Comprehensive investigation of gene expression regulation under temperature stress is very important to understand the biochemical and physiological adaptation processes in invasive insect pests16. In this study, a comprehensive transcriptome analysis and characterization of the gene expression profiles of S. invicta under cold and high temperature stress were evaluated. Through the analysis of DEGs, transcriptome changes in S. invicta adult ants were revealed. Using RNA-seq techniques four transcriptomes was de novo assembled from the adult stages of RIFA which exposed to four different temperatures (10, 20, 30, and 40°C), and 19,154 unigenes (19.33%) were successfully annotated from at least one public databases (UniProt) (Table 1). The results are in line with other transcriptome projects using Illumina technology25–27. 56.80% of the unigene sequences were most similar to gene sequences from Solenopsis invicta and more than 70% similarity with ant genus were observed. In this study, DEGs from adult RIFA which were exposed to different treatment temperatures (10, 20, and 40°C) were compared to that of a 30°C control group. The majority of DEGs were observed at T10, follow by T40, both of which expressed a greater DEG distribution than T20 (Fig. 3A). As mentioned earlier, this is consistent with proteomics data from Locusta migratoria under high and low temperature stress28. To identify specific genes associated with response to temperature, the number of unigenes with log2FC ≥ 10 was clarified by Venn diagram, and KEGG analysis was conducted to determine the probability of function in pathway enrichment. KEGG analysis revealed that from 203 specific cold-regulated DEGs (associated with T10), 41 DEGs were enriched in the KEGG pathways, and most of them were classified to following pathways: ‘Metabolic pathway’, ‘Carbon metabolism’, ‘Citrate cycle (TCA)’, ‘RNA transport’, and ‘Lysosome’. Interestingly in T20 and T40, ‘Metabolic pathway’ included more DEGs than other pathways (Fig. 5). ‘Purine metabolism’, ‘Spliceosome’, ‘Lysosome’, ‘RNA degradation’, ‘Glycolysis/Gluconeogenesis’, ‘Pyruvate metabolism’, ‘Phagosome’, ‘Sphingolipid metabolism’, ‘RNA transport’, ‘Glycerolipid metabolism’, ‘Carbon metabolism’, ‘ECM-receptor interaction’ are pathways that demonstrate similar results as those of investigations on transcriptome responses to cold stress in the carpenter moth, Eogystia hippophaecolus25, and the a chrysomelid beetle, Galeruca daurica16. Transcriptome analysis revealed that the expression of ‘Glycolysis’ and ‘TCA cycle pathways’ are up-regulated in a similar manner as the braconid wasp, Aphidius colemani, when exposed to low temperatures29. When RIFA was exposed to the highest temperature (40°C), ‘Tyrosine metabolism’, ‘Phenylalanine metabolism’, ‘Cysteine and Methionine metabolism’, ‘Spliceosome’, ‘Protein processing in endoplasmic reticulum’, and ‘Metabolic pathway’, were found to be enriched pathways that are similarly enriched in three species’ of rice plant hopper when exposed to 37°C22. ‘Fatty acid synthase’ and ‘Fatty acid metabolism’, are two of the main pathways of RIFA when exposed to high temperature. Expression of fatty acids as hydrophobic agents allows insects to avoid water loss in warmer regions of the globe30. At high temperatures, Gomphocerus sibiricus are known to increase their levels of oleic acid, linoleic acid, linolenic acid and glycerin, and phenomenon can suppress mortality due to excessive evaporation of body moisture31. In our study on RIFA, ‘Amino acid metabolism’ was clearly up-regulated during high-temperature stress. It is suggested that amino acid metabolism provides heat resistance in RIFA similar to that of results that have been reported for Locusta migratoria28 when exposed at 40°C. Due to the synthesis of immune proteins and defense enzymes, insects seek out and consume numerous free amino acids when coping with stress conditions such as high temperature, low temperature and fungal invasion32. The synthesis and metabolism of amino acids are necessary to produce a significant number of amino acids, which make available the raw materials necessary for the synthesis of heat-resistant proteins28.
In this study, two cuticular protein unigenes were identified from 203 co-regulated DEGs under cold temperature stress (Table S9). Cuticular protein gene expression has been observed in studying other insects studies such as beetles, moths, planthoppers, and stick insects when exposed to cold temperature stress16,22,25,33. Although the physiological role of cuticular proteins in insect cold hardiness has not yet been identified, it seems insect cuticle may play an important role in insects when coping with low temperature16,19,22,34,35.
According GO analysis (Fig. 6A), ‘Antioxidant activity’ was enriched at low temperatures. Suggesting that is might contribute to RIFA ability to resist oxidative stress damage at low temperature16, or their potential for cell preservation via antioxidant defense when in challenged by environmental complexity36. In addition, one cytochrome P450 was identified that up-regulated exclusively under low temperature (Table S9). Meanwhile NADH dehydrogenase subunit 1 was up-regulated at all treated temperature (Fig. 5C, Table S8). These two proteins are main enzymes at antioxidant activity pathway25. In comparative analysis of the transcriptional responses to low and high temperatures in three rice planthopper species, some cytochrome P450 genes were up-regulated under both low and high temperatures, which suggests cold and heat stress increase oxidative stress in the insect body22.
Heat shock proteins (HSPs) are another important protein that insects use as critical physiological products when under abiotic stress conditions28. From earlier studies it was believed that Hsp is associated with biological cold and heat resistance37,38. HSPs are molecular chaperones, which play important physiological roles including: correct folding of proteins, prevention of protein denaturation, and degradation of misfolded or condensed proteins and maintenance of correct protein conformation39,40. In this study, we identified one specific Hsp70 that up-regulated about a 12-fold change when RIFA was exposure at 10°C (Table S9). Two pathways, including ‘Protein export’ and ‘Protein processing in endoplasmic reticulum’, were enriched under Hsp70 gene expression at low temperatures (Fig. 5A, Table S12). Interestingly, heat shock protein 83 was found only in the 40°C treatment group that was up-regulated about 11 times (Table S11). Many studies have confirmed that the expression of Hsp genes can be up-regulated by cold and heat stimulus39,41. To assist the resistance to temperature stress, the Hsp60 gene expression in Stegobium paniceum significantly increases under high- and low-temperature stress42. Three Hsp90 and four Hsp70 were up-regulated by cold stress and were differentially expressed at desert beetle, Microdera punctipennis21. The differences in Hsp, insect species, sex of organism, and intensity of temperature are important factors related to Hsp expression level in insects16,43.
In conclusion, we compared the transcriptomes of S. invicta under high- and low-temperature stresses using RNA-Seq technology based on the high-throughput sequencing. Comparative transcriptome analysis identified many genes, and a large number of changes were discovered in the metabolic pathways through the GO and KEGG enrichment analysis. Our data will facilitate further molecular investigations and genomic research. Many novel relationships between high- and low-temperature and significantly up-regulated genes were identified in this study (Table S7-11). These newly found genes may be important to RIFA overwintering and adaptation potential in new environments as well as quarantine area.