Graphical abstract graphic file with name fx1.jpg [41]Open in a new tab Highlights * • Constructed a regulatory network for heat stress responses * • Identified XHSR as a key regulator, validated via knockdown experiments * • Confirmed XHSR’s role in heat stress via CRISPR/Cas9 knockout in Drosophila __________________________________________________________________ Biological sciences; Entomology; Genetics; Molecular genetics Introduction Global warming poses a significant threat to terrestrial, aquatic, and marine ecosystems. The increased temperatures can lead to severe impacts on water availability and agricultural production, resulting in steep declines in total food production, some of which may appear as early as 2040.[42]^1 The warming also leads to the extinction and migration of key species, which causes serious changes in community structures.[43]^2^,[44]^3^,[45]^4 Insects are particularly vulnerable to the impacts of global warming, because their complex physiological systems are heavily impacted by climate.[46]^5^,[47]^6 These negative impacts translate into reduced abilities to carry out essential life functions, such as foraging, mate-finding, mating, and producing viable progeny. Generally, parasitoid wasps are more susceptible to thermal stress than their hosts.[48]^7 As the most diverse group of insects, parasitoid represent a tremendous potential for biological control of insect pests.[49]^8 They are reared, transported among many countries, and applied in many cropping systems. They provide agricultural benefits at the levels of food security and environmental stewardship because they contribute to reducing the broadcast use of chemical insecticides. Pteromalus puparum is a cosmopolitan parasitoid wasp that uses numerous pest species as hosts, including the pupal stage of the butterfly Pieris rapae.[50]^9 Field surveys documented high rates of parasitism by P. rapae in East China, with up to 90% during early summer and 59–62% during winter.[51]^10 However, elevated environmental temperatures exert serious negative influence on P. puparum, recorded as reduced developmental rates, altered sex ratios, and reduced offspring density per host pupa. These data document diminished potential for pest population control.[52]^11 Elevated environmental temperatures do not influence parasitoid species and other insect species in a uniform manner. The aphid Sitobion avenae demonstrated increased heat tolerance and longevity in response to higher temperatures.[53]^12 Alternatively, the adult lifespan of the true bug, Diaphorina citri, was reduced by 74% at 41°C.[54]^13 Such variations may, in some species, be related to durations of heat exposure, as well as genetic and ecological factors. Insects employ a variety of genetic responses to cope with heat stress, including the regulation of HSPs, antioxidant enzymes, and lipid metabolism-related genes.[55]^14^,[56]^15^,[57]^16 The crucial role of HSPs in insects’ responses to heat stress is frequently emphasized, as they function as stress proteins or molecular chaperones.[58]^17^,[59]^18 Transcriptome analyses indicate that the expression of genes encoding heat shock protein 70 (HSP70) acts to prolong the lifespan of Drosophila melanogaster under short-term heat stress.[60]^19 However, longer exposures to severe heat stress inhibited HSP expression and activated the expression of genes that lead to apoptosis, causing irreversible tissue damage.[61]^20^,[62]^21 Another study showed that repeated episodes of mild heat stress in female Drosophila increased their resistance to acute heat stress via a decrease in dopamine level,[63]^22 contributing to the regulation of energy and lipid metabolism.[64]^23 Despite these findings, the overall pattern of insect responses and potential mechanisms for elevated environmental temperatures remain unclear.[65]^16 Based on the significance of parasitoid wasps in agriculture, meaningful clarity on how specific parasitoids respond to environmental temperatures and the range of survivable environmental changes these insects can withstand is necessary. In this study, we examined the genetic regulatory network and the function of hub genes involved in the effects of heat stress on the fitness traits of P. puparum. Results Heat stress induces distinct genetic network responses The biological fitness parameters of Pteromalus puparum undergo significant changes as the environmental temperature rises. After adult wasps were subjected to heat stress (35°C), their daily food intake increased by a factor of 1.52 compared to the control group ([66]Figure 1A). The number of offspring per female was reduced to only 11.6% of the control group ([67]Figures 1B and 1C). The successful parasitism rate witnessed a significant decrease, and their lifespan was drastically reduced by 40% ([68]Figures 1D–1I). Figure 1. [69]Figure 1 [70]Open in a new tab The effects of heat stress on fitness parameters and gene expression in Pteromalus puparum Means ± SEM. ∗∗ means p < 0.01, ∗∗∗ means p < 0.0001, ns means p > 0.05. (A) Daily food intake per wasp during the adult stage from eclosion to death, under 25°C and 35°C. The dotted line indicated median intake; (B) Daily number of offspring each female wasp produced from two days after eclosion to death, under 25°C and 35°C; (C) Total offspring number per female wasp produced throughout its adult stage, under 25°C and 35°C; (D) Successful rate of parasitism by wasps under 25°C and 35°C; (E) Degree of infestation by wasps under 25°C and 35°C; (F) Survival of adult female wasps under 25°C and 35°C, with hosts provided from two days after eclosion; (G) Lifespan of adult female wasps under 25°C and 35°C, with hosts provided from two days after eclosion; (H) Survival of adult female wasps during adult stage from eclosion to death under 25°C and 35°C. Wasps had never mated or oviposited. (I) Lifespan of adult female wasps during adult stage from eclosion to death under 25°C and 35°C. (J) Heatmap and cluster analysis. Each row includes 100 genes from a transcriptome sample selected using a random function, and the grouping information is labeled as “Age" and “Group" on the right side of the graph. Brown blocks from light to dark indicate 3 h, 6 h and 12 h, while green blocks from light to dark indicate 5 days, 10 days and 15 days, respectively. The blue blocks indicate the 25°C group, labeled "25°C," and the red blocks indicate the 35°C group, labeled "35°C." Each column represents one gene. Each cell represents the expression of a gene in a sample, expressed as log[2](FPKM+1), from 0 to 14 corresponding to the color from blue to red; (K) Graphs show the first two principal components (PC) explaining gene expression levels in all female wasps. Each data point represents a biological replicate, with PC coordinates determined using regularized log transformed read counts; (L) Venn diagram depicting the definition of genes altered by mild heat stress. DEGs are classified as genes responding to acute and chronic heat stress and are displayed according to up- or down-regulation of gene expression; (M) Top 10 GO terms of GO enrichment of up-regulated DEGs; (N) Top 10 GO terms of GO enrichment of down-regulated DEGs; (O) Results of the KEGG pathway enrichment analysis of DEGs, with circles indicating the number of DEGs significantly enriched to this pathway. To investigate the impact of heat stress on gene expression and adaptability in the context of aging, as well as to elucidate the mechanisms through which parasitoid wasps modulate gene expression to cope with elevated temperatures, we conducted transcriptome sequencing at various age stages of the adult parasitoid wasps. A total of 1,567,502,192 clean reads were obtained from 36 samples ([71]Table S1). The PCA and cluster analyses revealed two distinct gene expression patterns in response to heat stress: an acute response within the first 12 h, and a chronic response after 5 days ([72]Figures 1J and 1K). Chronic heat stress led to larger gene expression alterations than acute heat stress. We identified 29 up-regulated and 24 down-regulated genes, mainly HSPs, as differentially expressed under acute heat stress. Meanwhile, chronic heat stress resulted in the identification of 323 up-regulated and 354 down-regulated genes, including numerous transcription factors (TFs) ([73]Figure 1L). Top GO terms for up-regulated differentially expressed genes (DEGs) were enriched in peptide activity, while down-regulated DEGs were enriched in binding ([74]Figures 1M and 1N). DEGs were significantly enriched in signaling pathways including “longevity regulating pathway-multiple species,” and “Foxo signaling pathway” ([75]Table 1; [76]Figure 1O). By cross-referencing DEGs with evolutionarily conserved lifespan-related genes,[77]^24 we found that 135 DEGs predictably operate in aging ([78]Table S2). Table 1. KEGG pathway enrichment of DEGs under acute heat stress KEGG Pathway -LOG[10](FDR) __________________________________________________________________ DEGs __________________________________________________________________ 3 h 6 h 12 h 3 h 6 h 12 h Antigen processing and presentation 4.89 2.80 2.24 CTSL, HSP70, HSP90A CTSL, HSP70, HSP90A CTSL, HSP70, HSP90A Longevity regulating pathway - multiple species 4.57 2.80 2.02 HSP70, CRYAB HSP70, CRYAB HSP70, CRYAB Prion diseases 4.06 ns 1.70 HSP70, STIP1 0 HSP70, STIP1 Legionellosis 3.76 ns 1.53 SdhA, HSP70 0 SdhA, HSP70 Protein processing in endoplasmic reticulum 3.76 1.73 1.40 HSP70, HSP90A, CRYAB, BAG2 HSP70, HSP90A, CRYAB HSP70, HSP90A, CRYAB, FBXO2 Measles 2.51 ns ns HSP70 0 0 Estrogen signaling pathway 2.51 ns ns HSP70, HSP90A 0 0 Toxoplasmosis 2.13 ns ns HSP70 0 0 Influenza A 1.75 ns ns trypsin, HSP70 0 0 Pancreatic secretion 1.61 ns ns amyA, trypsin, PNLIP 0 0 Caffeine metabolism 1.61 ns ns xdh, uaZ 0 0 Starch and sucrose metabolism 1.59 ns ns amyA, malZ 0 0 Steroid hormone biosynthesis 1.56 ns ns UGT, CYP3A 0 0 Chemical carcinogenesis 1.52 ns ns UGT, CCBL, CYP3A 0 0 Quorum sensing ns ns 1.70 0 0 chitinase, GAD [79]Open in a new tab We employed network analysis to explore the complex interplay between gene expression, environmental temperature, and age. In the construction of the co-expression network, highly related genes were clustered together and assigned distinct colors, yielding 36 color modules ([80]Figure S1A). The blue and turquoise modules, as indicated by the red squares, had more co-expression clustering than the yellow squares ([81]Figure S1B). To examine the association with traits, which included temperature groups and age, the correlation coefficient between traits and the first principal component of the module, i.e., module eigengene (ME), was calculated. Six candidate modules were significantly correlated with temperature and age (p < 0.05, [82]Figure 2A). Five modules, blue, brown, pink, purple, and yellow modules, were positively correlated with heat stress. There was a significant correlation between age and the turquoise, brown, and purple modules ([83]Figure 2A). Cluster analysis among the modules revealed that the blue and brown modules clustered together, as did the purple and pink modules, while the yellow and turquoise modules were distinct from the others ([84]Figure S1A). The best hub gene of each candidate module was determined, and the one with the highest connectivity in the blue module was named Xap5 Heat Stress Regulator (XHSR) ([85]Table 2). Figure 2. [86]Figure 2 [87]Open in a new tab Weighted gene co-expression network analysis (A) Heatmap of module-trait correlations. Each row is a module eigengene (ME). The first column represented different groups. The 25°C group was set as 0, and the 35°C group was set as 1. The second column represented different ages of wasps, from 3 h to 15 days. The correlation coefficient and p value of MEs with traits were marked in each cell, where p values were indicated in parentheses. Blue indicates negative correlation (∼−1) and red indicates positive correlation (∼+1) between the ME and the trait; (B–G) Scatterplot of correlations between GS and MM of candidate modules. Each point indicates a module member (i.e., gene) assigned to each module. Table 2. The most highly connected genes in the candidate modules Module Gene ID Description blue [88]PPU15815 XHSR; Protein FAM50 homolog brown [89]PPU08660 RNA polymerase II elongation factor ELL pink [90]PPU12978 Venom serine protease BiVSP purple [91]PPU16576 60S ribosomal protein L8 yellow [92]PPU08277 Piwi-like protein Ago3 turquoise [93]PPU05818 Inner nuclear membrane protein Man1 [94]Open in a new tab We calculated gene significance (GS) to evaluate the genes’ relevance to group or age categories and module membership (MM) to measure the extent of association between genes and modules. Pearson correlation coefficients between GS and MM were obtained for six modules. Notably, the scatterplot of genes in the blue module presented a linear trend that closely approximated a slope of 1, suggesting a strong positive correlation between GS and MM (cor (GS vs. MM) = 0.7, p < 0.05, [95]Figure 2B). This indicates a strong relationship between genes in the blue module and both the trait and the module itself. Likewise, the correlation between GS and MM for the brown, yellow, and purple modules reached a significant correlation ([96]Figures 2C–2G). Since the brown and purple modules were strongly connected with age traits, genes in these two modules may be linked to the age effect of the heat treatment. This analysis solidly supports the blue module as a highly reliable representation of the transcriptional signatures induced by heat stress. To further explore the attributes of the blue module, we utilized Cytoscape for network visualization, employing a weight threshold of >0.2, resulting in the identification of 113 core members ([97]Figure 3A). The network is naturally divided into two subgroups, each with a central gene of the highest connectivity: XHSR and PpPPP6A. XHSR had previously been determined as the best hub gene within the blue module. Genes were ranked based on their Maximal Clique Centrality (MCC) score and color-coded, with the top 20 genes predominantly up-regulated ([98]Table S3).[99]^25 Expression patterns of top adjacency genes indicated an age-related increase in log[2]FoldChange values ([100]Figure 3B). The XHSR gene maintained elevated expression levels in the chronic heat stress period, and its expression gradually increased. The fold change values at various ages were 1.3, 1.6, 1.9, 2.4, 2.7, and 3.4 times higher than the baseline ([101]Figure 3B). The KEGG pathway analysis highlighted enrichment of four pathways by six genes, including PpCTSL, PpHSPs, PpP38, and PpGST ([102]Figure S2). Integration of ME_blue >0.8, GS_group >0.2, and weight >0.2 identified 55 candidate hub genes ([103]Table 3). These core candidates were likely to constitute the regulatory network employed by the parasitoid wasp in response to high temperatures. In order to explore the degree of conservation and potential variations in the critical regulatory genes, we conducted a phylogenetic analysis. This analysis confirmed the conservation of XHSR homologs in Hymenoptera, Coleoptera, Lepidoptera, Diptera, and Hemiptera, including D. melanogaster, despite dissimilarity with the XHSR homolog of P. rapae ([104]Figure 3C). Figure 3. [105]Figure 3 [106]Open in a new tab Regulatory and evolutionary characteristics of blue module candidate hub genes (A) The protein-protein interaction (PPI) network for hub genes in the blue module. Genes with weight <0.2 in the network were filtered out and hub genes were predicted. The importance of genes in the network is ranked by MCC and is represented in red, yellow, and blue from highest to lowest; (B) Heatmap of top hub genes ranked by MCC. Each row represented a gene, and each column represented an age. Each cell represented the differential expression of genes in stressed and unstressed conditions for one age group, expressed as log[2]FoldChange values. ∗ means p < 0.05; (C) Phylogenetic analysis of the homologous genes of XHSR. The maximum-likelihood tree was constructed using IQ-TREE software with 1000 ultrafast bootstrap replicates. XHSR is highlighted with a red star. Circles on the tree branches represent ultrafast bootstrap supports ≥50. Table 3. Hub genes in the blue module Gene ID Gene Name Description kME GS [107]PPU15815 XHSR Protein FAM50 homolog 0.9358 0.8330 [108]PPU04131 PpCRYAB1 Protein lethal (2) essential for life 0.9240 0.8816 [109]PPU01716 PpYNG2 Chromatin modification-related protein 0.9115 0.7003 [110]PPU04112 PpAKR1B1 Aldo-keto reductase AKR2E4 0.9095 0.7602 [111]PPU05064 PpRAB28 Ras-related protein Rab-28 0.9044 0.8286 [112]PPU16943 PpSEC11C Signal peptidase complex catalytic subunit 0.9030 0.7666 [113]PPU00892 PpPER2 Period circadian protein 2 0.8944 0.7966 [114]PPU04336 PpCHCHD2 Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 0.8942 0.7030 [115]PPU09664 PpCYP6 Probable cytochrome P450 6a14 0.8903 0.7697 [116]PPU08466 PpH2B Histone H2B 0.8891 0.7451 [117]PPU10167 [118]PPU10167 Protein singed wings 2 0.8833 0.8245 [119]PPU07654 PpEEF1A1 Elongation factor 1-alpha 0.8812 0.6521 [120]PPU15477 PpISYNA1 Inositol-3-phosphate synthase 1-A 0.8799 0.7611 [121]PPU16233 PpHSPE1 10 kda heat shock protein, mitochondrial 0.8782 0.8719 [122]PPU06466 PpPSMD1 26S proteasome non-atpase regulatory subunit 1 −0.8763 −0.7653 [123]PPU03831 PpHSPA8 Heat shock 70 kda protein cognate 4 0.8759 0.8579 [124]PPU00866 PpCTSL Cathepsin L 0.8752 0.6994 [125]PPU11226 PpHSD17B8 Estradiol 17-beta-dehydrogenase 8 0.8706 0.7439 [126]PPU04477 PpLCA5 Lebercilin 0.8638 0.7469 [127]PPU00422 PpPOLG DNA polymerase gamma 1 0.8613 0.8944 [128]PPU08409 PpSREBF1 Sterol regulatory element-binding protein 1 0.8589 0.7299 [129]PPU16234 PpHSPD1 60 kda heat shock protein, mitochondrial 0.8582 0.8685 [130]PPU07257 PpP4HA2 Prolyl 4-hydroxylase subunit alpha-2 0.8576 0.6892 [131]PPU02108 PpTXNL4A Thioredoxin-like protein 4A 0.8562 0.8028 [132]PPU15773 PpFIG4 Phosphatidylinositol 3 0.8508 0.7616 [133]PPU03107 PpPRSS1 Trypsin-1 −0.8504 −0.6260 [134]PPU07010 PpUTP23 U3 small nucleolar RNA-associated protein 23 0.8467 0.7283 [135]PPU07079 [136]PPU07079 Cuticle protein 10.9 0.8458 0.7281 [137]PPU05443 [138]PPU05443 Cuticlin-1 0.8457 0.7024 [139]PPU03644 PpRRM1 Ribonucleoside-diphosphate reductase large subunit 0.8438 0.8396 [140]PPU06969 PpVIPAS39 Spermatogenesis-defective protein 39 homolog 0.8407 0.6972 [141]PPU00804 PpHSP90A1-1 Heat shock protein 83 0.8406 0.8710 [142]PPU02432 PpPEBP1 Phosphatidylethanolamine-binding protein homolog F40A3.3 0.8364 0.7970 [143]PPU07658 [144]PPU07658 Actin-binding Rho-activating protein 0.8346 0.7253 [145]PPU12736 PpMAFK Transcription factor mafk 0.8335 0.8292 [146]PPU03960 PpDESI1 Desumoylating isopeptidase 1 0.8308 0.6305 [147]PPU07935 [148]PPU07935 Protein disabled 0.8268 0.6469 [149]PPU15624 [150]PPU15624 Uncharacterized protein CG1161 0.8255 0.7972 [151]PPU13156 [152]PPU13156 Protein FAM151A 0.8251 0.6285 [153]PPU01583 [154]PPU01583 Cyclic GMP-AMP synthase 0.8235 0.7991 [155]PPU03794 PpDDX5 ATP-dependent RNA helicase p62 0.8228 0.9310 [156]PPU14775 PpCLUAP1 Clusterin-associated protein 1 0.8220 0.8103 [157]PPU08124 PpGST Glutathione S-transferase 1, isoform C 0.8220 0.7618 [158]PPU06685 PpPPP6A Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit A −0.8204 −0.6491 [159]PPU01592 PpPRUNE1 Exopolyphosphatase PRUNE1 0.8139 0.8059 [160]PPU10765 PpHAX1 Myeloid leukemia factor 2 0.8126 0.6594 [161]PPU08531 PpALG6 Dolichyl pyrophosphate Man9GlcNAc2 alpha-1,3-glucosyltransferase 0.8122 0.7335 [162]PPU06370 PpKANK1 KN motif and ankyrin repeat domain-containing protein 0.8092 0.7196 [163]PPU10036 PpPQLC1 PQ-loop repeat-containing protein 1 0.8068 0.6560 [164]PPU10302 [165]PPU10302 Cerebellar degeneration-related protein 2-like 0.8060 0.7467 [166]PPU11519 PpBCAP31 B-cell receptor-associated protein 31 0.8044 0.7427 [167]PPU10709 PpECI2 Enoyl-coa delta isomerase 2, mitochondrial 0.7964 0.7496 [168]PPU10178 PpNMUR1 Pyrokinin-1 receptor −0.7756 −0.6206 [169]PPU04656 PpPEB3 Ejaculatory bulb-specific protein 3 0.7736 0.7250 [170]PPU11653 PpVA3 Venom allergen 3 0.7706 0.8186 [171]Open in a new tab Functional information on homologs of core module members was obtained using STRING and ChEA3. The STRING database integrates experimentally validated gene function data from model organisms and generates functional interaction networks in silico. Core module memberships of the blue module were inputted into STRING to identify enriched GO terms and pathways ([172]Figure 4A). Using Markov clustering (MCL), 62 Homo sapiens homologs of putative hub genes were identified and grouped into eight clusters. Cluster 1 showed intense interactions related to stress response, chaperone function, and acetylation ([173]Figure 4B). Two HSPs and EEF1A1 were enriched for chaperone mediated autophagy (FDR = 0.0060, [174]Table S4). Cluster 2 included genes involved in fatty-acyl-CoA biosynthesis and the acetyl-CoA metabolic process ([175]Figure 4C). SREBF1 and FASN were enriched by the AMP-activated protein kinase (AMPK) pathway (FDR = 0.0472, [176]Table S5), known for its role in longevity regulation. KANK1 was identified as an interacting partner of FAM50A, the human homolog of XHSR (interaction score >0.3). Gene expression analysis revealed differential expression of HSPs in cluster 1 after acute heat stress, while lipogenesis genes in cluster 2 were not differentially expressed until chronic heat exposure ([177]Figure S3). Key TFs associated with the core module memberships included SREBF1, PEBP1, HSPs, EEF1A1, and FASN ([178]Figure S4A).[179]^26 The TF co-regulatory network of the top 20 TFs indicated an interaction between TFs JUN and SREBF1 ([180]Figure S4B). Similarity-based text mining results supported the functional significance of PpSREBF1, PpHSPs, PpEEF1A1, and PpFASN. Figure 4. [181]Figure 4 [182]Open in a new tab Interactions between human homologs of candidate hub genes based on STRING database (A) The functional network was constructed from genes with interaction scores >0.3, and disconnected nodes were hidden. Edges indicated both functional and physical protein associations, and their thickness indicated the strength of data support. Inside the node bubbles is a preview of the structure of each gene. Nodes were clustered into 8 clusters using MCL clustering, distinguished by color. (B and C) Interactions between genes in Clusters 1 and 2. The GO enrichment results for the top 5 biological process genes in which nodes were involved were highlighted. The vital role of Xap5 heat shock regulator in the genetic regulatory network A set of genes identified from DEG and WGCNA analyses underwent quantitative real-time PCR (qPCR) validation after heat stress treatment. The overall expression trends were consistent, despite slight differences compared to transcriptome sequencing results ([183]Figure S5). RNA interference (RNAi) experiments targeting the hub gene XHSR were performed at 35°C, using a reporter gene, Luciferase, as a control. Major biological parameters, including adult lifespan, intake, offspring number, and %DI, were compared between wasps injected with dsLuc and dsXHSR. Wasps injected with dsXHSR showed a significant 69.3% decrease in XHSR expression compared with dsLuc (p = 0.0322, [184]Figure 5A a). Their mean lifespan significantly decreased at both 35°C and 25°C. At 35°C, dsXHSR wasps exhibited a mean lifespan of 7.0 days, about half of the dsLuc control group (p < 0.0001, [185]Figure 5A b). While under non-stress conditions at 25°C, dsXHSR wasps had a mean lifespan of 19.2 days, significantly shorter than the 40.1 d observed in the dsLuc control group (p < 0.0001, [186]Figure 5A c). However, no significant changes were observed in food intake and %DI following dsXHSR injection (p > 0.05, [187]Figure 5A d-e). Figure 5. [188]Figure 5 [189]Open in a new tab Effects of RNAi-mediated knock-down of hub genes on wasp fitness and gene expression (A) Effects of down-regulation of XHSR expressions by RNAi on regulatory networks and fitness of wasps. Means ± SEM. ∗ means p < 0.05, ∗∗ means p < 0.01, ∗∗∗ means p < 0.0001, ns means p > 0.05. (a) Changes in the relative expression levels (REL) of XHSR after injection of dsXHSR; (b) Survival and lifespan of wasps after injection of dsXHSR at 35°C; (c) Survival and lifespan of wasps after injection of dsXHSR at 25°C; (d) Daily food intake per wasp after injection of dsXHSR and dsLuc at 35°C; (e) Degree of parasitism after injection of dsXHSR and dsLuc at 35°C; (f-l) Changes in the REL of other candidate hub genes after injection of dsXHSR. f: PpCRYAB1; g: PpHSPE1; h: PpHSPD1; i: PpCTSL; j: PpEEF1A; k: PpSREBF1; l: PpFASN1. (B) Effects of down-regulation of expressional levels of three candidate hub genes by RNAi on wasps’ lifespan and regulatory networks. Means ± SEM. ∗ means p < 0.05, ∗∗ means p < 0.01, ∗∗∗ means p < 0.0001, ns means p > 0.05. (a) Changes in the REL of PpPPP6A after injection of dsPpPPP6A; (b) Survival and lifespan of wasps after injection of dsPpPPP6A at 35°C; (c) Changes in the REL of PpCTSL after injection of dsPpCTSL; (d) Survival and lifespan of wasps after injection of dsPpCTSL at 35°C; (e) Changes in the REL of PpSREBF1 after injection of dsSREBF1; (f) Survival and lifespan of wasps after injection of dsSREBF1 at 35°C; (g-m) Changes in the REL of candidate hub genes after injection of dsSREBF1. g: PpFASN1; h: PpHSD17B8; i: PpPER2; j: PpPRUNE1; k: PpFIG4; l: PpSPTLC1; m: PpMVK. We examined the subnetwork of XHSR further because the inhibition of PpPPP6A expression had no effect on the mean lifespan of the wasps, compared to the control group (p = 0.9172, [190]Figure 5B a-b). Lifespan analysis was conducted on wasps injected with dsPpCTSL at 35°C, considering the up-regulation of PpCTSL and its close relationship to XHSR. dsPpCTSL had 66.9% interference efficiency (p = 0.0063, [191]Figure 5B c), resulting in an average lifespan of 9.4 days, 18% shorter than the dsLuc group (p < 0.0001, [192]Figure 5B d). Following dsXHSR treatments, qPCR was performed on genes adjacent to PpCTSL in the co-expression network. The expression of 12 genes was significantly changed, with several genes in cluster 1 of the STRING functional network also significantly changed. No significant changes were observed in the genes of cluster 2, except for PpSREBF1 ([193]Figure 5A f-l). Based on the STRING database, we hypothesized that PpFASN1 is related to PpSREBF1, which was verified by changes in mRNA levels of PpFASN1 following dsPpSREBF1 injection ([194]Figure 5B e-g). The mRNA levels of PpHSD17B8, PpPER2, PpFIG4, PpPRUNE1, and PpSPTLC1 were likewise significantly reduced, but to a lesser degree ([195]Figure 5B h-m). The mRNA encoding PpMVK remained unchanged. Functional implications of the Xap5 heat shock regulator homolog gene knockout in Drosophila Given the observed conservation of XHSR across species ([196]Figure 3C), we conducted a knockout experiment on the XHSR homolog CG12259 in D. melanogaster using the CRISPR/Cas9 system to investigate the response of the mutant flies to high temperatures. The selection of D. melanogaster as our model stemmed from the hypothesis that XHSR’s remarkable conservation across diverse species implies its potential pivotal role in regulating thermal responses. A segment of CG12259 containing the XAP5 domain on the third chromosome was knocked out and replaced with RFP ([197]Figure 6A). Positive mutants were selected and crossed to obtain heterozygotes (ΔCG12259^+/−) with an RFP marker on one chromosome and homozygotes (ΔCG12259^−/−) with RFP markers on both chromosomes. A pair of primers designed upstream of the 5′ homology arm and on the RFP were used to perform diagnostic PCR, with the wild type as a control. This documented that the target region of the CG12259 locus had been knocked out ([198]Figure 6B). At 35°C, all the flies died within two days, with a one-day survival rate of 66.7% (n = 60) for wild-type flies and 33.3% (n = 60) for ΔCG12259^−/− flies. At 32°C, the survival rate of mutants was significantly lower compared to wild-type flies. The survival rate of ΔCG12259^−/− flies was lower than ΔCG12259^+/− flies ([199]Figure 6C, Log rank test, p < 0.0001). The median survival of homozygous individuals was 11 days with a mean lifespan of 10.1 days, while heterozygous individuals had a median survival of 12 days with a mean lifespan of 11.2 days. This demonstrates that CG12259 knock-out significantly compromised the survival of Drosophila under high temperatures (32°C and 35°C). This observation is similar to the effect of XHSR knockdown in P. puparum, which increased the susceptibility of wasps to high temperatures. These findings suggest that the physiological functions of XHSR are conserved across different species and can be extrapolated to other organisms. Figure 6. [200]Figure 6 [201]Open in a new tab Knock-out of Drosophila XHSR homolog CG12259 using CRISPR/Cas9 system, and the survival of mutants at 32°C (A) Schematic representation of the specific recognition of the CG12259 locus, causing double-strand breaks (DSBs), knock out, and replacement of the red fluorescent protein (RFP). A pair of primers for diagnostic PCR was designed upstream of the 5′ homology arm on the locus and on the RFP, respectively. The PCR product is 2390bp in length. (B) Diagnostic PCR using a primer set (arrows in A) in homozygous (ΔCG12259^−/−), heterozygous (ΔCG12259^+/−) and wild-type (WT) individuals. (C) Survival of homozygous, heterozygous and wild-type female flies at 32°C. Discussion Heat stress led to impaired fertility shortened lifespan, and compromised ability for host control in Pteromalus puparum. In this study, we delved into the effect of high temperature (35°C) on the gene expression profiles in Pteromalus puparum, focusing on the intricate regulatory network orchestrating the transcriptional changes. We determined the genetic mechanism(s) of how heat stress affects traits by identifying genes involved in the P. puparum heat stress response. A rapid and discernible change was observed in gene expression profiles in wasps as early as 6 h, following exposure to elevated temperatures. These changes likely signify the activation of stress response mechanisms and the initiation of adaptive strategies aimed at survival. Notably, the major KEGG enrichment pathways among DEGs, especially longevity regulating pathway and the Foxo signaling pathway, suggest a link between these early gene expression changes and the aging regulation in response to heat stress.[202]^24WGCNA workflow was applied to simplify the transcript profiles and construct a co-expression network. We identified the blue module as the most closely related to the effects of heat stress and was not correlated to age. The hub genes were identified based on the interrelationships and topological structures of genes within the blue module. The best hub gene in the regulatory network was named XHSR. Heat stress induced the expression of XHSR, and the induction increased with the age of the wasps, while they were continuously exposed to high temperatures. XHSR encodes an XAP5-domin protein, the function of which is unknown. The functional STRING network exhibited the strength of interactions between human homologs of candidate hub genes. These genes primarily formed two distinct clusters, in which one contained HSPs involving protein folding and the other contained proteins relating to fatty acid metabolism. Based on the STRING database, there is no evidence suggesting that FAM50A, the human ortholog of XHSR, is directly involved in the regulation of cellular responses to heat stress or lipogenesis. The distinct cluster characterized by the presence of HSPs within the network aligns with previous studies conducted in other organisms, where experiments have consistently highlighted the induction of HSPs, in particular HSP70 and HSP90 under high temperature conditions.[203]^27 HSP70 facilitates cellular processes through substrate regulation for unfolding, disaggregation, refolding, or degradation.[204]^28 HSP90 plays a role in signal integration and substrate targeting for proteolysis, primarily acting during the later stages of substrate folding, which are critical for cellular signaling and development.[205]^29 In addition, our findings suggest a critical role for CRYAB in the heat stress response. CRYAB is a small heat shock protein that modulates cellular processes related to survival and recovery during stress.[206]^30^,[207]^31 The induction of CRYABs by Foxo and HSF contributes to the lifespan extension of Caenorhabditis elegans.[208]^32 The function of hub genes was verified by assessing survival and lifespan changes alongside changes in mRNA levels of downstream genes following knockdown or knockout of selected genes. Suppression of XHSR led to a substantial reduction in wasp lifespan, thus documenting the positive role of XHSR in resistance to heat stress. Furthermore, the reduced lifespan of dsXHSR wasps in normal conditions suggests that XHSR has a broader role beyond the heat stress response, encompassing the regulation of longevity and highlighting its significance in multiple biological processes. While hub genes belonging to cluster 1 of the functional STRING network generally responded to the suppression of XHSR, those belonging to cluster 2 did not, except for PpSREBF1. Genes directly down-regulated by repression of XHSR include several HSPs, including PpEEF1A, PpCTSL, PpSREBF1, and among others. This suggested that XHSR may act as an upstream regulator of these genes and respond to acute heat stress by inducing the transcription of HSPs and other genes in cluster 1. Further examination of the mRNA results of hub genes in cluster 2 after injecting dsPpSREBF1 revealed that four genes were directly regulated by PpSREBF1. Pathway enrichment analyses suggested that genes in cluster 2 are involved in lipid metabolisms. Based on the expression patterns of genes in cluster 2 ([209]Figure S3), we inferred that these genes respond to chronic heat stress. Other studies have reported possible relationships between these hub genes and stress responses. Mammalian eukaryotic elongation factor 1A (EEF1A) has a thermo-sensing capacity.[210]^33 PpCTSL encodes a cysteine protease, cathepsin L, whose homologs exist in diverse organisms. Cathepsins are members of a family of proteases that function to degrade intracellular and endocytosed proteins in the lysosome.[211]^34 Cathepsin L plays a crucial role in the apoptosis of midgut epithelium cells, contributing to the remodeling of the larval midgut during metamorphosis in Helicoverpa armigera.[212]^35 PpSREBF1 encodes a sterol regulatory element binding protein. SREBFs are conserved in most organisms and regulate the expression of genes required to maintain cellular lipid homeostasis.[213]^36 In D. melanogaster, SREBF is regulated by levels of phosphatidylethanolamine rather than cholesterol levels. Its targets include acetyl-CoA synthetase, ACC, and FASN.[214]^37 Taken together, it is inferred that the hub genes in cluster 1 are mediated by XHSR in response to acute heat stress, whereas the hub genes in cluster 2 respond to chronic heat stress through PpSREBF1, which is downstream of XHSR ([215]Figure 7). Figure 7. [216]Figure 7 [217]Open in a new tab A model of hub genes in regulatory networks in response to heat stress The color of a gene represents its adjacency degree in the regulatory network. Solid arrows indicate gene interactions for which there is experimental evidence, and dotted arrows indicate possible gene functions supported by references.