Nematode culturing and conditioning

Heterorhabditis megidis UK211 and Steinernema carpocapsae All were used. Nematodes were cultured in last instar Galleria mellonella larvae (Mealworm Company, Sheffield, UK) using methods outlined in Woodring and Kaya [33], at 20°C, with an inoculum density of 100 IJs/insect. Cadavers were placed on White traps and monitored daily. After first emergence of IJs, the White trap water was replaced with fresh sterile water. IJs were allowed to emerge into the water for 3–4 days and collected. IJs from successive harvests were pooled, rinsed 3 times by sedimentation and stored at 1000 IJs/ml in sterile tap water in 35 ml aliquots in lidded plastic tubs (9 cm diameter). Tubs were placed at 20°C and 9°C temperature-controlled rooms and sampled at intervals (3 or 6 weeks at 20°C and 3, 6 or 9 weeks at 9°C). In addition, unconditioned IJs (time 0) were also sampled. There were 5 replicate tubs for each storage time and temperature.

Sample preparation

The contents of a tub were sedimented in a 50 ml Falcon tube in their conditioning temperature. The pelleted IJs in approx. 150 μl were transferred to a 1.5 ml Eppendorf tube and snap frozen in liquid nitrogen.

Each sample was homogenised in lysis buffer, containing 6M urea, 2M thiourea, and a Protease Inhibitor Cocktail (cOmplete, Mini Protease Inhibitor Cocktail, Merck), centrifuged at 10000 x g for 1 minute, and snap frozen. This step was repeated 3 times to ensure complete homogenisation. Protein content was then quantified using Qubit (Invitrogen), following the manufacturer’s instructions. Protein (100 μg) was purified using a 2D Clean Up Kit (GE Healthcare) according to the manufacturer’s instructions. The resulting pellets were stored in the kit’s wash solution at -20°C until the last samples were collected, then all were centrifuged at 13000 x g for 5 minutes and the resulting pellets were resuspended in 50 μl of resuspension buffer (6M urea, 2M thiourea, 0.1M TrisHCl, pH8). A 20 μl aliquot was removed from each sample for reduction, alkylation and digestion. One hundred and five μls of ammonium bicarbonate (50 mM) and 1 μl of dithiothreitol (DTT) were added and samples were incubated at 56°C for 20 minutes. Once cooled, samples were alkylated with 2.7 μl of iodoacetamide (IAA) in dark conditions.

One μl of a 1% (w/v) solution of ProteaseMax (Promega) and 0.5μg/μl trypsin (Promega) were added to the samples and incubated at 37°C for a minimum of 16 hours. Samples were removed from 37°C, centrifuged briefly and acidified with 1 μl of trifluoroacetic acid (TFA) for 5 minutes at room temperature (20–25°C). Samples were centrifuged at 13000 x g for 10 minutes and the supernatant was purified using C18 Spin Columns (Pierce, Thermo Fisher Scientific) following the manufacturer’s instructions and then lyophilised in a Speedyvac concentrator (Thermo Scientific Savant DNA120). Samples were then resuspended in a loading buffer, (2% v/v acetonitrile, 0.05% v/v TFA) and 1 μg was loaded from each of 4 biological replicates per samples were run on a QExactive (Thermo Fisher Scientific) high-resolution accurate mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system. Peptides were separated over a 2% to 40% gradient of acetonitrile on a Thermo Fisher EASY-Spray, PepMap RSLC C18 column (500mm length, 75mm ID), using a reverse-phase gradient at a flow rate of 250nL min^-1 over 125 minutes. All data were acquired over 105 minutes with the mass spectrometer operating in automatic data dependent switching mode. A full MS scan at 140,000 resolution and range of 300–1700 m/z was followed by an MS/MS scan, resolution 17,500 and range of 200–2000 m/z, selecting the 15 most intense ions prior to MS/MS.

Data processing

Protein identification and LFQ normalisation of MS/MS data was performed using Max-Quant v1.6.3.3 (http://www.maxquant.org) following the general procedures and settings outlined in Hubner et al. [34]. The Andromeda search algorithm [35] incorporated in the MaxQuant software was used to correlate MS/MS data for S. carpocapsae and H. megidis against the predicted protein data sets derived from the S. carpocapsae [36] and H. bacteriophora [37] genomics initiatives, respectively.

Normalised LFQ intensities were used to quantify protein abundances, and the data was filtered to remove contaminants. The LFQ intensities were log₂ transformed, and each replicate was renamed to their respective groups (3wks9°C for proteins from IJs stored at 9°C for 3 weeks). Only proteins found in 3 replicates of at least 1 group were retained. A data imputation step replaced missing values with values of low abundant proteins chosen randomly from a distribution specified by a downshift of 2 times the mean standard deviation (SD) and a width of 0.3 times the SD.

A principal component analysis (PCA) was initially performed on the normalised intensity values of all replicates. However, a number of outliers were identified, resulting in 3 replicates in each sample in the final datasets for analysis.

An analysis of variance (ANOVA) was performed on all groups using a Benjamini-Hochberg false discovery rate (FDR) of <5% to select proteins for z-score normalisation. These ANOVA significant proteins were used for hierarchical clustering of samples using Euclidean distance and average linkage pre-processed with K means. Gene Ontology (GO) term enrichment was performed in Blast2Go v5.2 using a Fishers exact test (p<0.05) on each cluster relative to all ANOVA significant proteins.

Pairwise Student’s t-tests were performed for all samples relative to the week 0 samples to visualise the effect of time and temperature conditioning on the IJs proteome. Volcano plots were generated in Perseus by plotting negative log p values of the y axis and log₂fold transformed differences on the x axis for each comparison. Statically significant (SS; p< 0.05) and differentially abundant (DA; fold change of 1.5) proteins were identified as SSDAs and selected for further analysis.

All statistically significant proteins identified in pairwise-t-tests were grouped using Bioedit (v7.0.5.3) and uploaded in FASTA format to STRING: Protein-Protein Interaction Networks Functional Enrichment Analysis v11.0 with the highest confidence setting (0.9) and disconnected nodes were removed, to identify protein-protein interactions which were increasing and decreasing in IJs after storage at 9°C or 20°C over time.

Bioinformatics

The H. megidis genome has not been sequenced, and therefore there is a paucity in molecular data available for the species. The genome of the closely related H. bacteriophora is available [37] and allows for the detection of similar peptides from H. megidis. The genome of S. carpocapsae has been recently sequenced [36].

The MS proteomics data and MaxQuant search output files have been deposited to the ProteomeXchange Consortium [38] via the PRIDE partner repository with the dataset identifier PXD027608.

Translation

The most prominent difference between the two species is in the response of proteins related to translation, which were decreased in H. megidis after storage in both 9°C and 20°C (Fig 4) and were generally increased in S. carpocapsae (Fig 5; S1 Table). All ribosomal proteins decreased in abundance over time in H. megidis IJs (S2 Table) regardless of storage temperature. Protein production is energetically expensive, costing up to 75% of the cell’s energy [39]. As metabolic activity is reduced, reduction of energetically expensive processes would be advantageous. Walther et al. [22] reported extensive proteome remodelling in aging C. elegans worms, with reduced ribosomes and increased proteasome complexes. Most proteasome related proteins detected were increased at both temperatures in both species. Reduction of translation and mRNA production has shown to extend the lifespan of C. elegans [24, 40]. S. carpocapsae IJs did not exhibit a general decrease in ribosomal proteins over time, and translation related proteins were generally increased in abundance, regardless of storage temperature (S1 Table; Fig 5).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. String interactions of H. megidis proteins which are decreased (left) and increased (right) in abundance after storage at 9°C (top) and 20°C (bottom) for 6 weeks.

https://doi.org/10.1371/journal.pone.0266164.g004

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. String interactions of S. carpocapsae proteins which are decreased (left) or increased (right) in abundance after storage at 9°C (top) and 20°C (bottom) for 6 weeks.

https://doi.org/10.1371/journal.pone.0266164.g005

Chaperones

Most chaperone proteins that were detected were increased in abundance in S. carpocapsae IJs, and to a greater extent after storage at 9°C (S1 Table). These include chaperonins, prefoldins, heat shock proteins and late embryogenesis (LEA) proteins. One chaperonin was detected in S. carpocapsae, increasing in abundance at 20°C, and in H. megidis, decreasing at 9°C. Chaperonins are ATP-dependent chaperones which assist in the folding of nascent polypeptides [41]. Various heat shock proteins (HSPs) were detected in S. carpocapsae (S1 Table) and generally increased in abundance over time, and to a greater extent at 9°C. HSPs are molecular chaperones which aid folding of proteins, prevent stress induced aggregation or misfolding of proteins, and can revert misfolded proteins to their native conformation [42]. HSPs, despite their name, are protective against a wide array of stresses and can also have roles in maintaining cellular components such as the cytoskeleton [43]. Small heat shock proteins are typified by a conserved α-crystallin C terminal domain [42] and are involved in the prevention of toxic aggregates of protein [44], and have higher binding affinities than the larger, classical HSPs [42]. Prefoldins are molecular chaperones which detect, bind to and deliver unfolded proteins, especially actin, to cystolic chaperonins [45]. Prefoldin proteins were amongst the few chaperone proteins decreased in abundance in S. carpocapsae IJs, at both 9°C and 20°C.

Many late embryogenesis abundant (LEA) proteins were detected in S. carpocapsae IJs, and most were temperature specific: increasing by up to ~90 times abundance after storage at 9°C, but mostly decreased by ~2–4 times if stored at 20°C (S1 Table). LEA proteins were first discovered in the seeds of plants [46, 47] and confer desiccation tolerance to the seeds. Since their initial discovery, these proteins have been found in a variety of nematodes such as C. elegans [48], Steinernema feltiae [49] and Aphelenchus avenae [50]. LEA proteins are atypical molecular chaperones which can protect the structure of proteins [51], if present before the exposure to stress. Unlike heat shock proteins, LEA proteins alone cannot protect proteins from heat stress and cannot revert misfolded proteins back to their native state [52, 53]. LEA proteins enhance the organism’s survival in response to cold and freezing conditions [54, 55], oxidative stress [56] and salt stress [57]. LEA proteins, in conjunction with trehalose, facilitate the formation of “glass” [50], which is protective against desiccation [48] and freezing [58]. Trehalose-6-phosphate synthase, which was increased at 6 weeks in S. carpocapsae regardless of storage temperature (S1 Table), may be involved in this bioglass formation or could be a response to starvation.

All chaperone proteins detected as SSDAs in H. megidis were decreased in abundance, regardless of storage temperature (Fig 4; S2 Table). With the sharp decrease in translation, chaperone proteins may be redundant to H. megidis IJs without an immediate stressor such as heat or desiccation triggering their expression. Decreased translation would also reduce the level of misfolded proteins and toxic aggregates, which may reduce the need for protein chaperone activities.

Metabolism

Proteins associated with gluconeogenesis are amongst the few groups of metabolism related proteins which were increased in abundance in S. carpocapsae IJs (Fig 5; S1 Table) and their increase may be related to starvation of the worm. Gluconeogenesis is responsible for the generation of monosaccharides used to generate energy in subsequent metabolic processes. Sugars formed in gluconeogenesis are converted to pyruvate during glycolysis, which is then converted to acetyl-CoA by pyruvate dehydrogenase. Pyruvate formed by glycolysis is transported to mitochondria to be converted to acetyl-CoA in the citric acid cycle, an aerobic process which utilises acetyl-CoA to reduce NAD+ to NADH, producing carbon dioxide as a by-product. The NADH is then used in oxidative phosphorylation. While glucose is the classic sugar transport unit formed in this pathway, nematodes and insects also form trehalose, a disaccharide composed of 2 glucose molecules. This sugar is functionally similar but also plays a crucial role in nematodes’ resistance against desiccation, freezing [59], and other stresses [60]. This sugar, along with LEA proteins facilitates the formation of bioglass, which enhances nematodes’ freezing tolerance [50].

Few glycolysis-related proteins were detected as SSDAs in H. megidis and these were generally decreasing in abundance at both temperatures (Fig 4; S2 Table). Most glycolysis proteins detected in S. carpocapsae did not change significantly over time at either temperature (Fig 5; S1 Table). The glyoxylate pathway is an alternative pathway to the TCA cycle, which is not normally found in metazoans, but is present in nematode dauers [61]. Isocitrate lyase and malate synthase catalyse the conversion of coenzyme A to succinate and malate [62], which may then be processed by succinate dehydrogenase to form malate, which can then be processed by malate dehydrogenase. This pathway allows for the generation of glucose molecules from the β-oxidation of fatty acids. There is significant overlap between the enzymes in these two metabolic pathways, and most enzymes were increased in abundance during storage at 20°C and decreased at 9°C in S. carpocapsae IJs (S1 Table). All SSDA proteins detected as part of both the citric acid cycle and the glyoxylate pathway were decreased in abundance regardless of storage temperature in H. megidis IJs (S2 Table).

Few oxidative phosphorylation enzymes were detected in H. megidis (S2 Table), but those that were detected increased in abundance at 20°C and decreased in abundance at 9°C (Fig 4; S2 Table). The exception to this was NADH-ubiquinone oxidoreductase ASHI subunit, which was increased by ~11 times at both temperatures. Proteins related to oxidative phosphorylation were abundant in a temperature specific manner in S. carpocapsae IJs, increasing at 20°C and decreased in abundance at 9°C (Fig 5; S1 Table).

Many fatty acid- and retinol-binding proteins (FARs) and nematode polyprotein allergens/antigens (NPAs) were detected in S. carpocapsae (S1 Table), most decreasing in abundance at both storage temperatures. FARs are a diverse family of proteins that are expanded in the S. carpocapsae genome [63]. NPAs are spliced to form many copies of nematode FARs [64]. Nematode FARs have structures similar to FARs found in other animals but have structures unique to nematodes, and therefore probably have nematode specific functions. FAR proteins may transport and store small quantities of lipids [65].

In general, S. carpocapsae proteins associated with metabolism decreased at 9°C and increased at 20°C (S1 Table). Proteins related to both gluconeogenesis and glycolysis were increased in abundance in S. carpocapsae IJs stored at 9°C, whereas proteins related to intermediary metabolism and oxidative phosphorylation were decreased to a greater extent at 9°C (Fig 5). This may be due to IJs being more active at higher temperatures and requiring more energy [18] and may be partially responsible for the IJs’ enhanced longevity at low temperatures [11, 12].

Cytoskeleton

Cytoskeletal proteins such as actin, myosin, collagen, and tubulin were amongst the highest abundance proteins detected in both species by raw LFQ intensity. Proteins associated with the cytoskeleton were generally decreased in abundance in both species over time (Figs 4 and 5; S1 and S2 Tables). EPN IJs tend to become less active over time [19], and therefore proteins associated with locomotion may be degraded. Collagen remodelling is also reported to be associated with lifespan-lengthening in C. elegans, regulated by the stress pathway SKN-1 [66]. Collagen was detected as decreased in abundance in S. carpocapsae IJs stored at 20°C, however it was increased in those stored at 9°C (S1 Table).

Stress/Detoxification proteins

String analysis identified a network of stress proteins which increased in abundance in S. carpocapsae at 9°C (Fig 5), although there was a tendency for stress/detoxification proteins to also increase in abundance at 20°C. Many stress proteins detected as SSDAs in this analysis in both species such as short chain dehydrogenases, thioredoxins, GSTs, catalase, oxidoreductases, aldehyde dehydrogenases (S1 and S2 Tables), are known to be regulated by SKN-1 [67], the pathway implicated in collagen remodelling mentioned above [66]. Proteins associated with the cell’s response to reactive oxygen species, such as catalase in H. megidis, and both catalase and copper oxide dismutase in S. carpocapsae, were increased to a greater extent at 9°C than at 20°C. Thioredoxin, an antioxidant, was decreased in abundance at both temperatures at all timepoints in both H. megidis and S. carpocapsae. Other stress proteins such as aldehyde dehydrogenase, which is involved in the stress response against by-products of anaerobic fermentation were increased to a greater extent at 20°C than at 9°C in S. carpocapsae (S1 Table).

Xenobiotic detoxification is generally divided into three distinct stages. Short chain dehydrogenases and reductases render xenobiotics less stable and represent phase I. Glutathione S transferase is involved in stage II, and transfers glutathione (an antioxidant) onto xenobiotics, increasing their solubility and facilitating its breakdown. Glutathione can also reduce free radicals generated during the stress response. UDP-glucoronosyl and UDP-glucosyl transferase increased in abundance in both H. megidis and S. carpocapsae IJs, at most timepoints and both temperatures (S1 and S2 Tables). UDP-glucoronosyl transferases add glucuronic acid to a xenobiotic, which may render it harmless, or aid in its excretion [68]. Glucosyltransferases may also be involved in the synthesis of trehalose [69], a sugar which enables survival in harsh conditions [60].

Fewer stress-related proteins were detected in H. megidis, and many of them were decreased in abundance after conditioning at both temperatures, except for catalase, which increased after storage at 9°C (S2 Table). Amongst these proteins, one of the few SSDA proteins that was constitutively increased in abundance over time was an autophagy related protein, and it increased to a greater extent at 9°C than at 20°C (S2 Table). Selective autophagy has been shown to improve C. elegans lifespan at low temperatures [70] and may have a similar role in H. megidis IJs.