Genome-wide identification and multi-level analysis of the HSP gene superfamily in Aphidoletes aphidimyza: sHSP gene family expansion and its role in diapause regulation

Abstract

Background

Insect diapause is a critical adaptive strategy for surviving unfavorable environmental conditions. The predatory insect Aphidoletes aphidimyza, widely used in biological pest control, relies on diapause for commercial storage and application. Heat shock proteins (HSPs), particularly small HSPs (sHSPs), are known to play pivotal roles in stress tolerance and protein homeostasis maintenance during diapause. However, the regulatory mechanisms of the HSP gene superfamily, especially the sHSP gene family, in A.aphidimyza diapause remain poorly understood. This study aims to comprehensively analyze the HSP gene superfamily in A.aphidimyza and elucidate the functional and regulatory roles of sHSPs in diapause.

Results

Our findings reveal that A.aphidimyza has evolved a multi-layered regulatory mechanism through selective expansion of the sHSP gene family. At the transcriptional level, specific transcription factors such as Ftzf1 bind to sHSP gene promoters, enhancing their expression during diapause. Post-transcriptionally, Among the 34 members of the sHSP gene family, 32 have only one exon, the intronless, single-exon structure of sHSP genes facilitates rapid mRNA maturation, enabling swift protein synthesis. Post-translational modifications, such as phosphorylation, regulate the oligomeric state of sHSP proteins, allowing them to dissociate into functional dimers and protect cellular protein homeostasis under stress. Notably, a unique branch of the sHSP gene family (CladeX) exhibited expansion, higher expression during diapause, relatively conserved sequences, suggesting its critical role in environmental adaptation.

Conclusions

This study provides a comprehensive analysis of the A.aphidimyza HSP gene superfamily, highlighting the pivotal role of the sHSP gene family, particularly CladeX, in diapause regulation. The multi-level regulatory mechanisms—transcriptional, post-transcriptional, and post-translational—enable A.aphidimyza to rapidly respond to adverse conditions like low temperature and short photoperiod, ensuring survival triggering diapause. These insights not only deepen our understanding of insect diapause but also offer potential applications for improving the storage and commercial use of A.aphidimyza as a biological control agent.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-12163-y.

Background

A.aphidimyza, belonging to the Diptera Cecidomyiidae family, is capable of preying on more than 60 species of aphids and has been commercialized as a natural enemy insect for aphid control since the 1980 s [1, 2]. A.aphidimyza undergoes complete metamorphosis, with only the larval stage being predatory on aphids [1, 2]. As a biological control agent, A.aphidimyza has many advantages, such as a wide prey range, strong searching efficiency, and a short life cycle [1, 3]. As a natural enemy insect, the sensitivity of A.aphidimyza to low temperature and other adverse environments has also become a key factor limiting its widespread application [4].

Many insects enter a state of developmental arrest called "diapause" when facing adverse environmental conditions such as extreme temperature and starvation [5]. For example, lower temperatures (e.g., 17 °C) significantly enhance winter diapause induction in Cydia pomonella. Whereas a photoperiod of 14-h light/10-h dark at 20 °C can efficiently induce summer diapause in pupae of Pieris melete [6, 7]. Insect diapause typically progresses through multiple phases: preparation (pre-diapause), initiation, maintenance, and termination. The initiation and maintenance of which are precisely regulated by hormone levels such as ecdysone and juvenile hormone [8]. These hormones transmit signals to downstream diapause-related genes through a series of transcription factors, thereby triggering a series of physiological changes, including increased expression of Heat shock proteins (HSPs). Studies in Drosophila melanogaster (Diptera: Drosophilidae) and Spodoptera litura (Lepidoptera: Noctuidae) have shown that 20-hydroxyecdysone (20E) can induce elevated expression of small heat shock proteins (sHSPs), promoting a state of low metabolism and high stress resistance, which enables long-term survival in harsh environments [8–11].

The diapause state can often last for months to years, so it is often used as an ideal storage stage for natural enemy insects [12]. When external environmental conditions become suitable again, insects will break diapause and re-enter the developmental state [12–14]. A.aphidimyza enters diapause at the prepupal stage after experiencing low temperature and short-day photoperiod treatments during the larval stage. Studies have shown that 20 °C and a 12.7-h short-day photoperiod are the critical conditions for inducing diapause in native Japanese A.aphidimyza [15], while the diapause induction conditions for A.aphidimyza in China are usually set at 8 h of light (25 °C) and 16 h of darkness (10 °C) [16]. Currently, commercial storage of A.aphidimyza mainly adopts low-temperature preservation of pupae, but this method has problems such as a short shelf life and low survival rate [17]. Given that diapausing insects have stronger tolerance to environmental stresses, studying diapausing A.aphidimyza is expected to provide new ideas for overcoming these problems. Therefore, in-depth research on the molecular mechanisms of diapause A.aphidimyza resisting environmental stress has important theoretical and applied value.

HSPs play a crucial role in insect responses to environmental stress and the maintenance of protein homeostasis. Recent studies have shown that the content of energy storage substances such as trehalose and glycerol significantly increases during the Early diapause phase in A.aphidimyza [16]. However, when insects enter diapause, they not only need to store energy but also undergo a series of physiological and biochemical adjustments to cope with the damage of adverse external environments to their protein homeostasis and cellular functions. HSPs can rapidly respond to adverse external conditions and protect the normal physiological activities of organisms, also playing an important role in this process [18]. HSPs are highly conserved in prokaryotes and eukaryotes. In addition to performing normal physiological functions such as folding newly synthesized polypeptides in conventional environments, HSPs can also assist in degrading misfolded proteins when organisms face severe environmental stresses, preventing them from causing damage to the organism [19–21]. Studies have found that the levels of HSP90, HSP70, HSP60, and sHSPs change significantly when insects face adverse environments or enter diapause [22]. Among them, HSP90, HSP70, and HSP60 can bind to specific substrate proteins in a manner dependent on adenosine triphosphate (ATP), changing their conformation, thereby affecting key processes such as protein folding, degradation, disaggregation, and cell localization [23, 24].

In contrast, sHSPs have a wider range of substrate binding and can rapidly exert their function in an ATP-independent manner in the absence of energy, preventing irreversible denaturation of substrate proteins, and are therefore regarded as the first line of defense for cells against adverse external environments [24, 25]. Since diapausing insects usually have low metabolic levels and limited energy supply, the ATP-independent action of sHSPs makes their protective effect particularly important during diapause. For example, expression analysis of the HSP gene superfamilies in diapausing larvae of Sesamia nonagrioides showed that the expression levels of SnoHsp19.5 and SnoHsp20.8 were significantly upregulated [26]. Similarly, in Pieris melete, compared with non-diapause pupae, the expression levels of PmHSP19.5 and PmHSP20.0 were also significantly upregulated in summer diapause and winter diapause pupae [27]. When energy is sufficient, ATP-dependent molecular chaperones such as HSP70 can release substrate proteins bound to sHSPs by binding to substrate proteins [28].

The unique structural characteristics of sHSPs enable them to play an important protective role during energy-limited diapause. In addition to their ATP-independent action, the unique structural features of sHSPs are also closely related to their advantages in the diapause process [29]. Most members of the sHSP gene family contain only one exon, and their amino acid sequences include a conserved α-crystallin domain of about 90 amino acids, a variable C-terminus, and an N-terminus with highly variable length and sequence [30, 31]. Two sHSP monomers combine through β-pleated sheets in the α-crystallin domain to form dimers, and multiple dimers further connect in different geometric configurations, eventually forming dynamic hollow spherical oligomeric structures composed of 12 to 48 sHSP monomers [25]. Furthermore, research has revealed that the oligomerization state of sHSPs is regulated by phosphorylation. Unphosphorylated HSP27 can form larger multimers, whereas phosphorylation induces conformational changes [32]. These changes, in turn, lead to the dissociation of the HSP27-formed complexes [32]. Structural studies on the 24-mer of archaeal Methanocaldococcus jannaschii HSP16.5 and the 12-mer of wheat Triticum aestivum HSP16.9 have shown that the disordered N-terminus may fill the central cavity of the sHSP oligomer, thereby affecting the stability of the oligomeric complex [33, 34].

The present study aims to comprehensively identify and phylogenetically analyze the HSP gene superfamilies in A.aphidimyza, and to deeply explore the response mechanism of the sHSP gene family during diapause in A.aphidimyza by integrating transcriptome data from diapausing and non-diapausing A.aphidimyza induced by low temperature and short photoperiod. This study will not only help to reveal the role mechanism of the HSP gene superfamilies in diapause insects such as A.aphidimyza to resist adverse environments, but also provide an important theoretical basis for developing new technologies for the storage and application of natural enemy insects based on diapause regulation.

Materials and methods

Insects and sample collection

The A.aphidimyza population used in this study originated from Zunyi City, Guizhou Province. The initial field-collected population was maintained under standardized laboratory conditions (25 °C, 16L:8D photoperiod) for six consecutive generations, feeding exclusively on Acyrthosiphon pisum. This rearing protocol aimed to minimize environmental and genetic variability, thereby establishing a genetically stable experimental population. To investigate the effect of diapause on gene expression, newly hatched (24 h) A.aphidimyza eggs were divided into two groups: The control group was reared under standard conditions (25 °C, 16L:8D photoperiod) and reached the prepupal stage in 7 days. The diapause induction group was reared under diapause-inducing conditions (25 °C during 8 h light, 10 °C during 16 h dark) and entered diapause at the prepupal stage in 10 days. All prepupal samples were collected at the same developmental stage (prepupal). According to previous experimental results, the diapause induction rate of A.aphidimyza under this low-temperature and short-day photoperiod condition can reach more than 80% (Diapause induction rate = number of individuals with arrested development at the prepupal stage/total number of individuals at the prepupal stage × 100%) [35]. Prepupal larvae from both groups were rapidly frozen in liquid nitrogen, with each sample containing 30 individuals and three biological replicates. In addition, to analyze gene expression patterns at different developmental stages, samples were collected from eggs, first-, second-, and third-instar larvae, pupae, and adults reared under standard conditions (25 °C, 16L:8D). Each sample contained 30 individuals, with three biological replicates set up for each stage. Three biological replicates were set up for each developmental stage, and all samples were snap-frozen in liquid nitrogen and stored in a −80 °C freezer.

RNA-seq and analysis

Total RNA was extracted from the aforementioned samples using Trizol reagent (Takara, Beijing, China). RNA concentration, purity, and integrity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Qualified RNA samples were sent to Berry Genomics (Beijing, China) for sequencing, where cDNA libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). After quality inspection, the libraries were sequenced on the Illumina NovaSeq 6000 platform using PE150 sequencing.

Raw sequencing data were first assessed for quality using FastQC (v0.11.9). Adapter sequences and low-quality reads were then removed using FastP (v0.39) [36, 37]. The clean reads were aligned to the reference genome of A.aphidimyza (NCBI accession number: GCA_030463065.1) using Hisat2 (v2.2.1) [38]. Transcripts were then assembled with the whole read set for each sample individually using StringTie (v2.1.7) [39], and subsequently merged across all samples to generate a comprehensive transcriptome assembly. Transcript quantification analysis was performed using Salmon (v1.5.2) [40], and reads counts were converted to Transcripts Per Million (TPM) values. The raw read counts were used as input for differential expression analysis using the DESeq2 (v1.34.0) [41] package, with a screening criterion of Adjusted p value < 0.05. To validate the transcriptome sequencing results, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) was used to detect the expression levels of genes within the sHSP gene family in diapausing and non-diapausing A.aphidimyza samples. Specifically, the RNA samples used for qRT-PCR were aliquots from the same total RNA extractions used for the corresponding transcriptome sequencing libraries. Specific primers for selected genes within the sHSP gene family were designed using Primer Premier 5.0 software, and Ribosomal protein L32 (RPL32) was selected as the internal reference gene [42]. The amplification efficiencies for all primer pairs, as determined by standard curve analysis, ranged between 90 and 110%. Primer sequences are listed in Table S1. qRT-PCR was performed using the TAKARA TB Green Premix Ex Taq kit (Takara, Beijing, China). Three biological replicates and three technical replicates were set up for each sample. The 2^−ΔΔCT method was used to calculate the relative expression levels of genes. Statistical analysis was performed using Student's t-test, with p < 0.01 indicating a significant difference.

Gene family identification and phylogenetic analysis

To identify members of the HSP gene superfamily in A.aphidimyza, BITACORA (v1.3) [43] tool was first used to perform homologous searches of the A.aphidimyza genome and protein sequence using the HSP protein sequences of D.melanogaster (HSP100, HSP90, HSP70, HSP60, sHSP) as query sequences. Subsequently, HMMER (v3.3) [44] was used to search for protein domains of candidate genes to confirm their HSP gene superfamily membership. To ensure the accuracy of gene annotation, the gene structure of each gene family was manually checked and corrected using the Integrative Genomics Viewer GSAman (v.0.8.2) (https://gitee.com/CJchen/IGV-sRNA). Gene annotation was conducted using MAKER (v3.01.03), combining RNA-seq to generate high-confidence gene models and ultimately produce a comprehensive, well-annotated gene set according to the methodology from our previous A.aphidimyza genome (GCA_030463065.1) [45, 46]. To identify potential errors within the gene family, we performed phylogenetic tree analysis. Specifically, multiple sequence alignment of the amino acid sequences derived from the coding sequences (CDS) of A.aphidimyza HSP gene superfamily members and D.melanogaster homologous genes was performed using the "L-INS-i" algorithm of MAFFT (v7.490) [47]. A maximum likelihood (ML) phylogenetic tree was constructed based on the alignment results using FastTree (v2.1.11) [48], employing the LG + GAMMA model. In addition, the HSP gene superfamily members of the closely related species Sitodiplosis mosellana were identified using the same method (NCBI accession number: GCF_009176505; SRA: SRP315051), along with those of Psylliodes chrysocephala (NCBI accession number: GCA_927349885.1; SRA: SRX27574413). HSP gene sequences of Aedes aegypti, D.melanogaster, were downloaded from the HSPIR database (https://pdslab.biochem.iisc.ac.in/hspir/predownload.php). Multiple sequence alignment of the HSP gene superfamily sequences of these five species was performed using MEGA11 (v11.0.13) [49]. Based on the alignment results, a maximum likelihood (ML) tree was constructed using MEGA11, and the phylogenetic tree was visualized and beautified using iTOL (v6) [50].

Expansion and selection pressure analysis of gene families

To analyze the evolutionary dynamics of the HSP gene superfamily, gene duplication and loss events were analyzed using Notung (v2.9.1.5) software based on The gene trees of sHSP, HSP40, HSP60, HSP70, HSP90, and HSP100 gene family members from five species, as follows: A.aphidimyza, S.mosellana, D.melanogaster, A.aegypti, and P.chrysocephala [51]. In addition, to explore the environmental adaptability of sHSP gene family in A.aphidimyza, the CodeML program in the PAML (v4.9j) package was employed to evaluate the sequence evolutionary rate of the sHSP gene family and to perform branch-site model analysis for sHSP genes. Likelihood ratio tests (LRT) were further conducted to assess the presence of positive selection in these genes of A.aphidimyza [52].

Analysis of sHSP gene family structure and function in A.aphidimyza

To investigate the structural characteristics of the A.aphidimyza sHSP gene family, conserved motifs in sHSP protein sequences were identified using MEME suite (v5.5.7) [53]. The motif prediction results from MEME, the phylogenetic tree of the sHSP gene family (constructed using MEGA11), and gene structure information (extracted from genome annotation files) were imported into TBtools (v2.149) for visualization and integration [54]. The Conserved Domain Database (CDD) search tool from NCBI was used to annotate the N-terminal domains of sHSP proteins. NetPhos (v3.1) software was used to predict potential phosphorylation sites in sHSP protein sequences, and sites with a predicted probability higher than 0.6 were retained [55]. To investigate the potential role of phosphorylation modification in regulating the function of sHSP proteins in A.aphidimyza, we selected two kinase genes—Protein Kinase C (pkc) and Calcium/Calmodulin-Dependent Protein Kinase II (CAMKII)—for experimental validation [56, 57]. As crucial intracellular signaling molecules, these two types of kinases have been extensively demonstrated to regulate the activity and conformation of small heat shock proteins (sHSPs) through phosphorylation in insects under stress conditions such as heat shock. Their expression levels were analyzed using Student's t-test, with the primer sequences listed in Table S1 and amplification efficiencies ranging from 90 to 110%. AlphaFold3 was used to predict the three-dimensional structures of sHSP protein monomers, dimers, and oligomers, and PyMOL (v2.6.0) was used to clearly display the three-dimensional structures of proteins by adjusting the viewing angle, using Cartoon mode, and coloring different domains [58, 59].

Analysis of hormone regulatory elements

We investigated the role of hormone signaling pathways in regulating A.aphidimyza sHSP gene expression by analyzing transcriptome data from diapausing and non-diapausing groups. Secondary transcription factors pivotal to ecdysone and juvenile hormone signaling pathways were screened using a criterion of |log2FC|> 2 and adjusted p value < 0.05 [59–62]. The sequence characteristics (position frequency matrices, PFMs) of these transcription factors were identified using the JASPAR database. Promoter sequences 2 kb upstream of A.aphidimyza sHSP genes were extracted, and PyJASPAR (v3.0.0) tool was used to scan for transcription factor binding sites based on the aforementioned PFMs [63]. GSDS (v2.0) was used to visualize the distribution of transcription factor binding sites in the promoter regions of sHSP genes [64]. Subsequently, the proportion of binding sites for the aforementioned transcription factors in the promoter regions of the sHSP gene family was calculated to evaluate the potential regulatory differences of hormone signaling pathways on different sHSP gene families.

Results

HSP gene superfamily identification and expression pattern of A.aphidimyza

Figure 1 shows the identification results and gene expression patterns of the HSP gene superfamily in A.aphidimyza. Through homologous comparison and domain analysis, a total of 100 HSP genes were identified in the A.aphidimyza genome, belonging to 6 gene families: sHSP, HSP40, HSP60, HSP70, HSP90, and HSP100 (Fig. 1a). Among these, the HSP40 gene family had the largest number of genes, with 44 genes identified; followed by the sHSP gene family (34 genes), while the HSP100 gene family had the fewest genes (2 genes). The number of members in the HSP60, HSP70, and HSP90 gene families were 11, 16, and 3, respectively. To analyze the expression changes of the HSP gene superfamily during diapause in A.aphidimyza, we plotted the log2FoldChange and adjusted p-values (p-adjust) of each HSP gene between diapause and non-diapause groups. The results indicated that most members of the HSP40 family were upregulated, the expression levels of the HSP60, HSP70, HSP90, and HSP100 families showed no significant changes or slight reductions in the diapause group. Notably, unlike the overall downward or stable trends observed in other HSP families, the most members of the sHSP gene family exhibited a upward trend in expression during diapause (Fig. 1b).

Fig. 1.

Composition, expression, expansion, and evolution of the HSP gene family in A.aphidimyza. a Number of genes in each HSP gene family. b Volcano plot showing differential expression of the HSP superfamily between diapause and non-diapause groups. The X-axis represents the log2 fold change, and the Y-axis represents the -log10(adjusted p-value) from DESeq analysis. Different colors represent different HSP families. The vertical dashed lines indicate the threshold of log2(Fold Change) = ± 1.0, and the horizontal dashed line indicates the threshold of adjusted p-value = 0.05. c Gene expansion analysis of the sHSP gene family across five insect species (P.chrysocephala, A.aegypti, D.melanogaster, S.mosellana, and A.aphidimyza). The numbers in black, red, and blue represent ancestral gene counts, gene duplication events, and gene loss events, respectively. d Maximum-Likelihood phylogenetic tree of sHSP genes based on amino acid sequences from five insect species. The red branches indicate the independently evolved CladeX in A.aphidimyza, which is supported by a bootstrap value of 97%. The lowest node supporting value is 46%. The outer blue bar chart shows the log2 fold expression values between diapause and non-diapause groups, with different background colors distinguishing the species

Expansion analysis of the HSP gene superfamily

Comparative analysis of the HSP gene superfamily across five species (A.aphidimyza, S.mosellana, A.aegypti, D.melanogaster, and P.chrysocephala) revealed differences in the number of members within each HSP gene superfamily. Among these, the HSP40 gene family consistently exhibited the largest number of members across all species, while the HSP100 gene family had the fewest members (Table 1). The results indicated that the sHSP gene family in the coleopteran insect P. chrysocephala evolved from 10 ancestral genes and underwent 8 gene duplication and 6 gene loss events in its lineage. In the lineage of dipteran insects, the sHSP gene family experienced 0 gene event. Notably, in the common ancestor lineage of Cecidomyiidae insects, namely S.mosellana and A.aphidimyza, the sHSP gene family underwent a significant expansion with 12 gene duplication and 3 gene loss events, indicating an expansion trend of this gene family in this family. Subsequently, in the S.mosellana lineage, the sHSP gene family further experienced 10 gene duplication and 2 gene loss events, while in the A.aphidimyza lineage, a more dramatic expansion occurred with 23 gene duplication and 4 gene loss events (Fig. 1c).

Table 1.

Number of genes in HSP gene families

	sHSP	HSP40	HSP60	HSP70	HSP90	HSP100
Psylliodes chrysocephala	12	40	10	17	3	2
Aedes aegypti	24	32	7	8	5	1
Drosophila melanogaster	18	57	25	41	8	2
Sitodiplosis mosellana	23	43	10	17	3	1
Aphidoletes aphidmyza	34	44	11	16	3	1

Evolution and expression of sHSP gene family in A.aphidimyza

To analyze the evolutionary relationships and expression characteristics of the sHSP gene family, a phylogenetic tree was constructed based on the sHSP amino acid sequences of five species (A.aphidimyza, S.mosellana, A.aegypti, D.melanogaster, and P.chrysocephala). The results showed that the A.aphidimyza sHSP gene family was divided into two distinct groups: one branch, which we named CladeX, contained 17 genes that formed a unique, species-specific cluster; the other branch, designated as Others, contained the remaining 17 genes (Fig. 1d). Differential expression analysis, conducted using DESeq and presented as a volcano plot, was based on the log2 foldchange and adjusted p-value of the genes. The results showed that CladeX branch exhibited significantly increased expression during diapause (Fig. 2a). Further comparative transcriptome analysis, confirmed this trend via an independent t-test, demonstrating that the overall expression level of CladeX genes was significantly higher than that of the Others group (p < 0.05) (Fig. 2b).

Fig. 2.

Differential expression and expression patterns of the A.aphidimyza sHSP gene family. a Volcano plot illustrating the expression differences of the CladeX and Others gene sets within the A.aphidimyza sHSP gene family between diapause and non-diapause samples. The X-axis represents the log2 fold change, and the Y-axis represents the -log10(adjusted p-value). The vertical dashed lines indicate the threshold of log2(Fold Change) = ± 1.0, and the horizontal dashed line indicates the threshold of adjusted p-value = 0.05. b Box plot comparing the log2 fold change values of the CladeX and Others gene sets, showing the significant difference between the groups (independent t-test, ** indicates p < 0.01). c qRT-PCR results showing the relative expression levels of CladeX genes in diapause and non-diapause samples. P-values were corrected for multiple comparisons. * indicates p < 0.05, and ns indicates no significant difference. d Line plot comparing the expression levels of the CladeX and Others gene sets across different developmental stages (egg, 1 st instar larva, 2nd instar larva, 3rd instar larva, pupae, and adult). log2(TPM + 1) values were used to represent expression levels. ***, **, and * indicate p < 0.001, p < 0.01, and p < 0.05, respectively

To validate the transcriptome analysis results, qRT-PCR was used to detect the expression levels of all 17 CladeX genes under diapause and non-diapause conditions (Fig. 2c). The p-values from this analysis were corrected for multiple comparisons. The experimental results showed that the expression levels of the majority of genes in the CladeX branch were significantly higher in the diapause group compared to the non-diapause control group (adjusted p-value < 0.05). Specifically, with the exception of the AaphsHSP08 and AaphsHSP17 genes, which showed no significant difference in expression, the expression levels of the remaining genes were significantly upregulated during diapause. This reconfirms the critical role of CladeX genes in the diapause process of A.aphidimyza (Fig. 2c).

To quantify the developmental expression dynamics of sHSP genes in A. aphidimyza, we statistically compared the LOG2(TPM + 1) values between the CladeX and Others groups across all stages (egg, L1-L3 larvae, pupae, and adult) using t-tests with p-values adjusted for multiple comparisons. Each developmental stage exhibited significantly lower expression in CladeX versus the Other group, with CladeX maintaining consistently low levels. This collectively confirms CladeX's basal suppression during development (Fig. 2d).

To investigate whether the sHSP gene family and its CladeX subfamily underwent positive selection in A.aphidimyza, we performed a branch-site model analysis using the CodeML program in the PAML package. Likelihood Ratio Tests (LRT) were conducted to detect signals of positive selection acting on specific sites along specific lineages. The results are summarized in Table 2 and TableS2.

Table 2.

Positive selection analysis of the sHSP gene family and CladeX in A.aphidimyza using branch-site model

	Likelihood Ratio Test (LRT)			Positively Selected Sites
	2ΔlnL	p-value	Significance	BEB Method
sHSP	−661.16	1	Not significant	Not found
CladeX	1.31	0.252	Not significant	Not found

The analysis revealed that neither the entire sHSP family nor the CladeX subfamily showed statistically significant evidence of positive selection. The LRT results for both groups were non-significant (p > 0.05). Although the NEB method identified two candidate positively selected sites (positions 30 and 50) in the CladeX subfamily, these sites were not supported by the more stringent BEB method (TableS2). The BEB method, which provides more reliable Bayesian posterior probabilities for site-specific selection, failed to confirm these sites. Furthermore, the overall non-significant LRT result suggests that the two candidate sites identified by the NEB method are likely false positives and not the result of pervasive positive selection. Therefore, we conclude that both the sHSP family and its CladeX subfamily are evolutionarily conserved and have not undergone significant positive selection in A.aphidimyza.

Structure and function analysis of sHSP gene family in A.aphidimyza

To further analyze the structural characteristics of the A.aphidimyza sHSP gene family, the conserved motifs in the sHSP protein sequences were first analyzed using MEME suite (5.5.7), and the motif analysis results, phylogenetic tree, and gene structure information were integrated (Fig. 3a). The results showed that the A.aphidimyza sHSP protein sequences contained multiple conserved motifs, while the specific biochemical functions of individual motifs (Motifs 1–5) require further experimental validation, their compositional patterns clearly distinguished the CladeX and Others branches. CladeX genes generally contained Motifs 1–5, while most Other branch genes lacked Motif 4 and Motif 5. In addition, AaphsHSP02 and AaphsHSP34 contained four exons each, while the remaining members of the A.aphidimyza sHSP gene family contained only one exon. Furthermore, NetPhos software was used to analyze the N-terminal sequences of sHSP proteins and predict potential phosphorylation sites. The results showed that phosphorylation sites of PKC were predicted in the Motif 5 sequences of the N-terminus of multiple sHSP proteins in CladeX (Fig. 3b). To investigate the potential impact of phosphorylation modification on sHSP function, we performed qRT-PCR validation for key protein kinases PKC and CaMKII identified from transcriptome screening. Statistical analysis by Student's t-test confirmed significantly elevated expression of both PKC and CaMKII in the diapause group compared to non-diapause controls (Fig. 3c), consistent with transcriptome trends.

Fig. 3.

Structural and functional analysis of the Aphidoletes aphidimyza sHSP gene family. a Schematic diagram of the motif and gene structure (exons, introns, and UTRs) of the A.aphidimyza sHSP gene family. Different colors represent different motifs; genes are categorized into two groups, CladeX and Others. b Amino acid sequence alignment of Motif 5 in the N-terminus of CladeX group sHSP proteins in the A.aphidimyza sHSP gene family. Red letters indicate predicted PKC phosphorylation sites. c qRT-PCR validation of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase II (CAMKII) gene expression under diapause and non-diapause conditions. The bar graphs show the relative expression levels determined by t-tests. p < 0.05 indicates a significant

To investigate the impact of N-terminal phosphorylation of the sHSP gene family on protein function, we used AlphaFold3 to predict the three-dimensional structures of proteins encoded by CladeX genes of the A.aphidimyza sHSP gene family. The structural prediction results showed that sHSP proteins within CladeX generally exhibited a core domain composed of β-sheets, while the N-terminal region showed structural diversity, containing multiple α-helix structures (Fig. 4a). Taking AaphsHSP20 as an example, its dimer structure prediction results showed that two monomers formed a stable dimer through the interaction of the β6 + 7 sheet region, while the N-terminal region was exposed to the outside of the dimer structure (Fig. 4b). sHSP proteins are typically assembled into dynamic hollow spherical oligomers composed of 12 to 48 monomers. To further investigate their oligomerization state and the effect of phosphorylation sites, we performed structural analysis using the dodecamer of sHSP proteins within CladeX as a model. Structural analysis indicated that these dodecamer models all exhibited similar assembly patterns: the N-terminal domains were clustered in the central region of the oligomer, while the hydrophobic β-sheet core domains were distributed on the outer surface of the oligomer, forming a hydrophobic shell (Fig. 4c). Notably, structural models showed that most of the predicted phosphorylation sites were precisely located in the central region of the oligomer, suggesting that phosphorylation modification may play a key role in regulating the assembly and function of sHSP protein oligomers.

Fig. 4.

Three-dimensional Structures and Hormone Regulation of A.aphidimyza sHSP Proteins. a Side views of different sHSP proteins within CladeX, showing 12 sHSP proteins (AaphsHSP08, AaphsHSP10-14, AaphsHSP17, AaphsHSP19-25, AaphsHSP29-31); yellow portions indicate the N-terminal sequences of the proteins. bView of the dimeric structure of AaphsHSP20; the two subunits are indicated in red and blue, respectively, with the β6 + 7 domain highlighted. c Views of dodecamer structures of different sHSP proteins within CladeX; yellow portions indicate the N-terminal sequences of the proteins. d Volcano plot showing differential expression of secondary transcription factors downstream of the Ecdysone and Juvenile Hormone pathways in diapause and non-diapause samples. The X-axis represents the log2 fold change, and the Y-axis represents the -log10(adjusted p-value). The vertical dashed lines indicate the threshold of log2(Fold Change) = ± 2.0, and the horizontal dashed line indicates the threshold of adjusted p-value = 0.05.The pink region indicates a |log2 fold change|> 2. e Distribution of predicted hormone-related transcription factor (Br, Foxo, Ftzf1, Hr3, Hr4) binding sites in the promoter region (2 kb upstream of the gene) of A.aphidimyza sHSP genes. Different colors and shapes represent different transcription factors. f Proportion of genes containing hormone response elements in the promoter regions of the CladeX group and the Others group (bar graph). Blue represents CladeX, and orange represents Others

Response of the sHSP gene family of A.aphidimyza to hormone signaling pathways

To investigate the role of hormone signaling pathways in the diapause regulation of A.aphidimyza sHSP genes, we selected 8 core secondary transcription factors downstream of the ecdysone and juvenile hormone pathways, namely Br, E74, E75, Foxo, Ftzf1, Hr3, Hr4, and Krh1, as well as the transcription factor Hsf of the HSP gene superfamily, and analyzed their expression patterns in the transcriptome data of the diapause and non-diapause groups [65, 66]. The results showed that the expression levels of a total of 5 secondary transcription factor genes changed by more than |log2fold|> 2. Among them, the expression levels of Br, Foxo, and Ftzf1 were up-regulated in the diapause group, while the expression levels of Hr3 and Hr4 were down-regulated in the diapause group (Fig. 4d). To investigate whether this hormone signaling pathway-related transcription factors could directly regulate the expression of the sHSP gene family, we predicted the potential binding sites of the above 5 transcription factors in the promoter regions of sHSP genes and visualized the analysis results. The results showed that binding sites for at least one hormone-related transcription factor were predicted in the promoter regions of most sHSP genes (Fig. 4e). To evaluate the potential regulatory role of hormone signaling pathway-related transcription factors on the sHSP gene family, we further statistically analyzed the proportion of promoter regions in CladeX and Others genes containing the binding sites of the aforementioned transcription factors. The results indicated that the proportion of promoter regions of CladeX group genes containing hormone-related transcription factor binding sites was higher than that of the Others group. The proportion of genes containing Ftzf1 binding sites in CladeX reached as high as 64.7%, and the proportion in Others also reached 29.4% (Fig. 4e). These results suggest that hormone signaling pathway-related transcription factors may play an important role in the diapause regulation of the A.aphidimyza sHSP gene family, and the potential regulatory effect on CladeX group genes may be stronger.

Discussion

When facing unfavorable environments such as low temperature and short photoperiod, the predatory insect A.aphidimyza typically adopts a diapause coping strategy. HSPs play a crucial role in organisms' responses to adverse environmental conditions [67]. To investigate whether HSP play an important role in the evolution and diapause process of A.aphidimyza, we induced diapause in A.aphidimyza under conditions of 8 h of light (25 °C) and 16 h of darkness (10 °C) and identified and analyzed the members of its HSP gene superfamily. The results showed that within the HSP gene superfamily of A.aphidimyza, only the sHSP gene family generally exhibited higher expression levels in diapausing individuals compared to non-diapausing ones. However, the expression level of sHSP33 showed a decreasing trend. Considering that the expression of the BmHsp19.5 gene in Bombyx mori initially increases and then decreases throughout the diapause process, we speculate that the expression of sHSP33 may be related to the diapause stage in A.aphidimyza [68]. It is possible that sHSP33 plays a role only during specific diapause stages. In this study, the expression levels of most sHSP genes in A.aphidimyza were elevated during the early diapause phase, which is consistent with findings reported during the diapause induction phase in Trogoderma granarium [69]. Similar sHSP upregulation patterns during diapause maintenance phases were also noted in Aspongopus chinensis [70]. So we speculate that the sHSP gene family plays a particularly critical role in the early phase of the diapause process in A.aphidimyza.

The sHSP gene family not only exhibited significantly increased expression levels in the diapausing A.aphidimyza species, but also, compared to S.mosellana, D.melanogaster, A.aegypti, and P.chrysocephala, the sHSP gene family in A.aphidimyza had undergone significant expansion. It is noteworthy that the sHSP gene family in S.mosellana, a closely related species belonging to the Cecidomyiidae family as A.aphidimyza, did not undergo expansion. Both A.aphidimyza and S.mosellana exhibit diapause characteristics, but their diapause strategies differ significantly: A.aphidimyza exhibits facultative diapause, which can be induced by low temperature and short-day photoperiod conditions. While S.mosellana adopts obligate diapause, the diapause process of which is not affected by external environmental factors [71]. Studies have shown that sHSP genes in insects can be induced by temperature and light [71–75]. This evidence suggests that A.aphidimyza may have responded to the facultative diapause process induced by low temperature and short-day photoperiod at the genomic level through the expansion of the sHSP gene family during evolution. To evaluate adaptive evolution in the A.aphidimyza sHSP gene family, we conducted branch-site model analysis with Likelihood Ratio Tests (LRT). Results showed no significant positive selection signals for sHSP, This evolutionary constraint reflects the conserved molecular chaperone functions essential for proteostasis maintenance [76].

Collectively, our results establish the critical role of the sHSP gene family during diapause in A.aphidimyza, although its precise regulatory mechanisms remain to be fully elucidated. Notably, the expression of CladeX—within the sHSPs—remains relatively low throughout various developmental stages, but is significantly upregulated upon diapause entry. Coupled with the previous finding that the sHSP family is evolutionarily conserved, it is plausible to conclude that this gene family is indispensable for survival under stressful conditions. Specifically, CladeX may adopt a strategy of maintaining constitutive low expression to minimize energetic costs under normal conditions, while being strongly activated during diapause to perform essential protective functions, thereby enhancing resilience against environmental challenges.

To further investigate the significance of the CladeX in diapause, we conducted an in-depth analysis of its structure. The results showed that the genes within CladeX do not contain introns and are composed of only one exon. This structural feature is consistent with the research results of Li et al. on the structure of sHSPs in Bombyx mori [77]. Li divided sHSPs into two categories: Orthologous clusters that contain at least one intron and are mainly involved in metabolic processes, and Species-specific clusters that are related to environmental stress [78]. They proposed that introns can hinder rapid gene regulation in organisms, and in genes that require rapid transcription to resist adverse environments, introns may be selected as unfavorable structures [78]. Based on this viewpoint, we speculate that when A.aphidimyza needs to rapidly synthesize sHSPs to respond to adverse external environments, the sHSP genes of CladeX, due to containing only one exon, can effectively reduce splicing time, thereby responding more quickly to environmental changes and subsequently protecting the organism from the impact of unfavorable conditions.

To further understand the functional characteristics of these genes, we analyzed the amino acid sequences of genes within A.aphidimyza CladeX. The results showed that PKC Ser phosphorylation sites were generally present in motif 5, and the expression level of PKC in diapausing A.aphidimyza increased, suggesting that the N-terminal sequences of genes within CladeX may be phosphorylated by PKC kinase. X-ray crystal structure analysis of the wheat Hsp16.9 dodecamer showed that its disordered and dynamic N-terminal sequence can fill the central cavity of the oligomer [79]. Combining this finding, we predicted the three-dimensional structures of genes within CladeX and surprisingly found that they not only had structurally diverse N-terminal domains, but also that the Motif 5 sequences containing predicted phosphorylation sites were mainly distributed within the central cavity of the dodecamer. Studies have shown that the structure of sHSP oligomers can be affected by the aggregation and dissociation of sHSP dimers, thereby achieving the conversion between active and inactive states. Dissociated sHSP dimers can bind to substrate proteins without relying on ATP, playing a role in protecting protein homeostasis. This conversion can be influenced by post-translational modifications such as temperature, pH, or phosphorylation [80, 81]. For example, phosphorylation of human Hsp27 (HSPB1) leads to the dissociation of Hsp27 tetramers into dimers [82]. Similarly, phosphorylation at homologous serine residues in D.melanogaster Hsp27 induces the disassembly of large oligomeric complexes into smaller species [83].Based on the above results, we speculate that members of the A.aphidimyza sHSP gene family within CladeX not only improve splicing efficiency through a single-exon structure, thereby accelerating the response speed of active proteins; but also, their proteins in an inactive state can exist in the form of larger oligomers such as dodecamers. Once their N-terminus is phosphorylated, the change in charge inside the oligomer will promote conformational changes, leading to the dissociation of sHSP dimers, which then perform the function of binding to and protecting substrate proteins.

Having understood the functional pattern of sHSPs during diapause in A.aphidimyza, we further aimed to investigate how sHSP expression is induced upon diapause entry. Insect diapause is known to be closely associated with the regulation of juvenile hormone (JH) and 20-hydroxyecdysone (20E). Diapausing insects precisely regulate the expression of diapause-related genes through transcription factors downstream of these hormones to adapt to harsh diapause environments [84, 85]. To investigate whether sHSPs can respond to juvenile hormone and ecdysone, we performed cis-acting element analysis on five transcription factors (Hr3, Hr4, Ftzf1, Foxo, and Br) that exhibited significantly changed expression levels in diapausing A.aphidimyza, using the JASPAR database. Particularly noteworthy is that cis-regulatory element analysis revealed that the CladeX genes of the A.aphidimyza sHSP gene family are enriched in Ftzf1 binding sites, with a proportion as high as 64.7%. These data suggest that ecdysone and juvenile hormone signaling pathways may be involved in the complex transcriptional regulation of sHSP gene expression in diapausing A.aphidimyza. As an important secondary transcription factor, Ftzf1 is not only regulated by ecdysone and juvenile hormone, but also plays multiple roles in insect development and physiological processes, such as regulating wing and leg expansion, programmed cell death of larval salivary glands, and larval fat body remodeling [86–89]. Ftzf1 can also bind to multiple chromosomal target sites to further regulate the expression of a series of genes related to diapause and development [90]. Therefore, we speculate that the up-regulation of Ftzf1 expression in diapausing A.aphidimyza is not only a characteristic of the diapause state, but also may be a central hub for regulating the expression of key diapause genes. From this, we propose the hypothesis that the increased expression level of Ftzf1 in diapausing A.aphidimyza may promote the transcription of CladeX of the sHSP gene family through mechanisms such as cis-regulatory element binding, thereby playing a protective role during diapause.

Conclusions

A.aphidimyza plays an important role in biological control. This study, combining bioinformatics and transcriptome analysis, revealed that the sHSP family within the HSP gene superfamily is significantly upregulated during diapause while remaining evolutionarily conserved. Further analysis indicated that the CladeX subfamily of sHSP genes shows particularly high expression and evolutionary conservation during diapause, suggesting its role in rapid environmental adaptation. At the transcriptional level, hormone signaling pathways may precisely regulate its transcription. At the post-transcriptional level, CladeX genes possess an intronless, single-exon structure, which may significantly accelerate mRNA maturation and protein synthesis in response to adversity. At the translational level, their N-terminal phosphorylation sites and related kinases were upregulated during diapause, possibly by altering sHSP oligomeric structure to promote its dissociation into active dimers, thereby protecting protein homeostasis. These multi-layered, flexible regulatory mechanisms enable A.aphidimyza to rapidly adapt to adverse conditions such as low temperature and short photoperiod, ensuring survival during diapause.

Abbreviations

HSP

Heat Shock Protein

sHSP

Small Heat Shock Protein

20E

20-Hydroxyecdysone

JH

Juvenile Hormone

qRT-PCR

Quantitative Real-Time PCR

PKC

Protein Kinase C

CaMKII

Calcium/Calmodulin-Dependent Protein Kinase II

MAPK

Mitogen-Activated Protein Kinase

ERK

Extracellular Signal-Regulated Kinase

AMPK

AMP-Activated Protein Kinase

Ka/Ks

Non-synonymous/Synonymous Substitution Rate

CK

Control Group

Authors’ contributions

Ziye Wang, Jiawen Qin, Xiaoxiao Zhu, and Hao Yang contributed to the experimental analysis. Xiaofei Yu and Huizi Wu provided the insects and assisted with the experimental design. Maofa Yang and Feng Zhang offered valuable revisions to the manuscript.

Funding

This work was supported by the Research on the Integration and Application of Key Technologies for Controlling Major Aphids in Agricultural and Forestry Plants Using Aphidoletes aphidimyza (2022XM11).

Data availability

The datasets generated and/or analyzed during the current study are available in the NCBI GenBank and NCBI BioProject databases. The nuclear mRNA sequences (CDS) and predicted protein sequences are available in GenBank with final accession numbers provided in Supplementary Table S3. The raw RNA sequencing data for the diapause and non-diapause *A. aphidimyza* samples is publicly available in the NCBI BioProject database under the final accession number PRJNA1293400. The publicly available RNA sequencing data for *A. aphidimyza* at different developmental stages, which was used in this study, is accessible from the NCBI BioSample database under Accession Number SAMN15005116.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.