Background
The term “heat shock protein” (HSP) represents a superfamily of genes, and their proteins as “molecular chaperons” increase in synthesis in response of unfavorable environmental condition in both prokaryotic and eukaryotic cells [
1]. Most of the HSPs have conserved sequences through bacteria to human, but their expression and function subject to environmental stress do vary from organism to organism [
2]. The HSPs are traditionally classified into eight families based on their molecular weight (MW) from 10 to 110 kDa and homology: HSP110 (HSPH), HSP90 (HSPC), HSP70 (HSPA), HSP40 (DNAJ), small HSP (sHSP, HSPB), and the chaperonin families HSPD (HSP60), HSPE (HSP10) and CCT (TRiC) [
3]. There have been a number of functional studies on HSPs in bacteria, algae, plant, amphibians, birds, and mammalian, especially in the model organisms of
Anopheles gambiae,
Drosophila melanogaster,
Arabidopsis thaliana,
Saccharomyces cerevisiae,
Caenorhabditis elegans,
Danio rerio and
Mus musculus [
4‐
7]. The HSP genes of insects encode molecular chaperones that help repair stress injuries via transportation and degradation of aggregated proteins, and they are susceptible to environment stresses, such as heat or cold stress, and insecticidal infection in insects [
8,
9]. The HSP expression patterns have been identified in
Drosophila species and some other insects [
10‐
13]; however, the information regarding HSP whole-genome diversity and association with insecticide resistance is still quite limited for insects as well as mosquitoes.
Recent research showed that
HSP90 was not only an important gene in
Apolygus lucorum adults in response to extremely high temperature, but was also associated with the resistance or tolerance to cyhalothrin, imidacloprid, chlorpyrifos, and emamectin benzoate and cyhalothrin [
14]. Two
HSP70s were upregulated in a chlorpyrifos-resistant population of
Plutella xylostella, whereas six
sHSPs were downregulated [
15]. The
sHSPs expression in responses of indoxacarb and cantharidin varied, and the exposure to beta-cypermethrin and chlorfenapyr resulted in an increase of 13
sHSP transcripts and a reduction of 12
sHSP transcripts in
Plutella xylostella, respectively, which indicates that different
sHSPs might play distinct roles in the development and regulation of physiological activities [
16]. One
HSP70 was highly expressed in the captafol-exposed larvae of
Drosophila melanogaster [
17]. In the imidacloprid and deltamethrin treatment groups, only one
HSP90 was upregulated more than two-fold in response to deltamethrin treatment in
Sogatella furcifera [
18]. Two
HSPs were up-regulated in DDT-resistant field isolates in
An. gambiae, supporting their link with insecticide resistance and/or stress response [
19].
HSPs are induced in response to stress induced by environmental factors and may be involved in adverse reactions, including insecticide resistance [
20].
Mosquitoes transmit disease resulting in around 700 million people of mosquito-borne illness each year and over one million deaths [
21]. Mosquito control relies primarily on the use of insecticides through insecticide-impregnated bed nets and indoor residual spray. In the past decades, pyrethroids have become the preferred insecticide because of their low toxicity to humans, high efficacy against mosquito vectors and short residual action. However, there has been increasing number of mosquitoes to have developed resistance to pyrethroids [
22].
Anopheles sinensis is a major malaria vector in China and other Southeast Asian countries [
23]. Because of extensive and continued application, pyrethroid resistance in
An. sinensis is now widespread in many regions of China [
24,
25]. However, the molecular mechanisms of pyrethroid resistance in
An. sinensis are not yet clearly understood and pose a challenge for the control of malaria in China. Recently,
An. sinensis genome was deeply sequenced and assembled, and are comprehensively conducting the research on the molecular mechanism of pyrethroid resistance using the species as model. Some families of genes have been identified at whole-genome level, and their association with pyrethroid resistance have been investigated or summarized, such as cytochromes P450s [
26] and carboxylesterases [
27]. However, there is still no report for the genome-wide identification of the HSP superfamily of genes in insects as well as in
An. sinensis. The association of
HSPs with pyrethroid resistance has been little known in insects as well as in
An. sinensis.
The present study identified and classified the HSPs superfamily of genes in An. sinensis genome, and analysed their phylogenetics and basic characteristics. More importantly, this study screened the HSP genes associated with pyrethroid resistance at whole-genome level through transcription comparison between resistant- and susceptible populations/strain from three geographical regions, quantitative PCR confirmation of candidate HSP genes, and expression changes of HSP genes in response to pyrethroid exposure. This study provides a comprehensive information frame for the HSP superfamily of genes in the An. sinensis and other insects, and lays a foundation for the functional study of HSPs in relation of pyrethroid resistance.
Discussion
In history, the HSP superfamily of genes across all organisms was classified into several families based on their molecular weights, HSP105/110, HSP90, HSP70, HSP60, HSP40, small HSP (sHSP) and HSP10 [
44,
45]. This is the first report for whole-genome identification of HSP superfamily of genes in the four species as well in insects. However, the HSP110 family does not exist in insect [
45], and the HSP10 (HSPE) and HSP60 (HSPD) families have been degraded as subfamilies in insects [
46]. The HSP gene number (72) of
An. sinensis is comparative with that of
An. gambiae (69) and
Culex quinquefasciatus (69), but much less than that of
Aedes aegypti (88). HSPs have been shown to be markedly associated with the resistance to heavy metals, pesticides and oxidative stress in insects, and the difference of gene number might result from the adaptation to different environment [
40,
46]. The much larger number in
Aedes aegypti appears due to the expansion of sHSP family (24 genes in
Aedes aegypti, and 10–14 in other three species) and HSP90 family (8 genes in
Aedes aegypti, and 4–5 in other three species).
The established phylogenetic relationship of HSP90 family genes are consistent with earlier studies, in which the HSP90A is a sister with the HSP90B, and the TRAP originated earlier than both HSP90A and HSP90B [
48,
49,
69]. Both the HSP90A and HSP90B exist in all eukaryotic kingdoms, and the TRAP also in bacteria [
49]. In this study, the four mosquito species investigated are lack of HTPG and HSP90C subfamilies of genes, like in
Drosophila melanogaster [
49]. In the HSP70 family, the C-terminal motif contains with diverse subcellular localizations signatures [
50]. In HSP110 subfamily have similar domains as canonical HSP70 subfamily but have long insertions and C-terminal extensions [
50]. The ATPase domain and the C-terminal helical lid of HSP110 subfamily were thought to mediate the interaction with HSP70 subfamily [
50]. In the chaperonins family, the CCT subfamily of genes in cytoplasm is a multi-subunit protein complex which functions in cytoskeletal protein folding in all eukaryotes [
51], and the HSPD and HSPE subfamilies in the mitochondria are orthologs of the
Escherichia coli GroEL (HSP60) and GroES (HSP10), respectively [
3]. It has been reported that HSPE (HSP10) proteins serve as the co-factor of HSPD (HSP60) to assist in the folding of newly synthesized proteins imported into mitochondria [
3]. In the HSP40 family, classified into three subfamilies, DNAJA, DNAJB and DNAJC are in accordance with those of previous studies,
Bombyx mori [
52]. For sHSP family, four orthologous clusters and four species-specific clusters was found. This clustering pattern is similar to those in
Bombyx mori and
An. gambiae [
47].
Previous studies showed that HSP superfamily members were differently expressed in diverse insecticides of insects. This may be correlated with the facts that HSP genes were significant in response to insecticides resistance of insects. This study checked the expression profiles of HSP genes in three field pyrethroid-resistant populations against the laboratory susceptible strain of
An. sinensis, and the expression patterns of HSP genes verified by qPCR. Similarly, the gene has been reported to be significantly overexpressed in chlorpyrifos-resistant resistance strains in
Plutella xylostella (HSP90) [
53], and in response of DDE induces in
Ruditapes decussatus (HSP90) [
54] and abamectin treatment in
Tetranychus cinnabarinus (HSP90) [
65]. In addition, the increased expression of HSP90 has been associated with pesticide exposure in
Apis mellifera [
55]. The transcriptional expression profile results also indicated that the expression of
AlHSP90 in female adults treated with chlorpyrifos and emamectin benzoate and in male adults treated with cyhalothrin were higher than that with other treatments [
14]. Earlier studies show that one
HSP70 gene was significantly upregulated in the DDT-resistant strains of
Aedes aegypti [
56], two
HSP70s in a chlorpyrifos-resistant population of
Plutella xylostella [
15], one
HSP70 in organophosphorus insecticide resistant
Chironomus yoshimatsu [
20], and two
HSP70s in chlorpyrifos-resistant
Laodelphax striatella [
57]. One
HSP70 gene has been reported to involve in cellular damage in reproductive tissues induced by cypermethrin insecticide in
Drosophila melanogaster [
58], and Colorado potato beetles produce more HSP70 in response of imidacloprid [
59]. The
AsHSP70-
2 is significantly up-regulated in AH-FR, and a number of HSP70 family of genes were significantly upregulated in populations/strains in a number of species. These findings suggest that the HSP70 families of genes might be also involved in stress response process. These results suggest that HSP70 expression be a sensitive indicator of exposure to certain insecticides and in conjunction with other biomarkers, and it may be useful for assessing exposure to environmental stressors ecosystems [
20]. However, HSP70 genes involved might be different along species, geographical populations and insecticides. It is significantly upregulated in response of induce of cypermethrin insecticide in
Caenorhabditis elegans (
HSP16) [
60]. The expression levels of
sHSP19.7 and
sHSP20.7 in cultured cells of
Mamestra brassicae were significantly up-regulated in response to high concentrations of chlorfenapyr [
61]. However, six
sHSPs were downregulated in a chlorpyrifos-resistant population of
Plutella xylostella [
15], and one sHSP was downregulated in response of imidacloprid treatment of
Sogatella furcifera [
18]. It appears that some sHSP genes are responsible for the defense against insecticide stress, but the gene response vary upon insecticide and insect species. The
AccDnaJB12 was upregulated from 1 to 1.5 h in response of lambda-cyhalothrin and paraquat treatment in
Apis cerana cerana [
62]. The
OcHsp40 mRNA levels had no significant difference observed with Cd concentrations of
Oxya chinensis [
63].
In arthropods, HSP90 proteins have been shown to be involved in tolerance and resistance to pesticides [
64,
65]. HSP90 family of genes has been known to play a role in protein folding and posttranslational regulation, in particular in steroid hormone targeting and cell death and apoptosis regulating [
66]. This study reveals that the
AsHSP90AB is significantly upregulated in the all three pyrethroid-resistant populations investigated and through 1 h to 48 h post pyrethroid exposure, and this result suggests that the
AsHSP90AB be the essential HSP gene for pyrethroid stress response. The
AsHSP90AA and
AsTRAP in HSP90 family might also be involved in pyrethroid stress response. However, three
HSP90 genes and two sHSP genes down-regulated during permethrin exposure on
Anopheles stephensi third instar larvae [
70]. The
AsHSP21.7 is significantly upregulated in CQ-FR, and a number of sHSP family of genes are significantly over-expressed over the detoxification process post pyrethroid exposure. Twelve of 14 sHSPs genes were significantly up-regulated in the fourth instar larvae of
Plutella xylostella after beta-cypermethrin exposure [
15]. These findings suggest that the sHSPs families of genes might be also associated with insecticide stress process. HSP90 and sHSP families genes expression patterns were different in different insecticides and different concentration in different insects.
HSPs are highly demanded by the insects to combat environmental stresses, and are associated with excess expressions of apoptotic genes under insecticide stress, which results in higher apoptosis [
53]. In the presence of abiotic and biotic stressors, HSPs upregulated are thought to participate in stress tolerance and promote cell survival mainly through refolding proteins and preventing their denaturation [
67,
68]. The difference in expression pattern of these HSPs may be due to a compensation effect among the
HSP genes [
53]. The insect HSPs response to insecticide stress has received increasing attention [
49,
53,
62]. In addition, inducible HSPs as playing a vital role in the insecticide resistance phenotype [
9]. Previous studies showed that blood feeding increases HSP expression in mosquito, that may suggest a role for HSPs in blood meal-induced reduction of insecticide toxicity [
4]. As molecular chaperones, HSPs may be induced by insecticides, and they also contribute to insecticide resistance [
20].