Regulatory Mechanism of Transcription Factor AhHsf Modulates AhHsp70 Transcriptional Expression Enhancing Heat Tolerance in Agasicles hygrophila (Coleoptera: Chrysomelidae)

Agasicles hygrophila is a classical biological agent used to control alligator weed (Alternanthera philoxeroides). Previous research has indicated that the heat shock factor (HSF) is involved in regulating the transcriptional expression of Hsp70 in response to heat resistance in A. hygrophila. However, the regulatory mechanism by which AhHsf regulates the expression of AhHsp70 remains largely unknown. Here, we identified and cloned a 944 bp AhHsp70 promoter (AhHsp70p) region from A. hygrophila. Subsequent bioinformatics analysis revealed that the AhHsp70p sequence contains multiple functional elements and has a common TATA box approximately 30 bp upstream of the transcription start site, with transcription commencing at a purine base approximately 137 bp upstream of ATG. Promoter deletion analyses revealed that the sequence from −944 to −744 bp was the core regulatory region. A dual-luciferase reporter assay indicated that overexpressed AhHsf significantly enhanced the activity of AhHsp70p. Furthermore, qPCR showed that AhHsp70 expression increased with time in Spodoptera frugiperda (Sf9) cells, and AhHsf overexpression significantly upregulated AhHsp70 expression in vitro. Characterization of the upstream regulatory mechanisms demonstrated that AhHsf binds to upstream cis-acting elements in the promoter region of AhHsp70 from −944 to −744 bp to activate the AhHSF–AhHSP pathway at the transcriptional level to protect A. hygrophila from high temperature damage. Furthermore, we proposed a molecular model of AhHsf modulation of AhHsp70 transcription following heat shock in A. hygrophila. The findings of this study suggest that enhancing the heat tolerance of A. hygrophila by modulating the upstream pathways of the Hsp family can improve the biocontrol of A. philoxeroides.


Introduction
Insects are poikilothermal organisms that adapt to different environmental temperatures through a variety of physiological and biochemical responses, and this ability directly influences their population expansion and distribution [1,2]. Moreover, climatic adaptability can drive insect adaptive evolution, promoting population differentiation and even the formation of new species [3][4][5].
Organisms respond to the chemical and physiological stresses associated with elevated temperatures by increasing the synthesis of heat shock proteins (Hsps) [6][7][8][9][10], which protect cells from hyperthermic stress by binding to denatured proteins and facilitating their correct refolding [8,11,12]. Under normal conditions, the expression of Hsp is typically maintained at very low levels, although expression levels can be rapidly upregulated in response to stress conditions such as heat shock [13,14]. In terms of insect thermotolerance, Hsp70, a member of the highly conserved chaperone class of proteins, is a typical representative of the heat shock gene family [15,16]. The Hsp70 promoter has beenis used extensively in transgenic and gene therapy studies to drive the expression of exogenous genes [14,17], and numerous in vitro studies have sought to elevate its transcriptional activity for selected transgenes [18]. In this regard, the regulation of transcriptional gene expression via DNA elements such as promoters and enhancers playsan important role in controlling the expression of genes associated with stress resistance [19][20][21][22].
The heat shock response represents a typical case of inducible gene expression [15,23] that involves transcriptional activation mediated by the heat shock factor (HSF), which binds to specific elements in the heat shock gene [24][25][26][27]. Among the stress-related proteins, members of the most abundant and conserved Hsp70 family have frequently been proposed as potential biomarkers of cellular toxicity [28]. Li et al. found that AtHsf regulates the expression of stress-responsive genes (Hsps) to enhance tolerance to heat and other abiotic stresses in Arabidopsis by functional analysis of AtHsf knockout mutants and AtHsf overexpressing transgenic plants [25]. In Caenorhabditis elegans, Baird et al. found that HSF-1 modulates the calcium-binding protein PAT-10 to increase thermotolerance and longevity during thermal stress [23]. In addition, it has been reported that post-translational modifications of Hsp70 family proteins including phosphorylation, acetylation, ubiquitination, aminoylation, and ADP ribosylation play an important role in regulating Hsp70 activity [29].
The flea beetle Agasicles hygrophila Selman & Vogt, which is used as an important biological control agent for alligator weed (Alternanthera philoxeroides [Amaranthaceae]) is markedly influenced by temperature [30][31][32]. Previous field surveys in Hunan Province, China have revealed that the population density of A. hygrophila decreases sharply during mid-summer from July to September, thereby limiting the control of A. philoxeroides population growth at this time of the year [33,34]. The correlation between high temperatures (>36 • C and even above 39 • C) and the population decline of this beetle species indicates that extremely high temperatures may be the primary factor suppressing the control of alligator weed during mid-summer [35][36][37]. In addition, temperature data for Changsha City collected from 2003 to 2013 indicated that in July and August, the frequencies of extremely high temperatures exceeding 36 • C were 42.5% and 32.1%, respectively, and the daily maximum temperatures recorded were often above 39 • C [37]. (Supplementary  Table S1; [China Meteorological Data, http://data.cma.gov.cn/, accessed on 6 August 2020]). Alternanthera philoxeroides (Mart.) Griseb., an aquatic amaranth native to South America [38], was introduced into China in the 1930s as a forage crop [39], and has spread throughout the southern regions of the country, becoming one of the most noxious weeds in China [39,40]. This alien species has been recognized as a serious aquatic pest problem threatening aquatic ecosystems worldwide and is currently listed as one of the 16 most serious invasive species in China [31,32,41].
In our efforts to elucidate the molecular mechanisms underlying the heat resistance of A. hygrophila, we previously demonstrated the importance of the AhHsp70 in the thermotolerance of this beetle [42] and subsequently isolated and identified a heat shock factor (AhHsf ) and its putative downstream target gene, AhHsp70. Our findings provide evidence that AhHsf is involved in regulating the transcriptional expression of AhHsp70 in response to the thermotolerance of A. hygrophila [43]. However, the mechanism by which AhHsf regulates the expression of AhHsp70 remains poorly understood.
In the present study, we sought to verify our hypothesis that AhHsp70 plays a proactive role in A. hygrophila stress resistance by interacting with AhHsf. To this end, we determined the sequence of the AhHsp70 promoter (AhHsp70p) using the inverse PCR (I-PCR) technique of chromosome walking, and performed subsequent bioinformatics analysis. In addition, we characterized the AhHsp70 core promoter region by promoter deletion analysis. Finally, using a dual-luciferase reporter (DLR) assay system, we determined the interaction between AhHsf and AhHsp70p and established that AhHsf directly targets the AhHsp70 core promoter region to activate the AhHSF-AhHSP70 signaling pathway by regulating the transcriptional expression of AhHsp70. These findings provide important insights into the regulatory mechanisms associated with the response of A. hygrophila to high external temperatures, and will potentially contribute to predicting the efficacy of biocontrol using this beetle in the face of ongoing climate change.

Analysis of the AhHsp70p Sequence in A. hygrophila
In this study, we isolated and identified a 944-bp upstream promoter sequence of the AhHsp70 gene (AhHsp70p) from A. hygrophila using I-PCR which was deposited in the NCBI database (GenBank accession number: MZ351037) ( Figure 1). Bioinformatics analysis revealed that the AhHsp70p sequence contains multiple functional elements including a common TATA box, which is a DNA sequence recognized by transcription factors, located approximately 30 bp upstream of the transcription start site. Berkeley Drosophila Genome Project: Neural network promoter prediction indicated that AhHsp70p transcription commences from a purine base approximately 137 bp upstream of the ATG coding region ( Figure 1). Furthermore, JASPAR and Tfsitescan predictions indicated that AhHsp70p has transcription factor binding sites at approximately −850 bp (Figure 1), whereas MethPrimer analysis revealed that AhHsp70p does not contain CpG islands (Supplementary Figure S1).
active role in A. hygrophila stress resistance by interacting with AhHsf. To this end, we determined the sequence of the AhHsp70 promoter (AhHsp70p) using the inverse PCR (I-PCR) technique of chromosome walking, and performed subsequent bioinformatics analysis. In addition, we characterized the AhHsp70 core promoter region by promoter deletion analysis. Finally, using a dual-luciferase reporter (DLR) assay system, we determined the interaction between AhHsf and AhHsp70p and established that AhHsf directly targets the AhHsp70 core promoter region to activate the AhHSF-AhHSP70 signaling pathway by regulating the transcriptional expression of AhHsp70. These findings provide important insights into the regulatory mechanisms associated with the response of A. hygrophila to high external temperatures, and will potentially contribute to predicting the efficacy of biocontrol using this beetle in the face of ongoing climate change.

Analysis of the AhHsp70p Sequence in A. hygrophila
In this study, we isolated and identified a 944-bp upstream promoter sequence of the AhHsp70 gene (AhHsp70p) from A. hygrophila using I-PCR which was deposited in the NCBI database (GenBank accession number: MZ351037) ( Figure 1). Bioinformatics analysis revealed that the AhHsp70p sequence contains multiple functional elements including a common TATA box, which is a DNA sequence recognized by transcription factors, located approximately 30 bp upstream of the transcription start site. Berkeley Drosophila Genome Project : Neural network promoter prediction indicated that AhHsp70p transcription commences from a purine base approximately 137 bp upstream of the ATG coding region ( Figure 1). Furthermore, JASPAR and Tfsitescan predictions indicated that AhHsp70p has transcription factor binding sites at approximately −850 bp (Figure 1), whereas MethPrimer analysis revealed that AhHsp70p does not contain CpG islands (Supplementary Figure S1).

Analysis of AhHsp70 Promoter Reporter Plasmid Activity
To determine the optimal co-transfection efficiency of the recombinant plasmid (pGL3basic-AhHsp70p) and internal reference plasmid (PRL-TK), we assessed different ratios of the recombinant plasmid and the internal control vector plasmid used for co-transfection and established that the optimal transfection efficiency was obtained at a ratio of 10:1 (Supplementary Figure S2, F(144) = 48.01, p < 0.0001; F(344) = 425.03, p < 0.0001; F(544) = 44.49, p < 0.0001; F(744) = 124.66, p < 0.0001; F(944) = 133.42, p < 0.0001). Thereafter, we analyzed the activity of the AhHsp70p reporter plasmids in Spodoptera frugiperda (Sf9) cells using thea TransDetect ® DLR assay system. The results indicated that, with the exception of pGL3-basic-AhHsp70p-144, the reporter plasmids showed significantly higher activity than the control plasmid (  In this experiment, Spodoptera frugiperda (Sf9) cells were used for the stable expression of AhHsp70 in vitro. Sf9 cells were transfected with recombinant plasmids containing AhHsp70p sequences of differing deletion length or pGL3-Basic and pRL-TK as controls for 48-96 h and the cells were harvested for the luciferase activity assay. qdz represents the AhHsp70 promoter. All values are shown as the mean ± SD. The data were analyzed using the Student's t-test. ** p < 0.01, extremely significant; *** p < 0.001, extremely extremely significant; ns, not significant.

Characterization of the Interaction between Transcription Factor AhHsf and AhHsp70p
Sf9 cells were co-transfected with an AhHsf overexpression vector (PIZ/V5-His-Hsf) ( Figure 3C and Supplementary Figure S3F) and AhHsp70p expression vector, and a DLR assay was used to determine the influence of AhHsf overexpression on the activity of the target gene promoter AhHsp70p ( Figure 3B). The DLR assay results revealed that compared to cells transfected with the control vector, AhHsf overexpression significantly enhanced the activity of AhHsp70p in vitro( Figure 3A  In this experiment, Spodoptera frugiperda (Sf9) cells were used for the stable expression of AhHsp70 in vitro. Sf9 cells were transfected with recombinant plasmids containing AhHsp70p sequences of differing deletion length or pGL3-Basic and pRL-TK as controls for 48-96 h and the cells were harvested for the luciferase activity assay. qdz represents the AhHsp70 promoter. All values are shown as the mean ± SD. The data were analyzed using the Student's t-test. ** p < 0.01, extremely significant; *** p < 0.001, extremely extremely significant; ns, not significant.

Characterization of the Interaction between Transcription Factor AhHsf and AhHsp70p
Sf9 cells were co-transfected with an AhHsf overexpression vector (PIZ/V5-His-Hsf) ( Figure 3C and Supplementary Figure S3F) and AhHsp70p expression vector, and a DLR assay was used to determine the influence of AhHsf overexpression on the activity of the target gene promoter AhHsp70p ( Figure 3B). The DLR assay results revealed that compared to cells transfected with the control vector, AhHsf overexpression significantly enhanced the activity of AhHsp70p in vitro ( Figure 3A

Expression Levels of AhHsp70 Following In Vitro Transfection
The level of AhHsp70 mRNA expression following in vitro transfection was determined by RT-qPCRThe results indicated that the levels of  All values are shown as the mean ± SD. The data were analyzed using the Student's t-test. ** p < 0.01, extremely significant; ns, not significant.

Expression Levels of AhHsp70 Following In Vitro Transfection
The level of AhHsp70 mRNA expression following in vitro transfection was determined by RT-qPCRThe results indicated that the levels of

Expression Levels of AhHsp70 Following In Vitro Transfection
The level of AhHsp70 mRNA expression following in vitro transfection was determined by RT-qPCRThe results indicated that the levels of   Relative mRNA levels were determined using the 2 −∆∆Ct method and normalized to those of the β-actin. The figure shows data on the relative AhHsp70 gene expression levels analyzed using one-way ANOVA followed by the least significant difference (LSD) test and bars with different letters indicate significant differences (p < 0.05). All values are shown as the mean ± SD of three replicates and pGL3-Basic was used as a control.

Transcription Factor AhHsf Upregulates the Expression of AhHsp70 In Vitro
The expression of AhHsp70 and AhHsf in co-transfected cells was determined using RT-qPCR, and the results indicated that there was a significant increase in AhHsf and AhHsp70 expression compared to the control group ( Figure 5, t (AhHsf ) = −9.50, p = 0.0109; t (AhHsp70) = −7.16, p = 0.0020). These findings indicate that overexpression of AhHsf can significantly promote the expression of AhHsp70 and that this gene plays a regulatory role in the activation of AhHsp70.
The expression of AhHsp70 and AhHsf in co-transfected cells was determined using RT-qPCR, and the results indicated that there was a significant increase in AhHsf and AhHsp70 expression compared to the control group ( Figure 5, t(AhHsf) = −9.50, p = 0.0109; t(AhHsp70) = −7.16, p = 0.0020). These findings indicate that overexpression of AhHsf can significantly promote the expression of AhHsp70 and that this gene plays a regulatory role in the activation of AhHsp70. Figure 5. Effect of AhHsf overexpression on the level of AhHsp70 expression. Relative mRNA levels were determined using the 2 −ΔΔCt method and normalized to those of the β-actin gene. The data were analyzed using the Student's t-test. * p < 0.05, significant; ** p < 0.01, extremely significant; ns, not significant. All values are shown as the mean ± SD of three replicates and pGL3-Basic was used as a control.

A Simple Model for Regulation of Transcriptional Gene Expression Following Heat Shock
Based on the findings of the present study and the Kyoto Encyclopedia of Genes and Genomes pathways, we propose a model illustrating how gene transcription might be regulated following heat shock ( Figure 6). In this model, an increase in environmental temperature initiates a signal transduction pathway that activates the transcription factor AhHsf and upregulates the expression of AhHsf target genes. Subsequently, AhHsf activates the expression of its target AhHsp70 thereby enhancing thermotolerance. Figure 5. Effect of AhHsf overexpression on the level of AhHsp70 expression. Relative mRNA levels were determined using the 2 −∆∆Ct method and normalized to those of the β-actin gene. The data were analyzed using the Student's t-test. * p < 0.05, significant; ** p < 0.01, extremely significant; ns, not significant. All values are shown as the mean ± SD of three replicates and pGL3-Basic was used as a control.

A Simple Model for Regulation of Transcriptional Gene Expression Following Heat Shock
Based on the findings of the present study and the Kyoto Encyclopedia of Genes and Genomes pathways, we propose a model illustrating how gene transcription might be regulated following heat shock ( Figure 6). In this model, an increase in environmental temperature initiates a signal transduction pathway that activates the transcription factor AhHsf and upregulates the expression of AhHsf target genes. Subsequently, AhHsf activates the expression of its target AhHsp70 thereby enhancing thermotolerance.

Discussion
Heat shock factor 1 (Hsf1) plays an essential role in protecting cells from proteindamaging stress associated with protein misfolding [27]. Previous studies have indicated that Hsf's plays a central role in remodeling the chromatin structure of Hsp promoters via constitutive interactions with a high-affinity binding site, the heat shock element (HSE) [44][45][46][47][48]. By binding to a gene promoter, transcription factors are typically assembled to form "transcriptional switches" that are capable of controlling gene expression [49].
In our previous study, based on RT-PCR and RNAi analyses, we found that the tran-

Discussion
Heat shock factor 1 (Hsf1) plays an essential role in protecting cells from proteindamaging stress associated with protein misfolding [27]. Previous studies have indicated that Hsf's plays a central role in remodeling the chromatin structure of Hsp promoters via constitutive interactions with a high-affinity binding site, the heat shock element (HSE) [44][45][46][47][48]. By binding to a gene promoter, transcription factors are typically assembled to form "transcriptional switches" that are capable of controlling gene expression [49].
In our previous study, based on RT-PCR and RNAi analyses, we found that the transcription factor AhHsf regulates the transcriptional expression of AhHsp70 and plays a key role in the thermotolerance of A. hygrophila [42,43]. In the present study, we isolated and determined the sequence of the promoter region of this gene (AhHsp70p) based on I-PCR. Subsequent bioinformatics analysis revealed that the sequence had certain characteristics common to promoter regions, although it appeared to be deficient in CpG islands. In contrast, other studies have found that some promoters contain CpG islands, which are believed to play an important role in the epigenetic regulation of these genes and are generically equipped to influence local chromatin structure and assist in the regulation of gene activity [50][51][52][53]. Moreover, promoters serve as key cis-acting elements that regulate gene expression, and it is generally believed that the cisand trans-regulatory machinery in promoter regions are basic requirements for gene expression [54]. In 2020, Jia et al. identified six AhHsp70s (hsp70-1, hsp70-2, hsp70-3, hsp70-4, hsp70-5, hsp70-6) in A. hygrophila [55]; all six Hsp70s of A. hygrophila had a non-organellar consensus motif RARFEEL [56], and the C-terminal sequences included the EEVD motif for cytoplasmic localization.
Promoter deletion analysis is one of the primary and most widely used techniques employed to determine whether they are cis-acting elements or specific transcription factor binding sites within a promoter that are primarily responsible for the transcriptional regulation of a particular gene [49,57]. Luciferase (firefly and Renilla luciferases) genes have been used extensively as reporter genes because of their sensitivity and efficiency [18,54,58]. For example, Apriana et al. demonstrated the root-specific expression of an alkenal reductase gene (OsAER1) in Oryza sativa, based on the deletion analysis of the OsAER1 gene promoter [59]. Through deletion analysis in transgenic rice, Chen et al. reported that the OsHAK1 promoter (Dp3037 sequence) a potentially suitable candidate for regulating the expression of osmotic/drought stress-responsive transgenes [60]. Similarly, Yao et al. used deletion analysis to identify a 22-bp DNA cis-element in the SPHK1 promoter that plays an essential role in transcriptional activation [57]. In the present study, we used the same analytical approach and our results indicated that cis-acting elements controlling AhHsp70 transcriptional expression in response to heat stress are located within a promoter region between base pairs −944 and −744. However, to facilitate more accurate localization of these cis-element positions, we will need to conduct further deletion analysis that entails progressively finer truncations.
To elucidate the mechanisms by which the heat resistance in A. hygrophila is regulated, we previously identified a heat shock factor (AhHsf ) and its downstream target gene AhHsp70 and established that they play important roles in the thermotolerance of A. hygrophila [43]. In the current study, we further demonstrated that transcription factor (AhHsf ) binding to the AhHsp70p sequence (from −944 bp to −744 bp) activated its transcriptional expression in vitro based on promoter deletion, the DLR system, and RT-qPCR analyses. Collectively, the in vitro results obtained in the present study and in vivo results obtained from previous work [43] provide convincing evidence that AhHsf binds to AhHsp70p to activate the AhHSF-AhHSP signaling pathway, thereby promoting transcriptional expression of AhHsp70, which in turn contributes to the enhancement of heat tolerance in A. hygrophila. These findings are similar to those reported in Drosophila melanogaster and yeast, in which cooperative interactions between Hsf's and its target binding sites (promoter and heat shock elements) regulate the transcriptional expression of heat shock protein genes [26,48,61]. Accordingly, we inferred that AhHsf acts directly on the AhHsp70 gene promoter to induce the transcriptional expression of AhHsp70, thereby enhancing the tolerance of A. hygrophila to high environmental temperatures. Nevertheless, we have established that overexpressed AhHsf can promote AhHsp70 gene expression at the transcriptional level, and further analyses will be needed to confirm whether our observations can be replicated at the protein level.

Experimental Insects and Host Plants
Adult A. hygrophila were collected in July 2018 from an alligator weed covered pond in Changsha (28 • 11 49 N, 112 • 58 42 E), Hunan Province, China, using a sweeping method. These specimens were maintained on alligator weed plants in a laboratory at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, and the Langfang Experimental Station of the Chinese Academy of Agricultural Sciences, Hebei Province, under controlled conditions (temperature, 26 ± 2 • C; relative humidity, 75% ± 5%; photoperiod, 12:12 h light:dark regime) [62]. To eliminate maternal effects, the flea beetles were cultivated for three generations before commencing the experiments. Groups of five females and five males were each placed in circular containers of 8 cm diameter and 12 cm height containing fresh A. philoxeroides stems. The gender of the experimental insects was determined based on the presence of a groove at the end of the abdomen, which is present in males but absent in females [37].
Roots of A. philoxeroides were collected from standing water at the Institute of Plant Protection, Hunan Academy of Agricultural Sciences, and planted in plastic boxes (25 × 25 × 20 cm) containing sterilized soil. The plants were subsequently grown in a greenhouse at the Langfang Experimental Station, with daily watering.

Expression Vector and Cell Lines
In the DLR assay system (Transgen, Beijing, China) thea firefly (Photinus pyralis) luciferase gene vector pGL3-basic (Promega, Madison, WI, USA) was used as a reporter plasmid, with the Renilla (Renilla reniformi) luciferase gene vector pRL-TK (Promega) as an internal control plasmid. The plasmids were kindly donated by Professor Ganqiu Lan of the College of Animal Science and Technology, Guangxi University. One element of the pIZ/V5-His vector (catalog number: V8000-01, Invitrogen, Carlsbad, CA, USA), an insect overexpression vector used for the transcription factor, was purchased from Invitrogen, and the other was kindly donated by Professor Changyou Li of the Laboratory of Biological Control for Insect Pests, Center for Advanced Invertebrate Cell Culture and Cell Engineering, Qingdao Agricultural University. All vector sequences were confirmed by Sangon Biotech Co. Ltd. (Shanghai, China). Sf9, donated by Professor Changyou Li, were cultured in TNM-FH insect medium (Hyclone, Gibco, New York, NY, USA) supplemented with 10% fetal bovine serum (Gibco, New York, NY, USA) in humidified air containing 5% CO 2 at 27 • C in a biochemical incubator (BPC-250F; Yiheng, China). These cells were subcultured at 3-to 5-day intervals and used for transfection experiments when cell densities reached approximately 80-85% confluence (at approximately five days) (Supplementary Figure S4).

Sample Collection and In Vitro Experiments
To determine the AhHsp70p sequence via reverse PCR, each group comprising five pairs of newly emerged (<12 h following eclosion) A. hygrophila adults was placed together in a circular container (12 cm in height and 8 cm in diameter) provisioned with fresh alligator weed leaves. The beetles were exposed daily to a temperature of 33 • C for a 4-h period (10:00 to 14:00) in a constant temperature incubator (RPX-450; Colin, Beijing, China). The sampled individuals were immediately frozen in liquid nitrogen for DNA or RNA extraction, or stored in a −80 • C freezer (DW-86L628; Haier, Tsingtao) until further analyses.
For the DLR assays, vectors with different AhHsp70p promoter lengths (pGL3-basic-AhHsp70p-144, pGL3-basic-AhHsp70p-344, pGL3-basic-AhHsp70p-544, pGL3-basic-AhHsp70p-744, and pGL3-basic-AhHsp70p-944) were used and the vector pIZ/V5-His-AhHsf was used to overexpress the transcription factor AhHsf. After transfection of the dual-luciferase reporter vector into Sf9 cells, the activity of AhHsp70p was analyzed using the DLR system, and the level of AhHsp70 expression was determined using qPCR. After co-transforming Sf9 cells with pIZ/V5-His-AhHsf and the AhHsp70p dual-luciferase reporter gene vector, the AhHsp70p activity was analyzed compared with that of a control group lacking the transcription factor overexpression vector, and the levels of AhHsp70 and AhHsf expression were determined based on RT-qPCR.

DNA or RNA Extraction and Reverse PCR
Agasicles hygrophila genomic DNA was extracted using the phenol-chloroform method and total RNA was extracted using TRIzol (Invitrogen) in accordance with the manufacturer's instructions [63]. The isolated total RNA was either stored at −80 • C for further use or converted to first-strand cDNA, synthesized using a reverse transcription kit (TransScript ® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR [One-Step gDNA Removal], AT341-02, TransGen Biotech, China) for subsequent RT-qPCR. The isolated DNA was used to determine the AhHsp70p sequence by I-PCR (Supplementary Figure S5). To characterize the promoter by PCR, primers were designed using Primer 5.0 (Table 1), and the products were cloned into a pEASY-T3 vector (TransGen, Beijing, China) and then sequenced.

Relative Quantitative Real-Time PCR
The AhHsp70 and AhHsf expression levels were assessed via RT-qPCR using a TransStart Green qPCR SuperMix Kit (AQ141-04-p, Transgen, Beijing, China) and an ABI Prism 7500 Real Time PCR System (Applied Biosystems, New York, NY, USA). All PCR reactions were performed in triplicate using the primers listed in Table 1. Reactions were performed as 20 µL reaction mixtures comprising 10 µL of 2× TransStart R Tip Green qPCR SuperMix, 0.4 µL of Passive Reference Dye II, 0.4 µL of forward and reverse specific primers, 1 µL of cDNA template, and 7.8 µL of ddH 2 O. β-actin was used as an internal reference standard and relative expression levels were determined using the 2 −∆∆Ct method with the following formula: ∆∆ Ct = (Cp target − Cp reference ) treatment − (Cp target − Cp reference ) control

Synthesis of AhHsp70p Insertion Fragments
To generate an AhHsp70p target luciferase reporter, we designed five deletion AhHsp70p specific primers (Table 1) using the CE V1.04 software. A KpnI restriction site sequence with approximately 15-25 bp homologous to the vector region was added to the 5 end of the forward primer and an XhoI restriction site with approximately 15-25 bp homologous to the vector was added to the 5 end of the reverse primer. The target fragment was obtained by PCR amplification using genomic DNA as a template. PCR amplification reactions were performed using a total reaction volume of 25 µL comprising 2.5 µL of 10× PCR buffer (10 µM), 0.5 µL of dNTPs (2.5 mM), 0.5 µL of Taq DNA polymerase (TransGen Biotech, China), 1 µL of each gene-specific primer pair, 0.5 µL of genomic DNA template, and 19 µL ddH 2 O. The PCR products were purified using an AxyPrep TM DNA Gel Extraction Kit (Axygen) and cloned into a pEASY-T3 vector (TransGen, Beijing, China). The veracity of the plasmids was confirmed using commercial sequencing (Sangon Biotech, Shanghai, China).

Construction of Luciferase Reporter Plasmids and AhHsp70p Luciferase Activity Assays
The target plasmids described in Section 4.5 were digested using KpnI and XhoI, and the resulting PCR products were subcloned into the KpnI and XhoI sites of the luciferase reporter vector pGL3-basic using a homologous one-step cloning kit (Trelief TM SoSoo Cloning Kit, TSV-S2; TsingKe Biotech, Shangai, China) according to the manufacturer's instructions, with pRL-TK being used as a control vector. Digestion reactions were performed using a total reaction volume of 50 µL containing 1 µg of pGL3-basic vector or insert fragment plasmids, 5 µL of 10× Cutsmart buffer (NEB), 1 µL of KpnI-HF, and 1 µL of XhoI, made up to the final volume with ddH 2 O. Following activation, the mixture was incubated for 4 h at 37 • C, after which XhoI was inactivated at 65 • C for 20 min, and 5 µL 10× gel loading dye was added to inactivate KpnI-HF. Recombinant DNA was transformed into Escherichia coli DH5α cells (TsingKe), and plasmids were sequenced (Sangon Biotech, Shanghai, China).

Assays for the Interaction between Transcription Factor AhHsf and AhHsp70p
To determine whether the transcription factor AhHsf directly regulates the expression of AhHsp70p in vitro, we constructed the overexpression vector pIZ/V5-His-AhHsf harboring AhHsf and co-transfected this into sf9 cells with AhHsp70p reporter plasmids. After co-transfection, AhHsp70p-related luciferase activity was measured using the DLR system following the manufacturer's protocol, and AhHsp70 and AhHsf expression levels were determined based on RT-qPCR analysis.

Statistical Analysis
Statistical analyses were performed using SAS software v8 for Microsoft Windows and GraphPad Prism software (version 6.0; GraphPad Software Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA; SAS Institute Inc., USA) was used to analyze the differences among treatments, followed by a least significant difference (LSD) test for multiple comparisons. AhHsp70 and AhHsf gene expression levels and AhHsp70p relative luciferase activity after co-transfection of cells with the AhHsf overexpression and AhHsp70p reporter plasmids were analyzed using the Student's t-test. The results are presented as mean values ± standard deviation (SD); p-values of 0.05 or lower were considered significant (* p < 0.05; ** p < 0.01).

Conclusions
In summary, we isolated AhHsp70p from A. hygrophila, determined its sequence using I-PCR, and characterized its composition based on bioinformatics analysis. We subsequently examined the activity of AhHsp70p via cell transfection and promoter deletion analyses, and systematically studied the interaction between co-transfected AhHsf and AhHsp70p using a dual-luciferase reporter assay system, with the levels of AhHsp70 and AhHsf expression in co-transfected cells being determined based on RT-qPCR analysis. Our findings indicated that the upstream sequence of the AhHsp70 promoter may contain core functional regions between base pairs −944 and −744. The DLR assay system results revealed that overexpression of AhHsf could significantly enhance the activity of AhHsp70p, and qPCR assays indicated that the level of AhHsp70 expression increased with the extension of Sf9 cell proliferation time within a certain limit, which is conceivably attributable to the fact that Sf9 cells cease proliferating after reaching a certain density or that cells are subject to a certain extent of apoptosis. Moreover, we found that AhHsf overexpression significantly enhanced the expression of AhHsp70 in transfected cells. Collectively, the findings of this study enabled a preliminary characterization of upstream regulatory mechanisms underlying the transcriptional regulation of AhHsp70 expression, which entailed binding of the transcription factor AhHsf to upstream cis-acting elements (promoter region from −944 bp to −744bp) of AhHsp70 to activate the AhHSF-AhHSP signaling pathway at the transcriptional level, thereby enhancing the transcriptional expression of AhHsp70 to protect A. hygrophila from high-temperature damage.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.