids form the lipid bilayers of cell membranes due to their amphiphilic structure. The major eukaryotic membrane phospholipids include sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI) [43]. The mitochondrial cell membranes of eukaryotes resemble prokaryotic cell membranes in that their major constituents are PE and phosphatidylglycerol (PG) [44]. In eukaryotic cells, the lipid bilayer is asymmetrical, with SM and PC making up the major portion of the outer (extracellular) leaflet [45], and PE, PS, and PI making up the major portion of the inner (cytoplasmic) leaflet [43, 46].

Levels of multiple phospholipid species from eukaryotic cell membranes (SM, PC, PE, PS, PI) were found to decrease in P. rapae fat bodies following infection, whereas levels of multiple phospholipid species from the same groups showed increased levels in the hemolymph (with the exception of PS) (Fig. 2). Of the 15 specific phospholipid species that both significantly decreased in the fat body and increased in the hemolymph, 4 were SMs, 2 were PCs, 7 were PEs, and 2 were ceramides (component

lipids that make up sphingomyelin) from the Cer-NS group (Fig. 3, Fig. 5A). These data imply a release of cell membrane phospholipids from the host fat body cells into hemolymph because of the destruction of fat body cell membranes, and the fat body itself, after infection.

Interestingly, the levels of several PSs decreased in the fat body without any concomitant increase in the hemolymph (Fig. 3, Fig. 5B). During conditions of stress, the cell membrane may lose its asymmetry and phospholipids normally found on the inner leaflet, like PSs, may become exposed on the outer leaflet. Lack of membrane asymmetry can lead to cell death, and PSs in the outer leaflet of cell membranes are specifically required for recognition by phagocytic cells [47-49]. Thus, it’s possible that when cells are lysed the loose PSs are quickly picked up by roving phagocytes.

Furthermore, PEs were the only major phospholipid group with members that showed decreased levels in the hemolymph following infection (Fig. 3). Surprisingly, all three PE species whose concentration decreased in the hemolymph were relatively unsaturated, while the only PE species that increased in the hemolymph while showing no loss in the fat body post-infection were relatively saturated (Fig. 5C). Previously, unsaturated PEs have been observed to be preferentially incorporated in the PE methylation pathway for PC synthesis in mammals [50]. Coincidently, numerous PC molecules had increasing concentrations in host hemolymph post- infection, but this conversion of PE to PC unlikely occurs in insects where no phosphatidylethanolamine N-methyltransferase (PEMT) has been identified [51]. Alternatively, these data suggest that certain PEs may convert into more saturated (more rigid) forms during infection. A reduction in cell membrane fluidity is a common membrane pathology [52, 53] and may be a passive consequence of infection, although it is unclear why this effect would only be observed in phospholipids in the hemolymph. Considering that some reduced PS species

mentioned above have the same acyl composition as those increased PEs in hemolymph (Fig. S3), we additionally propose a possible conversion of PS to PE. The PS decarboxylation pathway is one of the major pathways for PE biosynthesis [54]. It preferentially produces PEs with polyunsaturated acyl chains at the sn-2 position (via phosphatidylserine decarboxylase) [55]. However, in our analysis the increased PE species in host hemolymph mostly contained mono- or di-unsaturated acyl chains at the sn-2 position, suggesting that they were unlikely derived from PS decarboxylation. Instead, these PEs might be produced from a calcium-dependent base-exchange reaction, in which free ethanolamine is exchanged for the serine head group of PS (via phosphatidylserine synthase) [56]. Nevertheless, extra experiments are required to validate our hypothesis.

3.5 Cholesteryl esters dramatically increase in host hemolymph In animals, cholesterols are critical components of cell membranes and also act as precursors for many hormones (including the insect molting hormone ecdysteroid). However, insects are incapable of de novo synthesis of sterols because they lack the necessary squalene synthetase and lanosterol synthase enzymes, and thus have to acquire cholesterols from their food [57, 58]. The conversion of cholesterols into cholesteryl esters (cholesterol linked to a fatty acid) allows cholesterols to be transported more efficiently into and across cell membranes.

In the infected host hemolymph, levels of three cholesteryl esters (CEs) dramatically increased while cholesterol levels remained at consistently low levels (Fig. 2, Fig. 5D). In addition, no cholesterols or CEs showed significant concentration changes in the infected fat body. Interestingly, CEs have been identified as a main component in parasitoid wasp Dufour’s glands [59, 60], which is part of the ovipositor apparatus. Dufour’s gland secretions can be involved in nest building, serve as a larval food source, or act as pheromones involved in communicative functions [61], and can be

injected into hosts in a mixture with venom gland components. Our data imply that CEs in infected P. rapae hemolymph are derived from an outside source, namely the venom of P. puparum. Lipases with the predicted cholesteryl esterases function have been observed from the salivary glands of P. puparum larvae [62], suggesting wasp young hydrolyze the CEs released by their mothers into their hosts to gain essential sterols for development.

4 Discussion

In previous work, lipid concentrations in various hosts infected by parasitoid wasps were measured. In studies using the pupal ectoparasitoid N. vitripennis, lipid amounts rose in both host hemolymph and fat body of the host Sarcophaga bullata, but decreased in S. stemodontus, while no significant change was detected in either the hemolymph or fat body of Musca domestica. Lipid amounts increased and then decreased in hemolymph but continually decreased in the fat body of N. vitripennis- infected Phormia regine [63, 64]. Using the larval ectoparasitoid Euplectrus sp., lipid amounts increased in the hemolymph but decreased in the fat body of the host Pseudaletia separate after either parasitism or venom injection [65]. Using the larval endoparasitoid Cotesia flavipes, lipid amounts in the hemolymph of the infected host Diatraea saccharalis decreased but showed a non-significant change in the fat body [24]. It seems that the overall lipid concentrations in infected hosts can be quite variable and are dependent on the lipid-regulating strategies that each parasitoid uses to provide nutrition for their young, as well as the unique responses to infection by each host.

However, the methods used in previous studies to detect lipid concentrations in infected hosts were relatively limited. The vanillin-phosphoric acid method for assaying lipids [66, 67] can only detect the total amount of lipids, not the concentrations of individual lipid species. In addition, the infection protocols of

previous studies have their own caveats. Hosts injected with parasitoid venom may not display the integrated changes initiated in a live infection (in which the secretions of the venom gland and Dufour’s gland are mixed). Furthermore, lipid assays that occur later during the infection process include the effects of hatched parasitoid larvae on overall lipid concentrations. Consequently, we utilized a new lipidomics approach based on UHPLC-QTOF-MS to quantify individual lipid concentrations in P. rapae hosts naturally parasitized by the endoparasitoid P. puparum, at a time-point prior to the wasp eggs hatching. During our dissections, it was obvious to the eye that the fat body tissues of infected hosts became disassociated and the hemolymph became turbid, suggesting that infection induces the break-down of fat body components into the hemolymph.

Our lipidomics analyses revealed that the majority (76.07%) of lipid components that showed increased concentrations following infection were found in the hemolymph, while the majority (65.52%) of lipid components that showed decreased concentrations following infection were found in the fat body, especially for numerous phospholipids (SMs, PCs, PEs, PIs, and PSs). These data support the general view that the amount of stored lipids in parasitized host fat bodies tends to decrease [68]. They also suggest that parasitoid venom can induce the disintegration of infected hosts’ fat bodies to build up lipid levels in host hemolymph. Teratocytes, which are pathogenic cells released by parasitoid eggs to prep hosts for the developing larvae, have been shown to have similar effects in a variety of other parasitoid wasp species [23].

One of our main results was that the levels of many TAG species in the host fat body and hemolymph increased post-infection, while the levels of many DAG species in infected host hemolymph decreased post-infection. We suggest that there is a competition for TAGs in the infected host hemolymph between the host itself and the

parasitoid young. This causes the infected host to continuously biosynthesize TAGs from DAGs to hedge TAG consumption by the parasitoid. The concurrent accumulation of TAGs in both the parasitoid Lysiphlebia japonica larvae and its host Aphis gossypii shown in previous work, as well as the inability of L. japonica larvae and pupae to synthesize fatty acids on their own [69, 70] supports our hypothesis that parasitoids seek host TAGs.

For the infected host P. rapae, especially at its non-feeding pupal stage, TAGs are essential as energy reserves. For the young of the parasitoid P. puparum, TAGs are likely used to fuel the developing embryos and larvae, and the wasps may save a lot of energy by freeing host lipids for consumption rather than synthesizing them themselves. Interestingly, several lipases were identified as potential venom proteins in P. puparum [28]. These venom lipases have the inferred functions of catalyzing the hydrolysis of TAGs or binding and transporting lipids [38]. In order to compensate for the lost TAGs, we hypothesize that, infected P. rapae upregulate DGAT2 expression in order to boost conversion of DAGs into TAGs and maintain energetic homeostasis. This will be the subject of future work.

Our next major result was the observation that the overall saturation level of TAGs in the fat body decreases in infected hosts. Numerous individuals relatively unsaturated TAGs showed increasing concentrations at the same time that numerous individual relatively saturated TAGs showed decreasing concentrations, suggesting the saturated TAGs were converted to unsaturated TAGs. A newly identified venom protein in P. puparum, acyl-CoA desaturase (PPU08555), potentially performs this conversion function in infected hosts. Enhancing TAG fluidity could make it easier for the developing wasps to procure these host resources as dietary lipids. It is unclear why conversion to more unsaturated TAGs would occur in the fat body and not in the hemolymph, as young parasitoids consume hemolymph before they are

developmentally capable of consuming hard host tissues, but perhaps these unsaturated TAGs are released into the hemolymph at a later time point. Alternatively, the wasps may prefer to make host TAGs more unsaturated because unsaturated lipids are highly subject to lipid peroxidation, the oxidative degradation of lipids. In this process, free radicals “steal” electrons from the lipids, particularly in cell membranes, resulting in cell damage that may fatally upset host metabolism.

Another main result was the dramatic increase in CEs that we observed in host hemolymph post-infection, despite uniformly low host CE and cholesterol levels across other tissues and experimental conditions (as is expected for an herbivorous insect). These data suggest that the female wasp injects the CEs into the host. Insects have lost the ability to produce sterols themselves and instead must acquire them from food [57, 58]. Unlike with TAGs, it appears that wasp offspring cannot acquire cholesterol derivatives from their hosts, so that the adult female wasps must provide it to their offspring via injection into the host. CEs may be the preferred conformation of cholesterol because they are more efficiently transported than cholesterols [71, 72]. CEs were previously identified in the Dufour’s gland of some wasps but so far no metabolomics analysis has been done in the Dufour’s gland or venom gland of P. puparum.

In summary, we identified and quantified the lipid components in P. rapae hemolymph and fat body after infection by its endoparasitoid P. puparum. Our results provide novel insight into how parasitoids regulate host lipid metabolism to the benefit of their developing offspring. It will be important to identify the individual wasp venom proteins responsible for the beneficial host physiological changes that we observed.

Acknowledgments

The study was supported by the National Key R & D Program of China (2017YFD0200400), the Major International (Regional) Joint Research Project of the National Natural Science Foundation of China (31620103915), the Key Program of the National Natural Science Foundation of China (31830074), the Program for Chinese Innovation Team in Key Areas of Science and Technology of the Ministry of Science and Technology of the People's Republic of China (2016RA4008), the Program for Chinese Outstanding Talents in Agricultural Scientific Research of the Ministry of Agriculture and Rural Affairs of the People's Republic of China, the Natural Science Foundation of Zhejiang Province of China (LY18C140001), and the China Scholarship Council (201806320151).

Legends

Figure 1. PCA and OPLS-DA plots comparing lipid composition of parasitized and unparasitized P. rapae. Axes represent the two strongest discriminatory vectors from each model. (A-D) show PCA plots while (E-H) show OPLS-DA plots. (A, B, E, F) show fat body data while (C, D, G, H) show hemolymph data. FBU = fat

body/unparasitized, FBP = fat body/parasitized, HU = hemolymph/unparasitized, HP = hemolymph/parasitized.

Figure 2. Lipid groups differentially expressed following infection by P. puparum. Red bars represent the number of lipids with increased expression while blue bars represent the number of lipids with decreased expression. Dark colors represent the number of lipids that meet strict significance criteria for differential expression (VIP > 1 and p < 0.05) while lighter colors meet more relaxed criteria (p < 0.05). The total numbers of lipids in each tissue type are shown below. Acronyms represent different lipid groups (Supplementary file 1). Colored bars represent the lipid categories according to the Lipid Maps Classification System (www.lipidmaps.org). GL = Glycerolipids, FA = Fatty Acyls, SP = Sphingolipids, GP = Glycerophospholipids, ST = Sterol Lipids. Some of their functions are indicated at the far right in grey.

Figure 3. Comparison of lipids differentially expressed in both tissue types following infection by P. puparum. Specific lipids are plotted in a four-quadrant graph to show interrelated changes in expression levels across the fat body and hemolymph tissues. Orange squares represent lipids that meet strict significance criteria for differential expression (VIP > 1 and p < 0.05) in both tissues while green squares represent lipids that meet more relaxed criteria (p < 0.05) in both tissues. Orange bubbles reveal the numbers and identities of the different lipids in each quadrant.

Figure 4. Increased unsaturation of host fat body TAGs following infection by P. puparum. (A) The top panel shows the twenty TAGs with increased concentrations post-infection (FBU = fat body/unparasitized, FBP = fat body/parasitized), while the bottom panel shows the fourteen TAGs with decreased concentrations. The colored numbers highlight the number of double bonds found in each TAG. Error bars represent the means ± standard deviation from biological replicates. (B) Comparison of the expression of P. rapae desaturases in infected and control pupal transcripts. (C) Expression profile of the potential acyl-CoA desaturase venom gene PPU08555 in various tissues. Whole-body values are shown as columns whereas individual tissue values are shown as points (F = female, M = male, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5. Lipid levels changed in the fat body and/or hemolymph. (A) The concentrations of different SMs, PCs, PEs, and Cer-NSs whose levels decrease in the fat body and increase in the hemolymph following infection. (B) The concentrations of numerous PSs decrease in the fat body but show no increase in the hemolymph. (C) The concentrations of some highly unsaturated PEs decrease in the hemolymph following infection, with a concomitant increase in less saturated PEs. The numbers of double bonds in each PE are shown in green and red. (D) The concentrations of several CEs increase but remain low in hemolymph following infection. FBU = fat body/unparasitized, FBP = fat body/parasitized, HU = hemolymph/unparasitized, HP = hemolymph/parasitized. Error bars represent the means ± standard deviation from biological replicates (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure S1. The parasitized/fat body sample discarded as a statistical outlier (FBP2) displayed an unusual abundance of high molecular weight lipids.

Figure S2. Changes in P. rapae lipid metabolism enzyme expression and lipid levels post-infection by P. puparum. Lipid metabolism pathways are derived from KEGG pathway database (www.kegg.jp/kegg/pathway). Lipids and their precursors are shown in the normal black font: those with increased levels post-infection are in red boxes while those with decreased levels are shown in green boxes. Lipid metabolism enzyme names are shown in italics: those with increased gene expression post-infection are shown in red font while those with decreased gene expression are

shown in green font. Acronyms represent different lipid groups (Supplementary file

1).

Figure S3. PS and PE molecular species with significantly changed concentrations (VIP > 1 and p < 0.05) in the parasitized hosts. The yellow bar indicates each pair of PS and PE with the same acyl chain composition. The molecular species whose concentration increased and decreased are shown in green and red respectively.

Table S1. Characteristics of the OPLS-DA model between parasitized and unparasitized Pieris rapae using cross-validation and results of permutation.

OPLS-DA Permutation test

R2X R2Y Q2 R2 Q2

POS 0.821, 0.999 0.948 (0.0, 0.981) (0.0, -0.285) Fat body NEG 0.739 0.999 0.916 (0.0. 0.985) (0.0, -0.196)

POS 0.93 0.996 0.968 (0.0, 0.876) (0.0, -0.39) Hemolymph NEG 0.727 0.961 0.877 (0.0, 0.561) (0.0, -0.674)

R2X, R2Y, and Q2 represent the quality parameters of the OPLS-DA model, are related to statistical validity. Two hundred permutations were performed in permutation tests, and the resulting R2 and Q2 values were displayed.

Table S2. Lipids differentially expressed in each tissue type following infection (VIP > 1 and p < 0.05).

Fat body + Fat body - Hemolymph + Hemolymph -

TAG 36:0;TAG 12:0-12:0-12:0, TAG 38:0;TAG 12:0-12:0- 14:0, TAG 38:1;TAG 12:0-12:0-14:1, TAG 38:1;TAG 12:0- 12:1-14:0, TAG 39:0;TAG 12:0-13:0-14:0, TAG 39:1;TAG 12:0-12:1-15:0, TAG 40:0;TAG 12:0-12:0-16:0, TAG 40:0;TAG 12:0-14:0-14:0, TAG 40:1;TAG 12:0-12:0-16:1, TAG 40:1;TAG 12:0-14:0-14:1, TAG 40:2;TAG 12:1-12:1- 16:0, TAG 41:0;TAG 12:0-14:0-15:0, TAG 41:1;TAG 12:0- 13:0-16:1, TAG 42:0;TAG 12:0-14:0-16:0, TAG 42:1;TAG TAG 48:6;TAG 16:0-16:3-16:3, TAG 48:6;TAG 16:1-16:2- 12:0-12:0-18:1, TAG 42:1;TAG 12:0-14:0-16:1, TAG 16:3, TAG 49:6;TAG 15:0-16:3-18:3, TAG 50:3;TAG 15:1- TAG 48:1;TAG 14:0-16:0-18:1, TAG 48:2;TAG 42:2;TAG 12:0-12:0-18:2, TAG 44:0;TAG 12:0-14:0-18:0, 15:2-20:0, TAG 50:5;TAG 16:1-16:2-18:2, TAG 50:6;TAG 16:0-16:1-16:1, TAG 48:4;TAG 16:0-16:1-16:3, TAG 44:1;TAG 12:0-14:0-18:1, TAG 44:2;TAG 12:0-14:0- 14:1-18:2-18:3, TAG 50:6;TAG 16:0-16:3-18:3, TAG TAG 49:2;TAG 15:1-16:0-18:1, TAG 49:3;TAG 18:2, TAG 45:0;TAG 15:0-15:0-15:0, TAG 46:0;TAG 12:0- 50:8;TAG 16:2-16:3-18:3, TAG 50:9;TAG 16:3-16:3-18:3, 15:0-16:0-18:3, TAG 49:4;TAG 15:1-16:0-18:3, 16:0-18:0, TAG 46:1;TAG 12:0-16:0-18:1, TAG 46:2;TAG TAG 52:5;TAG 16:2-18:1-18:2, TAG 52:6;TAG 16:0-18:3- TAG 49:5;TAG 15:1-16:1-18:3, TAG 50:2;TAG TAG 12:0-16:0-18:2, TAG 46:3;TAG 12:0-16:1-18:2, TAG 18:3, TAG 52:6;TAG 16:1-18:2-18:3, TAG 52:8;TAG 16:3- 16:0-16:1-18:1, TAG 51:2;TAG 16:0-17:1-18:1, 47:0;TAG 15:0-15:0-17:0, TAG 47:1;TAG 13:0-15:0-19:1, 18:2-18:3, TAG 52:9;TAG 16:3-18:3-18:3, TAG 53:6;TAG TAG 51:4;TAG 15:0-18:1-18:3, TAG 51:5;TAG TAG 48:1;TAG 14:0-16:0-18:1, TAG 48:2;TAG 14:0-16:0- 17:0-18:3-18:3, TAG 54:8;TAG 18:2-18:3-18:3, TAG 15:1-18:1-18:3, TAG 52:2;TAG 16:0-18:1-18:1, 18:2, TAG 48:3;TAG 12:0-18:1-18:2, TAG 49:1;TAG 15:0- 54:9;TAG 16:0-18:3-20:6, TAG 54:9;TAG 18:3-18:3-18:3, TAG 52:3;TAG 16:0-18:1-18:2, TAG 53:3;TAG 16:0-18:1, TAG 50:1;TAG 16:0-16:0-18:1, TAG 50:2;TAG TAG 56:8;TAG 18:2-18:3-20:3, TAG 56:9;TAG 18:3-18:3- 17:0-18:1-18:2 16:0-16:1-18:1, TAG 50:3;TAG 16:0-16:0-18:3, TAG 20:3 50:4;TAG 16:0-16:3-18:1, TAG 51:1;TAG 16:0-17:0-18:1, TAG 51:2;TAG 16:0-17:1-18:1, TAG 51:3;TAG 16:0-17:0- 18:3, TAG 52:1;TAG 16:0-18:0-18:1, TAG 52:2;TAG 16:0- 18:1-18:1, TAG 52:3;TAG 16:0-18:1-18:2, TAG 52:4;TAG 16:0-18:1-18:3, TAG 52:4;TAG 16:0-18:2-18:2, TAG 52:5;TAG 16:0-18:2-18:3, TAG 54:2;TAG 18:0-18:1-18:1, TAG 54:3;TAG 18:1-18:1-18:1, TAG 54:4;TAG 18:1-18:1- 18:2, TAG 54:5;TAG 18:1-18:2-18:2 DAG 32:1;DAG 16:0-16:1, DAG 32:3;DAG 16:0-16:3, DAG 33:3;DAG 15:0-18:3, DAG 33:4;DAG 15:1-18:3, DAG 34:1;DAG 16:0-18:1, DAG 34:2;DAG 16:0-18:2, DAG 34:3;DAG 16:0-18:3, DAG 34:4;DAG 16:1-18:3 peak1, DAG 34:4;DAG 16:1-18:3 peak2, DAG 34:5;DAG 18:2-16:3, DAG 34:6;DAG 16:3-18:3, DAG 35:2;DAG 17:0-18:2, DAG DAG DAG 36:6;DAG 18:3-18:3 DAG 34:1;DAG 16:0-18:1 35:3;DAG 17:0-18:3, DAG 35:4;DAG 17:1-18:3, DAG 36:1;DAG 18:0-18:1 peak1, DAG 36:1;DAG 18:0-18:1 peak2, DAG 36:2;DAG 18:0-18:2, DAG 36:3;DAG 18:0-18:3, DAG 36:3;DAG 18:1-18:2, DAG 36:4;DAG 18:1-18:3, DAG 36:5;DAG 18:2-18:3, DAG 36:6;DAG 18:3-18:3, DAG 37:3;DAG 19:0-18:3 peak1, DAG 37:3;DAG 19:0-18:3 peak2 MGDG MGDG 36:5;MGDG 18:2-18:3 FA FA 18:1, FA 18:2, FA 18:3 FA 20:0, FA 22:0 Cer-NS d34:1;Cer-NS d14:1/20:0, Cer-NS Cer-NS d32:1;Cer-NS d14:1/18:0, Cer-NS d34:1;Cer-NS Cer-NS d34:2;Cer-NS d14:2/20:0, Cer-NS d36:1;Cer-NS d14:1/20:0, Cer-NS d36:1;Cer-NS d14:1/22:0 d14:1/22:0

Cer- Cer-NDS d32:0;Cer-NDS d14:0/18:0 NDS SM d32:1;SM d14:0/18:1, SM d32:1;SM d14:1/18:0, SM d33:1;SM d14:1/19:0, SM d34:1;SM d14:0/20:1, SM SM d34:1;SM d14:0/20:1, SM d34:1;SM d34:1;SM d14:0/20:1, SM d34:1;SM d14:1/20:0, SM SM d14:1/20:0, SM d36:1;SM d14:0/22:1, SM d34:2;SM d14:2/20:0, SM d35:1;SM d14:1/21:0, SM d36:1;SM d14:1/22:0 d36:1;SM d14:0/22:1, SM d36:1;SM d14:1/22:0, SM d36:2;SM d14:2/22:0, SM d37:1;SM d25:1/12:0, SM d38:1;SM d14:1/24:0 PC 34:0;PC 16:0-18:0, PC 34:1;PC 16:0-18:1, PC 34:2;PC 16:0-18:2, PC 34:3;PC 16:0-18:3, PC 35:1;PC 17:0-18:1, PC PC 34:2;PC 16:0-18:2, PC 36:4;PC 18:2-18:2, 35:2;PC 17:0-18:2, PC 35:3;PC 17:0-18:3, PC 36:1;PC 18:0- PC PC 36:5;PC 18:2-18:3 18:1, PC 36:2;PC 18:0-18:2, PC 36:3;PC 18:0-18:3, PC 36:3;PC 18:1-18:2, PC 36:4;PC 18:2-18:2, PC 37:2;PC 19:0- 18:2, PC 38:3;PC 20:0-18:3 PE 33:2;PE 15:0-18:2, PE 33:4;PE 15:1-18:3, PE 34:1;PE 16:0-18:1, PE 34:2;PE 16:0-18:2, PE PE 34:1;PE 16:0-18:1, PE 34:2;PE 16:0-18:2, PE 35:3;PE 35:1;PE 17:0-18:1, PE 35:2;PE 17:0-18:2, PE 17:0-18:3, PE 36:1;PE 18:0-18:1, PE 36:2;PE 18:0-18:2, PE PE 34:6;PE 16:3-18:3, PE 36:5;PE 18:2-18:3, PE 36:6;PE PE 35:3;PE 17:0-18:3, PE 36:1;PE 18:0-18:1, PE 36:3;PE 18:0-18:3, PE 36:3;PE 18:1-18:2, PE 36:4;PE 18:1- 18:3-18:3 36:2;PE 18:0-18:2, PE 36:4;PE 18:1-18:3, PE 18:3, PE 38:2;PE 20:0-18:2 37:3;PE 19:0-18:3, PE 38:2;PE 20:0-18:2, PE 38:3;PE 20:0-18:3

PS 34:1;PS 16:0-18:1, PS 36:2;PS 18:0-18:2, PS 36:2;PS 18:1-18:1, PS 36:3;PS 18:0-18:3 peak1, PS PS 36:3;PS 18:0-18:3 peak2, PS 36:4;PS 18:1- 18:3, PS 36:5;PS 18:2-18:3

PI 34:1;PI 16:0-18:1, PI 34:3;PI 16:0-18:3, PI 36:0;PI 18:0-18:0, PI 36:1;PI 18:0-18:1, PI PI 36:2;PI 18:0-18:2, PI 36:2;PI 18:1-18:1, PI 36:3;PI 18:0-18:3, PI 36:4;PI 18:1-18:3, PI 36:6;PI 18:3-18:3

LPC LPC 18:3 LPC 16:0, LPC 18:0 LPC 18:0

LPE LPE 18:2 peak1, LPE 18:2 peak2, LPE 18:3 LPE 18:1 LPI LPI 18:0 CE CE 18:1, CE 18:2, CE 18:3

Table S3. Lipids differentially expressed in both tissue types following infection (VIP > 1 and p < 0.05).

Fat body + Hemolymph + Fat body - Hemolymph + Fat body + Hemolymph- Fat body - Hemolymph -

TAG 48:1;TAG 14:0-16:0-18:1, TAG 50:2;TAG 16:0-16:1-18:1, TAG 51:2;TAG TAG 16:0-17:1-18:1, TAG 52:2;TAG 16:0-18:1-18:1, TAG 52:3;TAG 16:0-18:1-18:2 DAG DAG 36:6;DAG 18:3-18:3 DAG 34:1;DAG 16:0-18:1 Cer-NS Cer-NS d34:1;Cer-NS d14:1/20:0, Cer-NS d36:1;Cer-NS d14:1/22:0 SM d34:1;SM d14:0/20:1, SM d34:1;SM d14:1/20:0, SM d36:1;SM d14:0/22:1, SM SM d36:1;SM d14:1/22:0 PC PC 34:2;PC 16:0-18:2, PC 36:4;PC 18:2-18:2 PE 34:1;PE 16:0-18:1, PE 34:2;PE 16:0-18:2, PE 35:3;PE 17:0-18:3, PE 36:1;PE PE 18:0-18:1, PE 36:2;PE 18:0-18:2, PE 36:4;PE 18:1-18:3, PE 38:2;PE 20:0-18:2 LPC LPC 18:0

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