Characterization and Expression Patterns of Heat Shock Protein 70 Genes from Paracoccus marginatus in Response to Temperature and Insecticide Stress

Abstract

The objective of this study was to identify the Hsp70s in Paracoccus marginatus and explore their roles in P. marginatus’s resistance to temperature and insecticide stress. The full-length cDNA sequences of PmHsp70s were obtained by PCR cloning and sequencing. The physicochemical and structural characteristics of PmHsp70s were analyzed, and a phylogenetic tree was constructed. The gene expressions of PmHsp70s were detected using qRT-PCR to explore the impacts of temperature and insecticide stress on P. marginatus. A total of 12 PmHsp70s were identified and cloned. The amino acids encoded by PmHsp70s were found to contain highly conserved regions characteristic of the Hsp70 family. The subcellular localization results showed that the majority of PmHsp70s were located in the cytoplasm. A total of 13 unique conserved motifs were identified for the PmHsp70s, of which 9 were shared motifs. The phylogenetic tree showed that the 12 PmHsp70s could be clustered into five branches, with the closest evolutionary relationship observed with the Phenacoccus solenopsis. The expression of the majority of PmHsp70s was up-regulated in P. marginatus when subjected to heat stress, with the higher expression fold change observed for PmHsp70-9, PmHsp70-11, and PmHsp70-12. The expression of specific PmHsp70s was notably suppressed under cold stress, whereas the expression of others was markedly enhanced. Upon exposure to chlorfenapyr and lambda-cyhalothrin, the expressions of PmHsp70-11 and PmHsp70-12 were significantly up-regulated with the highest expression fold change, respectively. The results revealed the significance of specific PmHsp70s in the resistance of P. marginatus to temperature and insecticide stress. This study improved our understanding of the mechanisms underlying P. marginatus’s adaptive responses to unfavorable environmental conditions.

1. Introduction

The challenge of global climate change represents one of the most significant issues currently facing humanity. The resulting increases in average temperatures and the frequency of extreme heat events have important implications for the survival and distribution of organisms [1]. The global surface temperature increased by 1.1 °C between 2011 and 2020 in comparison with temperatures recorded between 1850 and 1900. Furthermore, the global atmospheric temperature increase is projected to exceed 1.5 °C by the end of this century [2]. Insects are ectothermic organisms whose growth, development, and life activities are affected by environmental temperatures [3,4]. Throughout evolutionary history, insects have evolved a range of adaptations to cope with environmental change, including behavioral, physiological, and molecular responses [5,6]. Of these, the heat shock protein is the most extensively studied molecular response mechanism in insects [6].

Heat shock protein in insects typically performs their functions when organisms are exposed to adverse environments, including extreme temperatures [7,8,9,10,11], UV radiation [12], and insecticide stress [13,14,15]. In response to stress, heat shock proteins act as molecular chaperones, facilitating the appropriate folding of other proteins, maintaining cellular and protein homeostasis, and reducing cellular damage caused by adverse environmental stresses, thereby protecting the organism [16]. Based on the homology and molecular weight, HSPs can be classified into the following categories: small heat shock proteins (sHSPs), Hsp40, Hsp60, Hsp70, Hsp90, and Hsp110 [17,18]. Among them, Hsp70 is one of the most highly conserved protein families in response to adverse environments [19]. The Hsp70 family comprises two distinct proteins: heat shock cognate protein 70 (Hsc70) and heat shock protein 70 (Hsp70). Hsc70 is constitutively expressed under normal conditions and has no notable response to external stressors; Hsp70 is expressed at low levels in organisms under normal conditions but is induced to express at significantly higher levels under external stress [20]. The induced expression of Hsp70 plays a critical role in enabling insects to cope with external temperature stress. For example, the expression of MaltHsp70s was markedly elevated in response to high-temperature stress in Monochamus alternatus, reaching a peak at 45 °C [21]. The majority of Hsp70 genes in Bemisia tabaci have been demonstrated to be induced by both high and low temperatures. Moreover, the silencing of BtHsp70-6 expression has been shown to result in a significant reduction in the survival rate of B. tabaci under high-temperature stress [22]. The inhibition of gene expression for Hsp70-01 and Hsp68 resulted in a reduction in the thermotolerance of adult Hippodamia variegata [23]. Furthermore, Hsp70 plays an important role in the insects’ response to insecticide stress [15,24], although the specific response varies across species. The expression of CpHsp70-2 was significantly up-regulated in Cydia pomonella when subjected to stress induced by acetamiprid, methomyl, carbaryl, and cypermethrin. However, imidacloprid and deltamethrin were found to have a less pronounced effect on its expression [25]. RNAi-mediated inhibition of the LbHsp70.4 and LbHsp70.5 gene expression in Liposcelis bostrychophila resulted in increased sensitivity to both beta-cypermethrin and malathion [14].

The papaya mealybug, Paracoccus marginatus (Williams and Granara de Willink), is a significant and harmful mealybug species native to Central America that has invaded several countries [26,27]. There is a tendency toward further spread [28]. P. marginatus is a polyphagous mealybug with a wide range of host plants [29], including Carica papaya L. (papaya), Solanum tuberosum L. (potato), Manihot esculenta (cassava), Ipomoea batatas (L.) Lam. (sweet potato), and Solanum melongena L. (eggplant). The mealybug infestation caused extensive damage to the plants, with the stems, leaves, and fruits being subjected to intense sucking. This caused the leaves to curl, yellow, and drop, leading to a reduction in crop quality and yield [30]. At present, chemical control is the primary method employed for the management of this mealybug. In the previous study, lambda-cyhalothrin, chlorfenapyr, beta-cypermethrin, pyridaben, and tolfenpyrad were found to be highly effective in the control of P. marginatus [31].

Papaya mealybugs are predominantly distributed in tropical and subtropical regions [27]. This mealybug is capable of establishing populations within a temperature range of 20 to 30 °C, with the highest net fecundity observed at 30 °C [32]. The upper temperature limit for the hatching of papaya mealybug eggs is 37 °C, while the optimal developmental temperature for adult females is 28.4 °C, with a maximum developmental temperature of 32.1 °C [33]. The papaya mealybugs have been observed to exhibit an increase in protective enzyme activity in response to fluctuations in temperature, both high and low [34]. However, the influence of Hsp70 on temperature and insecticide stress remains unclear in the context of climate change and extensive insecticide utilization.

In the present study, the Hsp70 genes in papaya mealybugs were identified in the preliminary transcriptome data. The full-length cDNA sequences of the Hsp70 genes were cloned and subsequently analyzed using bioinformatics. The expression pattern of PmHsp70s in papaya mealybugs under temperature and insecticide stress was also investigated. It was hypothesized that the expression levels of PmHsp70 genes in papaya mealybugs would be significantly regulated in response to temperature and insecticide stress. This study will provide a theoretical basis for the development of the molecular mechanism of papaya mealybugs in response to extreme temperature and insecticide stress.

2. Materials and Methods

2.1. Insect Rearing

The papaya mealybugs were collected from papaya trees at Fujian Agriculture and Forestry University, Fuzhou City, Fujian Province, China, in 2018. Subsequently, the specimens were maintained on potato sprouts in a climate incubator at the Institute of Plant Protection, Fujian Provincial Academy of Agricultural Sciences, Fuzhou City, Fujian Province, China. The climate incubator was maintained at a temperature of 26 ± 1 °C, a relative humidity of 60%, and a photoperiod of 12L:12D.

2.2. Identification of PmHsp70s

The papaya mealybugs transcriptome data obtained by sequencing were annotated according to the following publicly accessible functional databases: National Center for Biotechnology Information (NCBI), Gene Ontology (GO), Cluster of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Pfam, and Swiss-Prot. The annotated information was used to identify and localize the candidate PmHsp70s of the papaya mealybugs. Subsequently, multiple sequence alignments were conducted to remove duplicate transcripts from the sequencing and splicing processes. To further confirm the integrity of the conserved structural domains of the candidate PmHsp70s, the NCBI-CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 18 June 2024)), SMART database (http://smart.embl.de/ (accessed on 18 June 2024)), and ScanProsite online software (https://prosite.expasy.org/scanprosite/ (accessed on 18 June 2024)) were utilized. Consequently, the reference sequences of candidate PmHsp70s were obtained.

2.3. Gene Cloning

Total RNA was extracted from papaya mealybugs using Eastep^® Super Total RNA Extraction kit (LS1040, Promega, Madison, WI, USA) according to the manufacturer’s protocol. The quantity and quality of the extracted total RNA were determined using a NanoDrop Spectrophotometer (ND2000, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 2% agarose gel electrophoresis, respectively.

The cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (R312-02, Novizen, Nanjing, China) according to the following procedure: (A) The reaction mixture A contained 1000 ng of total RNA, with nuclease-free water added to make up a total volume of 8 μL. The reaction mixture A was incubated at 65 °C for 5 min and then placed on ice for 2 min. (B) The reaction mixture A was added to 2 µL of 5× g DNA wiper mix and incubated at 42 °C for 2 min. (C) A volume of 2 µL of enzyme mix and 1 µL of oligo dT was added to the reaction mixture A, followed by incubation at 25 °C for 5 min, 37 °C for 45 min, and 85 °C for 5 s. The cDNA was stored at −20 °C.

The cDNA was employed as a template for the amplification of the PmHsp70s in papaya mealybugs, utilizing the 2× Phanta Max Master Mix (P525-02, Vazyme, Nanjing, China). The primers specific to the target gene for polymerase chain reaction (PCR) were designed using Oligo 7 (Table S1). A 25 μL total reaction mixture was employed for the PCR, comprising 2.0 μL of cDNA, 12.5 μL of 2× reaction buffer, 0.5 μL of dNTP mix, 1.0 μL of each forward and reverse primer, 0.5 μL of DNA polymerase, and 7.5 μL of nuclease-free water. The procedure for PCR was as follows: (A) 95 °C for 5 min; (B) 95 °C for 15 s; the annealing temperature for each PmHsp70 is listed in Table S1 for 15 s; and 72 °C for 2 min, with 35 cycles in step B; and (C) finally, 72 °C for 5 min.

Subsequently, the PCR products were purified using a gel extraction kit (D2500-02, Omega BioTek, Norcross, GA, USA) following the manufacturer’s instructions. Then, the purified products were sent to BioSune Biotechnology Co., Ltd. (Shanghai, China) for sequencing.

2.4. Sequence Analysis and Phylogenetic Tree Construction

A physical and chemical property analysis was conducted on PmHsp70s. The protein sequence of PmHsp70s was obtained using ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/ (accessed on 20 June 2024)). The molecular weight, isoelectric point, instability index, aliphatic index, and grand average of hydropathicity of PmHsp70s were predicted using ProtParam (https://web.expasy.org/protparam/ (accessed on 20 June 2024)). Subcellular localization of PmHsp70s was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/ (accessed on 20 June 2024)).

To evaluate the structural diversity of PmHsp70 proteins, the distribution of conserved motifs was detected using the online program Multiple Expectation Maximization for Motif Elicitation (https://meme-suite.org/meme/index.html (accessed on 20 June 2024)) [35]. The following parameters were set: the motif number threshold was set to 20, the motif width was set to 30 to 70 residues, and the remaining parameters were set to the default values. The evaluation results were visualized using TBtools v 2.096 software [36].

To facilitate a comparison of the relationships of PmHsp70s in the papaya mealybugs with those identified in other insects, a phylogenetic tree was constructed. The amino acid sequence of Hsp70 in the following species was obtained from the NCBI database: Drosophila melanogaster, Bemisia tabaci, Phenacoccus solenopsis, Nilaparvata lugens, Laodelphax striatellus, and Sogatella furcifera. The phylogenetic tree was constructed using MEGA 11.0 software by the neighbor-joining method with 1000 bootstrap replications [37].

2.5. Expression Patterns of PmHsp70s Under Stress

2.5.1. Temperature and Insecticide Treatments

The impacts of high and low temperatures on the gene expression of PmHsp70s were examined. The papaya mealybugs were placed into a Petri dish (90 mm diameter) with several holes to allow for air circulation. The Petri dishes containing the papaya mealybugs were subjected to a series of temperature treatments. These treatments were defined based on the findings of preliminary experiments conducted to determine the survival rate of papaya mealybugs under high-temperature conditions. The lethal temperature limit of the papaya mealybugs was 48 °C (unpublished data), and thus, the temperature treatments were selected to be below this threshold. Furthermore, the potential for temperature stress to affect the papaya mealybugs in their natural habitat was also taken into consideration. The following temperature treatments were conducted: (A) blank control CK, 26 °C; (B) high temperature H1, 38 °C; (C) high temperature H2, 42 °C; (D) high temperature H3, 46 °C; and (E) low temperature L1, 4 °C. High- and low-temperature treatments were conducted in incubators and freezers, respectively. Each temperature was treated separately for 30 min, with three replicates of 10 papaya mealybugs performed for each treatment. Following the treatments, the samples were frozen in liquid nitrogen immediately and stored in a −80 °C refrigerator for subsequent RNA extraction.

The impact of insecticide stress on the gene expression of PmHsp70s was examined. The highly effective insecticides against papaya mealybugs, chlorfenapyr and lambda-cyhalothrin, which were identified in the previous study, were utilized [31]. The concentrations employed were the LC₅₀ (lethal concentration for 50% of the population) for female adult papaya mealybugs, which were 24.83 mg/L and 0.7821 mg/L [31]. The leaf dip method was utilized for the treatment of the papaya mealybugs. Once the mealybugs had fully colonized the leaves, they were dipped into the insecticide for 15 s. Following air drying, the potato leaves with papaya mealybugs were transferred to a clean Petri dish, which was placed in the climatic chamber. Three replicates of 10 papaya mealybugs per treatment were conducted. Samples were collected after 2 h of treatment, frozen in liquid nitrogen, and stored in a −80 °C refrigerator for subsequent RNA extraction.

2.5.2. cDNA Synthesis and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

The primers for the PmHsp70s were designed for use in qRT-PCR using the Oligo 7 software (Table S2). The reference gene employed was Pmβ-actin in papaya mealybugs [38]. The total RNA was extracted from the samples that had been treated with the temperature and insecticide stress. cDNA templates for qRT-PCR were synthesized using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (R323-01, Vazyme, Nanjing, China), as follows: (A) The reaction mixture comprised 1000 ng of total RNA, 4.0 μL of 4× gDNA Wiper Mix, and RNase-free water to make a total volume of 16 μL. The mixture was incubated at 42 °C for 2 min. (B) The reaction mixture A was added to 4 μL of 5× HiScript III qRT SuperMix and then incubated at 37 °C for 15 min and 85 °C for 5 s.

A total reaction mixture of 20 μL was used for qRT-PCR. The reaction mixture consisted of 10 μL of 2× ChamQ SYBR qPCR Master Mix (Q311-03, Vazymes, Nanjing, China), 0.4 μL of forward and reverse primers each, 7.2 μL of nuclease-free water, and 2.0 μL of cDNA template. The reaction was conducted using a QuantStudioTM 6 Pro Flex real-time PCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The procedure for the qRT-PCR reaction was as follows: The reaction was heated to 95 °C for 30 s, then to 95 °C for 10 s and 60 °C for 30 s, for a total of 40 cycles. It was then heated to 95 °C for 15 s, 60 °C for 1 min, and finally to 95 °C for 15 s. Three biological replicates were conducted, with three technical replicates included for each biological replicate.

2.6. Statistical Analyses

The relative expression of the PmHsp70s in papaya mealybugs under conditions of temperature and insecticide stress was normalized to the expression level of samples in the control group using the 2^−ΔΔCt method [39]. The normality of the data was evaluated using the Shapiro–Wilk test, as implemented in SPSS 25.0 software. When the data satisfied the criteria for normal distribution, the independent t-test was employed for the analysis of variance. Conversely, when the data did not align with the assumptions of normality, the Mann–Whitney test, a non-parametric alternative, was used for the analysis of variance. Values of p < 0.05 and p < 0.01 were used as criteria for determining whether the observed differences were statistically significant or highly significant.

3. Result

3.1. Identification and Gene Cloning of PmHsp70s

A total of 31 Hsp70 genes were identified from the annotation data of the papaya mealybugs transcriptome. Duplicated sequences, sequences with incomplete structural domains, and sequences with discrepancies in molecular weight were excluded from further analysis. Ultimately, a total of 12 PmHsp70s were identified in the papaya mealybugs.

The full-length cDNA sequences of the 12 PmHsp70s of papaya mealybugs were obtained through the process of PCR cloning and subsequent sequencing. The results of the NCBI-CDD database, SMART analysis, and ScanProsite analysis demonstrated that the proteins encoded by the 12 PmHsp70s all contained three unique complete and typical motifs of the Hsp70 family: GIDLGTTYS, IFDLGGGGTFDVSIL, and VGGSTRIPKVQ.

3.2. Sequence Analysis of PmHsp70s

The proteins encoded by PmHsp70s exhibited a range of amino acid lengths, from 625 to 771 amino acids; a range of molecular weights, from 69,022.98 to 86,467.32 kDa; and a range of theoretical isoelectric points, from 5.05 to 7.92. The instability indices of PmHsp70s were predicted to range from 23.74 to 44.12. The proteins encoded by PmHsp70-3, PmHsp70-4, PmHsp70-6, PmHsp70-7, PmHsp70-8, and PmHsp70-10 were predicted to exhibit stability, whereas other PmHsp70s were predicted to display instability. The aliphatic index of PmHsp70s was estimated to fall within the range of 80.83 to 86.28. The grand hydropathicity values of PmHsp70s ranged from −0.496 to −0.386, indicating a predominantly hydrophilic nature of HSP70 proteins. Subcellular localization predictions revealed that most PmHsp70s were located in the cytoplasm, with a small number also present in the mitochondrial matrix, nucleus, and endoplasmic reticulum (

Table 1. Physicochemical properties of the proteins encoded by PmHsp70s of P. marginatus.

Gene	Amino Acids	Molecular Weight (kDa)	Isoelectric Point	Instability Index	Aliphatic Index	Grand Average of Hydropathicity	Subcellular Location
PmHsp70-1	693	75,113.99	5.87	40.16	82.28	−0.395	Mitochondrial matrix
PmHsp70-2	771	86,467.32	7.92	41.28	85.14	−0.451	Nucleus
PmHsp70-3	653	71,683.10	5.33	35.11	80.83	−0.434	Cytoplasm
PmHsp70-4	684	77,139.05	7.10	40.40	83.71	−0.462	Cytoplasm
PmHsp70-5	698	78,290.62	5.05	35.61	85.93	−0.390	Cytoplasm
PmHsp70-6	686	76,761.65	5.93	35.74	86.28	−0.386	Cytoplasm
PmHsp70-7	686	76,685.21	6.29	26.08	83.16	−0.451	Cytoplasm
PmHsp70-8	660	72,820.58	5.31	23.74	84.77	−0.457	Endoplasmic reticulum
PmHsp70-9	625	69,036.93	5.68	40.35	85.68	−0.439	Nucleus
PmHsp70-10	653	71,741.18	5.33	34.73	81.12	−0.431	Cytoplasm
PmHsp70-11	644	71,583.82	5.50	44.12	82.86	−0.496	Cytoplasm
PmHsp70-12	625	69,022.98	5.63	41.12	83.97	−0.443	Nucleus

).

Conserved motifs of PmHsp70s were predicted using the MEME online program. A total of 13 unique conserved motifs were identified. PmHsp70s exhibited a variable number of motifs, ranging from 9 to 12. All PmHsp70s-encoded proteins were found to contain nine common motifs, including motif 1, motif 2, motif 3, motif 4, motif 5, motif 6, motif 7, motif 8, and motif 11. In comparison with the other PmHsp70s, PmHsp70-1 lacked motif 9 and motif 10. Both PmHsp70-3 and PmHsp70-10 exhibited a single specific motif 13, while PmHsp70-2, PmHsp70-4, PmHsp70-5, PmHsp70-6, and PmHsp70-7 all displayed a single specific motif 12 (Figure 1).

3.3. Phylogenetic Analysis of PmHsp70s

The phylogenetic tree showed that PmHsp70-2, PmHsp70-4, PmHsp70-5, PmHsp70-6, and PmHsp70-7 were clustered in a sub-branch (A-branch) and showed high homology with sequence similarity ranging from 0.506 to 0.719. The B-branch (PmHsp70-9, PmHsp70-11, and PmHsp70-12), C-branch (PmHsp70-3 and PmHsp70-10), D-branch (PmHsp70-1), and E-branch (PmHsp70-8) all showed the highest homology with the Hsp70s in P. solenopsis. In terms of evolutionary distance, branches A, B, and C formed a cluster with branches B and C being relatively closely related and branches D and E forming another cluster (Figure 2).

3.4. Expression Analysis of PmHsp70s Under Temperature Stress

To verify the effects of extreme temperature on papaya mealybugs, qRT-PCR was performed to profile gene expression of PmHsp70s under high- and low-temperature conditions. The expression of PmHsp70 genes in papaya mealybugs was observed to increase to varying degrees following exposure to elevated temperatures (38~46 °C) (Figure 3). The majority of PmHsp70 genes exhibited relatively modest up-regulated expression folds, ranging from 1.12 to 3.14 times that of the control group. The expression levels of PmHsp70-9, PmHsp70-11, and PmHsp70-12 were observed to be elevated, with the greatest expression of those genes occurring at 42 °C, exhibiting fold changes of 144.70 ± 7.19, 79.72 ± 1.49, and 221.43 ± 2.55, respectively. The expression of PmHsp70-12 was markedly increased with elevated temperature, exhibiting an initial increase followed by a decline (Figure 3). The fold change in the expression of PmHsp70-12 was observed to be 100.0 ± 1.15, 221.43 ± 2.55, and 187.59 ± 2.51 times higher than that of the control group at 38 °C, 42 °C, and 48 °C, respectively. The expression of specific PmHsp70 genes did not exhibit a statistically significant difference from that of the control group. No statistically significant difference was observed in the expression of PmHsp70-1 when compared with the control group at either 38 °C or 46 °C (38 °C: t = −0.749, df = 4, p = 0.495; 46 °C: t = −0.013, df = 4, p = 0.990). Similarly, the expression of PmHsp70-5 was not found to be significantly different from that of the control group at both 38 °C and 42 °C (38 °C: t = −0.081, df = 4, p = 0.939; 42 °C: t = −2.398, df = 4, p = 0.074).

The expression of each PmHsp70 demonstrated unique modifications in response to low-temperature stress. In comparison with the control group, no significant difference was observed in the expression levels of PmHsp70-1 and PmHsp70-6 at 4 °C (PmHsp70-1: t = −1.201, df = 4, p = 0.296; PmHsp70-6: t = 2.613, df = 4, p = 0.059). Furthermore, the expression of PmHsp70-2, PmHsp70-5, and PmHsp70-7 was markedly down-regulated at 4 °C (PmHsp70-2: t = 8.806, df = 4, p = 0.001; PmHsp70-5: t = 11.937, df = 4, p < 0.001; PmHsp70-7: t = 3.044, df = 4, p = 0.038). In contrast, the remaining PmHsp70 genes exhibited a notable up-regulation in expression, with PmHsp70-12 displaying the most significant increase, at a fold change of 3.41 ± 0.14 times relative to the control (Figure 3).

3.5. Expression Analysis of PmHsp70s Under Insecticide Stress

The effect of insecticides on the expression of PmHsp70s in papaya mealybugs was investigated. Some PmHsp70s exhibited a degree of expression change in response to the insecticides. The numbers of PmHsp70s exhibiting altered expression following treatment with chlorfenapyr and lambda-cyhalothrin were eight and six, respectively. The expression levels of PmHsp70-1, PmHsp70-5, and PmHsp70-10 were found to be significantly down-regulated after treatment with chlorfenapyr and lambda-cyhalothrin, in comparison with the control group. In contrast, the expression level of PmHsp70-8, PmHsp70-11, and PmHsp70-12 was significantly up-regulated in comparison with the control group. Following treatment with chlorfenapyr and lambda-cyhalothrin, the gene exhibiting the greatest fold increase in expression was detected in PmHsp70-12 and PmHsp70-11, which displayed 2.64 ± 0.41-fold and 2.30 ± 0.18-fold increases, respectively, in comparison with the control group (Figure 4).

4. Discussion

The papaya mealybug is a significant invasive alien insect, primarily distributed in tropical and subtropical regions. Previous studies have primarily concentrated on the effects of elevated temperature and insecticides on the biological traits [33,40], protective enzyme activity [34], and detoxification enzyme activities [40] of papaya mealybugs, and little information is currently available on the molecular mechanisms underlying the adaptation of the papaya mealybug to temperature and insecticide stress. Heat shock proteins play a pivotal role in insects’ response to external stresses, including extreme temperatures, ultraviolet light, pharmaceutical stress, and starvation stress [41]. In this study, we investigated the potential roles of Hsp70s in papaya mealybugs in response to temperature and insecticide stress. In total, 12 PmHsp70 genes of P. marginatus were identified and cloned. All of the genes exhibited the three distinctive sequences characteristic of the Hsp70 family, thereby confirming their classification as members of the Hsp70 family. The phylogenetic analysis revealed that the Hsp70s of diverse insect species exhibited a high degree of conservation. The PmHsp70s of P. marginatus exhibited a high degree of homology with those of P. solenopsis, which also belongs to the family Pseudococcidae. N. lugens, L. striatellus, and S. furcifera, which belong to the family Hemiptera, are more closely related to each other than B. tabaci, which belongs to the family Aleyrodidae. This suggests that the Hsp70s have experienced a significant degree of evolutionary conservation.

The frequency of extreme temperatures has increased as a consequence of climate change [2]. The impact of extreme temperatures on insects represents a significant challenge. Insects have evolved a series of strategies to cope with adverse environmental conditions [6]. Hsp70 plays a critical role in the insect’s response to extreme temperatures. Several studies have demonstrated that the expression of Hsp70 undergoes rapid changes in insects subjected to heat and cold stress, thereby reducing the damage to the organism [22]. Four Hsp70s in Aphis gossypii were observed to exhibit significant upregulation following exposure to high temperatures. Silencing of two Hsp70s was found to significantly enhance the sensitivity of A. gossypii to high temperatures [42]. In our study, the expression of PmHsp70s in P. marginatus was observed to be affected by high temperatures. Following exposure to elevated temperatures, the response of PmHsp70-9, PmHsp70-11, and PmHsp70-12 was markedly more pronounced, while the other genes displayed reduced sensitivity. In particular, the relative expression level of PmHsp70-12 at 42 °C was observed to be 221.43 ± 2.55 times higher than that of the control group. The expression of these three genes demonstrated a tendency to increase and subsequently decrease, reaching a peak at 42 °C. This is consistent with the expression response pattern to high temperatures observed in other insects. For example, TpHsp70-1 expression in Tyrophagus putrescentiae was highest at 39 °C and decreased at 42 °C [43]. This suggests that heat shock proteins can only provide a certain degree of protection to the organism [44].

Heat shock proteins can be induced in response to cold shock [45]. Hsp70 plays an important role in cold hardening in both Bactrocera dorsalis and Bactrocera correcta. A reduction in the expression of this gene resulted in a notable decline in survival rates of B. dorsalis and B. correcta under cold conditions [11]. The present study also revealed that low temperature induced a change in the expression of PmHsp70s. Compared with the consistent expression pattern of P. marginatus under high-temperature stress, some PmHsp70s were up-regulated and some genes were down-regulated in papaya mealybugs when exposed to low-temperature stress. The genes with the highest degree of up-regulation were both PmHsp70-11 and PmHsp70-12, when papaya mealybugs were exposed to high- and low-temperature stress. Nevertheless, the alteration in gene expression levels in response to low temperature (3.41 ± 0.14 times) was less pronounced than that observed in response to high temperature (221.43 ± 2.55 times). Similarly, the AaHsp70s in Aphis aurantii were much less responsive to low temperatures in comparison with high temperatures [7]. The findings in our study indicate the potential involvement of PmHsp70-11 and PmHsp70-12 in the response to extreme temperatures, including high and low temperature. The PmHsp70s in P. marginatus demonstrate greater sensitivity to high temperatures than to low temperatures.

The utilization of chemical insecticides represents a significant stressor for insects in their natural habitat. Our recent studies showed a change in the expression levels of P450, GST, carboxylesterase, and esterase genes in P. marginatus subjected to sublethal lambda-cyhalothrin exposure [40]. The detoxification metabolism plays a pivotal role in insect responses to insecticides. Furthermore, evidence indicates that the Hsp70 gene is induced in response to insecticide stress and may contribute to insecticide resistance [46], although the responses vary among insect species. The expression of Hsp70-1 in Bombyx mori was significantly increased in response to stress induced by dichlorvos and phoxim [47]. The gene expression of MpHsp70 was found to be significantly elevated in Myzus persicae when exposed to lambda-cyhalothrin stress [13]. RNAi-mediated knockdown of the AgHsp70 gene increased the susceptibility of Aphis gossypii to 2-tridecanone, gossypol, and flupyradifurone [48]. In this study, exposure of papaya mealybugs to chlorfenapyr and lambda-cyhalothrin resulted in the up-regulation of gene expression for three PmHsp70s and the down-regulation of the other three PmHsp70s. The genes that exhibited a significant increase in expression included PmHsp70-11 and PmHsp70-12, which were also found to be up-regulated in papaya mealybugs in response to high and low temperatures. These findings support the hypothesis that temperature and insecticide stress induce the PmHsp70s’ response, which may be a key factor in the adaptive response of P. marginatus to unfavorable environmental conditions. It was further hypothesized that PmHsp70-11 and PmHsp70-12 play a pivotal role in the response of P. marginatus to external stresses, including temperature and insecticide stresses. Further investigation into the roles of PmHsp70-11 and PmHsp70-12 in P. marginatus in response to temperature and insecticide stress can be conducted using RNAi or gene-editing techniques.

5. Conclusions

In this study, the potential mechanisms of Hsp70s in P. marginatus in response to temperature stress and insecticide exposure were investigated. The results revealed the identification of 12 PmHsp70 genes in P. marginatus and the expression change of certain PmHsp70s by temperature and insecticide treatments. Our findings indicate the importance of PmHsp70-11 and PmHsp70-12 in papaya mealybugs’ resistance to temperature and insecticide stress. The results of this study could provide new insights into the molecular basis of insect defense mechanisms and suggest potential target genes for papaya mealybug control using RNAi. Further functional analysis of Hsp70s and other heat shock proteins (such as Hsp60s and Hsp90s) may facilitate a better understanding of the mechanisms involved in extreme temperature and insecticide tolerance in P. marginatus.