Abstract
Background
Heat tolerance is a key parameter that affects insect distribution and
abundance. Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is a
devastating pest of mulberry in the main mulberry-growing regions and
can cause tremendous losses to sericulture by directly feeding on
mulberry leaves and transmitting viruses to Bombyx mori. Moreover, G.
pyloalis shows a prominent capacity for adaptation to daily and
seasonal temperature fluctuations and can survive several hours under
high temperature. To date, the molecular mechanism underlying the
outstanding adaptability of this pest to high temperature remains
unclear.
Results
In this study, we performed comparative transcriptome analyses on G.
pyloalis exposed to 25 and 40 °C for 4 h. We obtained 34,034 unigenes
and identified 1275 and 1222 genes significantly upregulated or
downregulated, respectively, by heat stress. Data from the
transcriptome analyses indicated that some processes involved in heat
tolerance are conserved, such as high expression of heat shock protein
(HSP) genes and partial repression of metabolism progress. In addition,
vitamin digestion and absorption pathways and detoxification pathways
identified here provided new insights for the investigation of the
molecular mechanisms of heat stress tolerance. Furthermore,
transcriptome analysis indicated that immune and phosphatidylinositol
signaling system have a close relationship with heat tolerance. In
addition, the expression patterns of ten randomly selected genes, such
as HSP and cytochrome P450, were consistent with the transcriptome
results obtained through quantitative real-time PCR.
Conclusions
Comparisons among transcriptome results revealed the upregulation of
HSPs and genes involved in redox homeostasis, detoxication, and immune
progress. However, many metabolism progresses, such as
glycolysis/gluconeogenesis and fatty acid biosynthesis, were partially
repressed. The results reflected that the heat tolerance of G. pyloalis
is a fairly complicated process and related to a broad range of
physiological regulations. Our study can improve understanding on the
mechanisms of insect thermal tolerance.
Electronic supplementary material
The online version of this article (10.1186/s12864-017-4355-5) contains
supplementary material, which is available to authorized users.
Keywords: Glyphodes pyloalis, Heat stress, RNA-Seq, Heat shock protein,
Redox homeostasis, Metabolism, Immune
Background
High temperature is an environmental factor that limits the
distribution and abundance of insects. Insects are sensitive to
temperature changes [[39]1]. Over the past 30 years, global warming has
led to significant changes in the number of insect species [[40]2].
Several insect species, such as Drosophila melanogaster (D.
melanogaster) and Bombyx mori (B. mori), are sensitive to heat stress,
and the mass mortalities of these insects are often caused by high
temperature [[41]3, [42]4]. Conversely, many pests have evolved
outstanding capability to adapt to high-temperature stimulations
[[43]5]. In recent years, many studies have focused on the effect of
temperature on these insects because of their adaptability to a broad
range of temperature [[44]6].
Heat stress and acclimation of insect are currently considered a
multistep process, involving a combination of behavioral,
physiological, and cellular responses [[45]7, [46]8]. Recent studies
showed that heat tolerance in certain organisms is related to protein
folding, degradation, transport, and metabolism [[47]9, [48]10].
Moreover, current studies on flies revealed that several signal
pathways, such as stress-responsive c-Jun N-terminal kinase signaling
pathway, play an important role in adaptive metabolic response to heat
stress [[49]11]. Thus, many biologists have attempted to uncover the
mechanisms underlying gene expression regulation under heat stress.
Recently, researchers have characterized proteomic responses induced by
heat stress in insects, such as Aphids, D. melanogaster, and B. mori.
These studies displayed many protein-determining thermotolerances, such
as chromatin remodeling and translation, iron ion and cell redox
homeostasis, and carbohydrate and energy metabolism [[50]12–[51]14].
Despite these findings, minimal information is available on the gene
expression profile of pest resistant to heat stress.
Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), a specialist pest
on mulberry, is widely distributed throughout Asia. This pest can
damage sericulture not only by feeding on mulberry but also by
transmitting viruses to B. mori. G. pyloalis encounters a wide range of
daily and seasonal temperature fluctuations. On a summer day, the
average temperature in Hangzhou (120.2′ E, 30.3′ N), Zhejiang Province,
China is about 33.8 °C, and the highest temperature can reach almost
42 °C. Temperature increases during the summer lead to adverse effects
on temperate bivoltine silkworm rearing and cause economic losses. In
contract, G. pyloalis adapt to relatively high temperatures (35–40 °C)
[[52]15]. However, previous studies of G. pyloalis have focused only on
single genes, such as α- and β-glucosidase [[53]16]; the molecular
mechanisms involved in the heat responses have not been explored
through the transcriptome method, which has been widely used in profile
gene expressions in insects. Uncovering the conundrum of the heat
tolerance of G. pyloalis through RNA-Seq technique is reasonable,
because the mechanism of G. pyloalis in managing heat stress remains
unknown.
Midgut is the primary site of digestion and absorption in insects
[[54]17]. This site not only controls food storage and nutrient
absorption but also maintains water, ion, and osmotic pressure balance
[[55]18]. Moreover, the midgut of an insect is involved in immunity and
detoxication of harmful substances during digestion and absorption
[[56]19]. Thus, it is a pivotal organ for the interaction between an
insect and external environment, and it represents a key target for the
heat tolerance of an insect. However, few studies have focused on
midgut transcriptome response to high temperature [[57]20].
In the present study, G. pyloalis, an important lepidopteran pest, was
assessed for the effects of heat acclimation. The results showed that
approximately one-seventh of the transcriptome are differentially
regulated upon heat acclimation. In addition, our results revealed that
genes, which are related to heat tolerance, received pronounced effect
after heat stimulation. This result showed that heat tolerance is a
more complex process than we previously assumed. Our study can improve
understanding on the molecular mechanism of heat tolerance in insects
and provide novel targets for pest prevention and control.
Results
Heat resistance of G. pyloalis Walker
G. pyloalis and B. mori are specialist insects on mulberry.
Morphological changes in the midguts of G. pyloalis and B. mori were
monitored after heat stress. Histological staining was performed to
determine whether G. pyloalis can elicit heat resistance in its
histomorphology and structure level under high temperatures
(Fig. [58]1). After exposure to 40 °C for 4 h, the midgut of B. mori
had a weaker condition than that exposed at 25 °C (Fig. [59]1a and b).
Heat stress resulted in significant changes, and a large number of
bubble-like structures were observed adjacent to the midgut contents of
B. mori (Fig. [60]1b). However, structures observed in the midgut of G.
pyloalis were fewer than those in B. mori after heat stress (Fig.
[61]1b and d).
Fig. 1.
Fig. 1
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Morphological changes of midgut with different treatments.
Morphological changes of B. mori (A–B) and G. pyloalis (C–D) were
observed. Each (a) is a picture with low magnification, and the
blank-lined area is shown in (b) with high magnification. Minimal
bubble-like structure was observed in G. pyloalis after 4 h of heat
treatment. However, many bubble-like structures (indicated by
arrowheads) were produced in B. mori. Bars: A–a, B–a, C–a, and D–a:
10 μm; A–b, B–b, C–b, and D–a: 3.3 μm
mRNA sequencing, assembly, and functional annotation
We performed RNA-Seq to quantify the expression of genes in G. pyloalis
to elucidate the molecular basis of heat stress response. RNA was
extracted from the midgut of G. pyloalis and the transcriptome was
sequenced using Illumina short reads. Approximately 13.37 Gb data and
34,034 unigenes were obtained (Additional file [63]1). The total
length, average length, N50, and GC content of unigenes were
36,437,658 bp; 1070 bp; 2000 bp; and 43.09%, respectively
(Table [64]1). We also detected the distinction of unigene length
distributions between the two treatments and found no significant
difference (Additional file [65]2). However, there were 22,729 unigenes
expressed in the normal condition (25 °C) while 23,051 unigenes
expressed after heat stress. We then annotated our unigenes with seven
functional databases and found that 19,653 (57.75%), 13,170 (38.70%),
15,353 (45.11%), 8354 (24.55%), 15,546 (45.68%), 3878 (11.39%), and
14,410 (42.34%) unigenes can be mapped to NR, NT, UniProtKB/Swiss-Prot
(Swiss-Prot), Cluster of Orthologous Groups of proteins (COG), Kyoto
Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and
Interpro databases, respectively. After performing functional
annotation, we detected 19,609 CDS. The remaining unaligned unigenes
were analyzed using ESTScan to predict the coding regions. Another 1821
CDS were obtained after the analysis. The G. pyloalis sequences showed
42.9% matches with B. mori, followed by Danaus plexippus (27.63%),
Papilio xuthus (2.2%), and Riptortus pedestris (2.13%)
(Additional file [66]3). Furthermore, 2497 unigenes were differentially
regulated after heat shock treatment with a criterion of |fold change|
≥4.0 and FDR ≤ 0.001 in DEGs definition. Of these DEGs, 1275 genes were
upregulated, and 1222 genes were downregulated (Fig. [67]2). Ten
significantly most upregulated genes that response to heat stress are
as follows: ribonuclease, thiolase 4, peroxisomal multifunctional
enzyme type 2-like, keratin-associated protein 10–7-like, aldehyde
dehydrogenase isoform 1, PGI4-45_10 phosphoglucose isomerase, Probable
cytochrome P450 304a1, Purine nucleoside phosphorylase associated with
the functions redox homeostasis, detoxication and many metabolisms
progresses (Additional file [68]4). This analysis showed heat stress
had significant effect on the gene expression.
Table 1.
Distribution of differentially expressed unigenes and Quality metrics
of Unigenes
Sample Total Number Total Length Mean Length N50 N70 N90 GC (%)
Control 27,703 27,143,070 979 1789 987 365 43.42
Heat-shock 29,106 27,433,438 942 1693 929 353 43.34
All-Unigene 34,034 36,437,658 1070 2000 1146 397 43.09
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N50: 50% of the Total Length is contained in Unigenes greater than or
equal to this value
Fig. 2.
Fig. 2
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DEGs in the midgut after different temperature treatments. The
variability pattern was displayed by volcano plot. Y axis represents
−log10 significance. X axis represents log2-fold change. Red points
represent upregulated genes. Blue points represent downregulated genes
based on the discriminative significance values (|fold change| ≥ 4.0
and FDR ≤ 0.001) adopted in this study
GO and KEGG analyses of DEGs
We focused on the 1275 induced and 1222 repressed genes to further
understand the overall biology of transcriptional response in G.
pyloalis to heat stress. For GO analysis, we annotated DEGs into three
GO categories, namely, cell component, molecular function, and
biological process (Fig. [71]3). In the cell component category, the
terms “glutamyl-tRNA amidotransferase complex” and “membrane” were the
enriched components. “Steroid hormone receptor activity” and “lipid
transporter activity” were the top two molecular function terms. The
most enriched components of the biological process category were
“steroid hormone mediated signaling pathway”, “response to a steroid
hormone” and “cellular response to steroid hormone stimulus”.
Fig. 3.
Fig. 3
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GO enrichment analysis revealed the biological processes most
associated with detected DEGs. Based on the GO results, cellular
progress, metabolic progress, binding, and catalytic activity were the
most enriched GO terms under heat stress
KEGG is a database for biological systems that integrates genomic,
chemical, and systemic functional information [[73]21]. In this study,
1666 DEGs were mapped to 291 pathways. Among these DEGs, “metabolic
pathways” was the most dominant pathway in the midgut. In addition,
many pathways, such as “sphingolipid metabolism”, “phenylalanine
metabolism”, “RIG-I-like receptor signaling pathway” and “Toll-like
receptor signaling pathway” were also enriched (Additional file [74]5).
These results indicated an integration between metabolic and immune
responses during heat tolerance, and this integration ensures energy
balance and permits growth and defense in G. pyloalis.
Expression of pathways and genes for heat tolerance
Heat shock proteins (HSPs)
The transcriptional response of insects to heat shock includes a large
number of HSPs, implying a universal mechanism of heat tolerance in
insects [[75]22]. We focused on genes that encoded HSPs to reveal the
generality and particularity of heat tolerance between G. pyloalis and
other insects. In this study, the data revealed upregulation or
downregulation of HSPs under heat stress conditions. As shown in
Fig. [76]4, 12 HSPs, including HSP70, HSP40, and small HSPs (sHSPs),
which were distributed in different families, were identified (|fold
change| ≥ 4.0 and FDR ≤ 0.001). Among these HSPs, five genes belonged
to the HSP70 family, whereas two and five genes belonged to the HSP40
and sHSP, respectively. In terms of gene expression levels, nine HSPs
displayed greater than fourfold increased expression, indicating a
vital role in protein processing and heat tolerance in G. pyloalis.
Fig. 4.
Fig. 4
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Comparative distribution of HSP coding genes between two samples. High
temperature resulted in gene expression related to HSP70, HSP40, and
small HSP. The color scale is displayed at the upper left, which
encompasses from the lowest (green) to the highest (red) RPKM value
Antioxidant and detoxication
Apart from genes encoding HSPs, several genes, which are involved in
antioxidant and detoxication and regulated by heat stress, were
identified. These genes included genes encoding two superoxide
dismutase (Unigene21826_All and CL573.Contig1_All), a peroxidase
(CL1988.Contig2_All), and a thioredoxin (CL1904.Contig1_All). In
addition, these genes are directly linked to the generation of reactive
oxygen species (ROS) under heat stress [[78]23, [79]24] (Table [80]2).
Detoxication-related genes were also identified in our study, including
genes encoding glutathione S-transferase (CL2634.Contig1_All,
Unigene19946_All, Unigene21727_All, and CL3231.Contig2_All) and
cytochrome P450s (CYPs). 14 of the 23 cytochrome P450 (CYPs) unique
sequences were induced, and most of these sequences were grouped into
CYP3 clade (five of seven sequences), CYP6 clade (four of eight unique
sequences), and CYP9 (three sequences; Fig. [81]5). Meanwhile, the
polypeptides of the two unique sequences, namely, CL716.Contig3_All and
Unigene19157_All, were determined as unigenes for aldehyde
dehydrogenase (ALDH). They have been considered effective detoxifying
enzymes and are involved in many fundamental biochemical pathways,
including reduced cytotoxic aldehydes triggered by lipid peroxidation
[[82]25]. Therefore, antioxidant and detoxication have a close
relationship with heat tolerance and even share a similar handling
mechanism.
Table 2.
Changes in the transcriptional expression of redox homeostasis and
detoxication-related genes in response to thermal stress (40 °C) for
4 h
Gene ID Control Heat shock Regulated P value Gene description
CL1904.Contig1_All 1.66 83.63 up 0 Thioredoxin
Unigene21826_All 0.29 4.95 up 7.98E-09 [Cu-Zn] SOD
CL573.Contig1_All 13.58 57.84 up 3.75E-84 [Cu-Zn] SOD
CL1988.Contig2_All 2.37 16.18 up 6.73E-122 POD
CL2634.Contig1_All 0.01 3.84 up 1.45E-08 GSTT1
Unigene19946_All 0.01 2.78 up 4.64E-06 GSTe4
Unigene21727_All 0.17 3.37 up 1.24E-05 GSTu1
CL3231.Contig2_All 1.05 4.24 up 1.16E-13 GSTT1
CL716.Contig3_All 0.01 35.38 up 2.09E-242 ALDH
Unigene19157_All 1.04 4.91 up 2.02E-05 ALDH
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Significant FDR ≤ 0.001 for mRNA expression is represented. SOD:
Superoxide dismutase; ALDH: Aldehyde dehydrogenase
Fig. 5.
Fig. 5
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Heatmap of the expression level of different pathways. Expression level
of some genes of fructose and mannose metabolism,
glycolysis/gluconeogenesis, fatty acid biosynthesis, and fatty acid
elongation was shown in a, b, c, and d, respectively. The color scale
is shown at the upper left, which encompasses from the lowest (green)
to the highest (red) RPKM value
Metabolism and protein turnover
Through KEGG pathway enrichment analysis, we found that many pathways
related to metabolic reactions were enriched, including carbohydrate
(14 pathways), lipid (15 pathways), and amino acid metabolisms (14
pathways). The genes associated with carbohydrate metabolism were
partially repressed, such as glycolysis/gluconeogenesis (16 DEGs,
0.96%), fructose and mannose metabolism (13 DEGs, 0.78%), starch and
sucrose metabolism (18 DEGs, 1.08%), galactose metabolism (15 DEGs,
0.9%), and oxidative phosphorylation (17 DEGs, 1.02%). A repression in
the processes involved in lipid metabolism, including sphingolipid
metabolism (23 DEGs, 1.38%), steroid biosynthesis (13 DEGs, 0.78%),
fatty acid degradation (21 DEGs, 1.26%), and glycerolipid metabolism
(25 DEGs, 1.5%), was also observed. The downregulated genes in fructose
and mannose metabolism, glycolysis/gluconeogenesis, fatty acid
biosynthesis, and fatty acid elongation pathways are illustrated in
Fig. [85]6. Significant changes in metabolism-related genes involved in
heat response were consistent with a previous report describing global
gene expressions in livestock [[86]26, [87]27]. In addition, unigenes,
which related to protein turnover processes, were induced in response
to heat stress. Among these unigenes, one proteasome-related unigene
(CL2694.Contig1_All) was significantly upregulated. In addition, 34 of
57 ubiquitin system-related unigenes were specifically induced in
response to heat stress, suggesting that the stress-related autophagy
mechanisms were induced after heat stress. 8 of 10 ribosomal proteins,
which were involved in protein translation and regulation of protein
formation, were upregulated. These results suggested that protein
turnover response was induced for protection against the detrimental
effects of protein misfolding.
Fig. 6.
Fig. 6
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Heatmap of the different expression of cytochrome P450 genes under
different heat treatment temperatures. The color scale is shown at the
upper left, which encompasses from the lowest (green) to the highest
(red) RPKM value
Immune response
Insects lack adaptive immunity and rely on innate immune reactions for
their defense [[89]28]. In the present study, a large number of
immune-related genes were enriched, and most of the gene sets
demonstrated an upregulated expression after treatment at 40 °C. These
genes were mainly involved in five pathways, namely, RIG-I-like
receptor signaling pathway (8 DEGs, 0.48%), Fc gamma R-mediated
phagocytosis (44 DEGs, 2.64%), chemokine signaling pathway (38 DEGs,
2.28%), Toll-like receptor signaling pathway (12 DEGs, 0.72%), and
antigen processing and presentation (6 DEGs, 0.36%) (Fig. [90]7a). Nine
unigenes involved in the Toll-like receptor signaling pathway were
upregulated and thus may play a central role in relaying intracellular
immune signals (Fig. [91]7b). Meanwhile, some unigenes participating in
immune defense mechanisms were also detected in our data, although they
do not belong to the pathways mentioned previously. Three unigenes
(CL1248.Contig8_All, Unigene8334_All, and Unigene6267_All) were found
to encode lectin, which is directly associated with the component of
the immune system. Five genes encoding lysozyme were also determined,
and three of them (CL623.Contig1_All, Unigene6122_All, and
Unigene5037_All) had high expression levels and two (Unigene12321_All
and CL623.Contig2_All) had low expression levels. Furthermore, some
antiviral-related genes, such as genes encoding hdd1
(Unigene19527_All), trypsin-like serine proteinase
(CL1159.Contig4_All), and scavenger receptor class B (Unigene7913_All),
were induced. The abundant expression of immune response-related genes
suggested that the immune responses were activated after heat shock.
Fig. 7.
Fig. 7
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General statistics on gene regulation under different temperatures and
heatmap of the expression level of Toll-like receptor signaling
pathways. a Numbers of upregulated and downregulated genes associated
with various immune events. b Heatmap of genes related to Toll-like
receptor signaling pathways. The color scale is shown at the upper
left, which encompasses from the lowest (green) to the highest (red)
RPKM value
Stress signal transduction
Stress signal transduction is the most important aspect for high
temperature response. Many pathways involved in stress signal
transduction were enriched in our analysis, such as
Phosphatidylinositol (PI) signaling system (25 DEGs, 1.5%), Notch
signaling pathway (16 DEGs, 0.96%), TNF signaling pathway (21 DEGs,
1.26%), Phospholipase D signaling pathway (34 DEGs, 2.04%), Jak-STAT
signaling pathway (15 DEGs, 0.9%) and AMPK signaling pathway pathways
(26 DEGs, 1.56%) (Table [93]3). The threonine protein kinase
(CL962.Contig1_All) was also upregulated, and its activation was
responsible for cellular stresses, such as heat shock [[94]29]. These
findings indicate the importance of signal transduction in the thermal
tolerance of G. pyloalis.
Table 3.
Significantly enriched signal pathways after heat treatment
Signal pathway DEGs genes with pathway annotation All genes with
pathway annotation P value Pathway ID
Phosphatidylinositol signaling system 25 (1.5%) 162 (1.04%) 0.03917114
ko04070
Notch signaling pathway 16 (0.96%) 94 (0.6%) 0.04112893 ko04330
TNF signaling pathway 21 (1.26%) 134 (0.86%) 0.04798497 ko04668
Phospholipase D signaling pathway 34 (2.04%) 239 (1.54%) 0.05239752
ko04072
Jak-STAT signaling pathway 15 (0.9%) 91 (0.59%) 0.05950994 ko04630
AMPK signaling pathway 26 (1.56%) 182 (1.17%) 0.07817966 ko04152
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Validation of data through quantitative real-time PCR (qRT-PCR)
Thousands of genes showed significantly different expression levels. In
this study, we randomly selected ten genes to confirm their expression
levels through qRT-PCR. Expression patterns were validated among the
ten annotated transcripts (CL2742.Contig1_All, Unigene4872_All,
Unigene7931_All, CL658.Contig2_All, CL2227.Contig1_All,
Unigene5878_All, CL887.Contig1_All Unigene6953_All, Unigene6918_All and
CL1060.Contig3_All). Some genes related to heat tolerance showed
upregulated expression levels at 40 °C. These genes included genes
encoding CYP9G3 (CL2742.Contig1_All), CYPB5 (Unigene6953_All), HSP19.7
(Unigene4872_All), and HSP16.1 (Unigene7931_All). Meanwhile, we found
that Ribosomal protein L32 (Rpl32) and β-actin were stable. The Rpl32
was used as an internal control. The changing trend of the qRT-PCR
results was consistent with the results obtained by DEG expression
profiling (Fig. [96]8).
Fig. 8.
Fig. 8
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qRT-PCR analysis of the expression levels of ten unigenes. X axis
represents different unigenes. CL2742.Contig1_All, cytochrome P450 9G3;
Unigene4872_All, HSP 19.7; Unigene7931_All, HSP 16.1;
CL658.Contig2_All, aldehyde dehydrogenase family of seven members A1;
CL2227.Contig1_All, acetyltransferase 1; Unigene5878_All, hemolymph
juvenile hormone-binding protein; CL887.Contig1_All, aldose
1-epimerase-like; CL1060.Contig3_All, 17-beta-hydroxysteroid
dehydrogenase; Unigene6918_All, Hematopoietically-expressed homeobox
protein HHEX homolog; Unigene6953_All, cytochrome P450 B5. Y axis
represents the relative expression levels of genes. Ribosomal protein
L32 (Rpl32) was used as an internal control
Discussion
Many insects develop various mechanisms, including morphological and
physiological adaptations, to cope with high-temperature stress. In
this study, the morphological features of G. pyloalis indicated heat
tolerance superior to that of B. mori and thus can contribute to its
effective survival under high temperature. The adaptation strategies of
G. pyloalis to heat stress may also be related to the expression of
some unique genes. We conducted high-resolution analysis on
transcriptome dynamics to identify the genes associated with excellent
heat tolerance of G. pyloalis. In total, we obtained 2497 DEGs (1275
upregulated and 1222 downregulated) under heat stress and analyzed a
large suite of biomarkers related to antioxidant, detoxication,
metabolism, protein turnover, immune, and stress signal transduction.
The GO analysis results showed that most of the DEGs after heat
treatment were significantly represented in “response to stimulus”,
“regulation of response to stimulus”, “response to steroid hormone” and
“cellular response to steroid hormone stimulus”. This finding suggested
that most of the DEGs at the early heat stress stages were mainly
associated with stress response and similar experiment results were
observed in the plant response to heat stress. Many genes associated
with “membrane”, which revealed that most of the cells required being
repaired [[98]30]. Moreover, many highly represented pathways, such as
sphingolipid metabolism, phenylalanine metabolism, metabolic pathways,
and PI signaling system, were enriched when exposed to heat stress,
indicating the importance of these pathways in establishing thermal
tolerance [[99]31].
In insects, HSPs act as molecular chaperones and participate in various
cellular processes, such as protein folding, proteolytic pathway, or
stabilizing denatured proteins [[100]32–[101]34]. HSPs can be divided
into several families, namely, HSP40, HSP60, HSP70, HSP90 and sHSPs
according to their molecular weights [[102]35]. Twelve HSPs distributed
in the HSP70, HSP40, and sHSPs were observed to respond to heat
treatment during the transcriptome analysis of G. pyloalis. The
preferential induction of HSPs upon heat stress has been widely
reported in numerous studies. In D. melanogaster, the accumulation of a
major HSP, namely, HSP70, affects inducible thermal tolerance in larvae
and pupae [[103]36]. In Ceratitis capitata, expression of Hsp70 and
thermal tolerance were assayed at a range of temperatures in several
stages of its development. In the current study, the induced expression
of five HSP70s in G. pyloalis possibly facilitated the refolding of
damaged proteins and prevented protein aggregation under heat stress.
In addition, five small HSPs of G. pyloalis were upregulated by heat
stress and likely cooperated with co-chaperones for the prevention of
cellular protein aggregation [[104]37]. sHSPs are abundant in nearly
all organisms and act as a response machine of organisms to some
extreme stresses [[105]38]. These results suggested that the induction
of HSP was an evolutionarily conserved mechanism, and the coordinate
upregulation of the molecular chaperones, including HSP70 and sHSP, was
an important factor in acquiring thermal tolerance and can render the
midgut cells of G. pyloalis resistant to heat stress.
Exposure to heat stress can result in ROS production, which can
directly lead to a variety of toxic effects, including protein
dysfunction, lipid peroxidation, and oxidative stress. In relation to
signal transduction of ROS, we detected upregulation of several genes
involved in vitamin digestion and absorption pathways. Many studies
have demonstrated the importance of vitamins in oxidative stress
responses. Sies [[106]39] mentioned that vitamins C and E can react
with free radicals and singlet molecular oxygen, which is the basis for
their functions as antioxidants. Notablely, the induction of vitamin
digestion and absorption pathway may suggest a specific function in the
adaption of insect to high temperature. To the best of our knowledge,
this result is some of the first evidence indicating the existence of
vitamin digestion and absorption pathway after heat stress in insects.
In Table [107]2, several antioxidant-related genes, including
superoxide dismutase (SOD), peroxidase (POD), and thioredoxin were
strongly upregulated after 40 °C treatment. Recent proteomic analysis
showed that SOD overexpression upregulated ROS scavenging in rice
grains under heat stress. The SOD knockout rice was susceptible to heat
stress, which suggested that SOD played an important role in adaptation
to heat stress [[108]40]. POD, as a regulator of ROS scavenger, is a
hematin-containing oxidase that can catalyze the oxidation of reduced
compounds [[109]41]. Meanwhile, thioredoxin can be potentially
responsive to ROS and can catalyze electron transport to ribonucleotide
reductase and other reductive enzymes and transcription factors
[[110]42]. The coordinate upregulation of the three antioxidant-related
genes after 40 °C treatment suggested that they were regulators of ROS
scavenger in G. pyloalis and were potentially generate ROS in response
to heat stress. Similar antioxidant-related genes were also observed in
Bactrocera dorsalis and Bemisia tabaci exposed to high temperatures
[[111]43, [112]44]. Our data indicated that a link between oxidative
stress and vitamin digestion and absorption was present in heat
tolerance of G. pyloalis.
In addition to ROS generation, heat stress can promote toxic substance
accumulation. In this study, we found that many detoxication-related
genes, such as genes coding CYP, glutathione-S-transferase (GST), and
ALDH, showed upregulated mRNA expression patterns after 4 h of heat
stress. In most species, CYP effectively catalyzes lipid peroxidation.
Meanwhile, the induction of a state of oxidative stress plays a central
role in CYP-dependent cytotoxicity [[113]45]. Apart from eliminating
drugs, cytochrome P450s catalyze a broad range of oxidative processes
involved in the metabolism of fatty acids and biosynthesis of sterols.
Therefore, the induction of 14 CYPs in G. pyloalis may be an essential
step in heat tolerance. In addition, we found 3 of 5 CYPs in B. mori
were significantly induced at 40 °C, suggesting several CYPs played a
conserved role in response to heat stress (Additional file [114]6).
However, further experiments are necessary to determine the exact
function of these proteins. Meanwhile, GSTs comprise a family of
isozymes and can convert toxic substances into reduced toxic
metabolites through chemical reactions involving the conjugation of
glutathione [[115]46]. They protect macromolecules from reactive
electrophiles, such as environmental carcinogens, ROS products, and
chemotherapeutic agents [[116]46]. Our observation suggested that the
upregulation of genes encoding GST was related to increased
intracellular oxidative stress and cellular toxic substance metabolism
[[117]47]. Detoxification pathways identified here provided new
insights for the investigation of the molecular mechanisms of heat
stress tolerance. ALDHs can eliminate toxic aldehydes by catalyzing
their oxidation to nonreactive acids, the enzyme activities of which is
mainly implicated in the metabolism of endogenous lipid peroxidation
products [[118]48]. Two mRNAs for ALDH were significantly upregulated
after heat shock, suggesting they contribute to the improved heat
tolerance through metabolic pathway.
Insects have developed a range of strategies, such as metabolism
adaptation, to manage heat stress. Our results indicated that the
metabolism of G. pyloalis in high temperature was weak. A similar
reduction was observed in livestock [[119]27]. The suppression of
metabolism progress may be a conservative progress and can reflect
cellular homeostasis or an energy-saving mechanism to manage heat
stress, because ATP generation is a highly-regulated process involving
three metabolism pathways, namely, glycolysis, tricarboxylic acid
cycle, and oxidative phosphorylation. In addition, the ROS generated by
heat stress can chemically modify proteins and alter their biological
functions. Therefore, the removal of damaged proteins is vital for the
maintenance of cellular homeostasis. Numerous studies have revealed
that the ubiquitin-proteasome system plays a vital role in recognizing
and degrading damaged proteins [[120]49, [121]50]. In the present
study, 34 ubiquitin-related unigenes were induced by heat stress. These
results revealed that the upregulation of protein degradation progress
was involved in stress response and adaptation.
For insects, the innate immune system is the major effector response
system [[122]51]. In our results, the activities of the RIG-I-like and
Toll-like receptor signaling pathways were modulated by heat stress.
The role of transcriptional activation of the Toll-like receptor in
heat shock response has been reported [[123]52–[124]54]. Several
reports have described some endogenous ligands, such as HSP60 and
HSP90, that activate TLR4 to regulate innate immune responses and are
also induced by high temperature [[125]55]. Frequent confrontation of
the immune system with HSPs can potentiate immunity. The upregulation
of lectin, lysozyme, Hdd1, trypsin-like serine proteinase, and
scavenger receptor class B under high temperatures further suggested
that heat stress facilitated the components of immune defense.
The sensing and transduction of intracellular stress signal are
critical for the adaptation and survival of insects under heat stress.
In plants, the involvement of calcium and calcium-activated calmodulin
in heat shock signal transduction was already investigated [[126]56].
However, the mechanisms that enable insects to sense heat signal and
trigger intracellular responses remain unclear. The PI signaling system
is vital during the progress of plant development and growth and
associated with cellular responses to environmental stress [[127]57].
Meanwhile, Notch signaling pathway participates in physiological and
stress erythropoiesis [[128]58]. In this study, the preferential
induction of PI signaling system and Notch signaling pathway under heat
stress was uncovered and the results suggested that gene expression in
the PI signaling system was related to the heat tolerance mechanism of
G. pyloalis. These results suggested that the PI signaling system and
Notch signaling pathway rapidly and effectively regulated downstream
signaling and gene expression in G. pyloalis in response to
high-temperature stress.
Conclusions
In the present study, we presented the first comprehensive evaluation
of the midgut transcriptome of G. pyloalis by using Illumina sequencing
technology and conducted a comparative expression analysis after heat
treatment. A total of 34,034 unigenes, which might provide a major
genomic resource for investigating the midgut of G. pyloalis, were
obtained. Most of these genes had an annotation with matches in the
seven functional databases. Furthermore, 2497 DEGs were identified in
the midgut after heat stress. The results of the GO and KEGG pathway
enrichment analyses indicated that the DEGs related to metabolism,
immunity, and signal transduction were enriched after heat stress. In
addition, the transcriptome results revealed a number of genes that are
potentially relevant to HSPs, antioxidant, and detoxication in G.
pyloalis. These genes could be major targets for thermal tolerance.
Many of which can be understood with deep functional studies.
Nonetheless, our study provides insight into the heat tolerance of
insects.
Methods
Biological materials and tissue collection
G. pyloalis larvae were collected from the mulberry fields of Zhejiang
University, Hangzhou (120.2′ E, 30.3′ N), Zhejiang Province, China. B.
mori (N4) larvae were reared as described previously [[129]59]. The
method of high temperature treatments or thermal exposure was modified
from methods of Xiao et al. (2016), Li et al. (2014) and Moallem et al.
(2017) [[130]20, [131]60, [132]61]. G. pyloalis and B. mori treated at
25 °C served as controls. For treatments, larvae on day 3 of the fifth
instar were selected and separated into two groups with 13 larvae each.
Larvae were reared in different chambers at 25 °C and 40 °C for 4 h,
respectively. They were supplied hourly with fresh mulberry leaves.
Three biological replications were performed with each treatment. Four
males and four females were selected for each treatment. Clean midgut
samples were collected from eight (four males and four females) G.
pyloalis and B. mori, respectively. The sample were freezed at −80 °C
immediately.
Histological staining
After being exposed to different temperature treatments as described
above, the samples of larval G. pyloalis and B. mori were collected for
hematoxylin–eosin (HE) staining. The midgut of G. pyloalis was
dissected and fixed in formalin-acetic acid-alcohol (FAA) liquid for
12 h. The samples were dehydrated through a series of graded ethanol
baths (70%–100%) to displace water, and then infiltrated with paraffin
wax, a finally cut into sections (5 μm). The obtained tissue sections
were stained with 2% Mayer’s hematoxylin and 1% eosin [[133]62]. Slides
were observed and image can be examined directly in the microscope
(Nikon Nis-Elements).
De novo assembly and gene annotation
Total RNA was extracted using RNAiso plus reagent (TaKaRa) according to
the manufacturer’s protocol. The concentration and quality of total RNA
was quantified using NanoDrop Spectrophotometer (Thermo Fisher
Scientific). For each group, total RNA from three replicates was pooled
together in equal quantities. Approximately 6 μg of RNA representing
each group were used for illumine sequence. After the high-throughput
sequencing (Illumina HiSeq 4000) of midgut samples, we used Trinity to
filter raw reads by discarding low-quality, adaptor-polluted, and
high-content unknown base reads and to generate 150 bp paired-end read
lengths. The clean reads were assembled into primary transcripts, and
overlapping transcripts were assembled into large contigs after
removing redundant transcripts. The final unigenes were obtained
through gene family clustering with Tgicl [[134]63]. Gene function was
annotated based on the following databases: NCBI non-redundant protein
sequences (NR); InterPro member database; Clusters of Orthologous
Groups of proteins (KOG/COG); Swiss-Prot (a manually annotated and
reviewed protein sequence database); Kyoto Encyclopedia of Genes and
Genomes (KEGG); and Gene Ontology (GO). The unigenes that were not
aligned to any database mentioned above were predicted by ESTScan
([135]http://sourceforge.net/projects/estscan).
Gene expression quantification
Bowtie2 software
([136]http://bowtie-bio.sourceforge.net/Bowtie2/index.shtml) was used
to map all the clean reads to the unigene library. Gene expression
level was then calculated using the RSEM software
([137]http://deweylab.biostat.wisc.edu/RSEM). The fragments per
kilobase of transcript per million (FPKM) mapped reads can be used to
quantify the unigene expression level. False discovery rate (FDR) was
used to determine the threshold P-value in multiple tests. Furthermore,
|fold change| ≥ 4.0 and FDR ≤ 0.001 were used as the parameters for
determining significant differences in gene expression. The DEGs were
then used for GO and KEGG enrichment analyses. The P-value calculating
formula in the hypergeometric test is:
[MATH: P=1−∑i=0
m−1MiN−Mn−iNn :MATH]
In this equation, N and n indicate the number of genes with GO/KEGG
annotations and the number of DEGs in N, respectively. The variables M
and m represent the numbers of genes and DEGs, respectively, in each
GO/KEGG term.
qRT-PCR validation
Ten annotated unigenes and five B. mori CYPs were randomly selected to
quantify by qRT-PCR and evaluate data level. RNAiso plus reagent
(TaKaRa) was used to extract total RNAs from three biological
replications, and Primer Premier 5.0 was used to perform the primer
design. qRT-PCR primers are displayed in Additional file [138]7 and
Additional file [139]8. The reverse transcription system was used to
synthesize cDNA as described previously [[140]64–[141]66]. qRT-PCR was
performed on a LightCycler® 480 real-time PCR system (Roche). The
reaction system consisted of 2 μl of cDNA, 10 μl of SYBR Green qPCR
master mix (TaKaRa), 0.5 μl of each of the primers, and sterile water
made up to 20 μl. The ΔΔCt method was used to analyze the relative
differences in transcript levels [[142]67]. Ribosomal protein L32
(Rpl32) was used for internal control, and the experiment was performed
on three biological replicates.
Additional files
[143]Additional file 1:^ (18KB, docx)
Distribution of base content and quality. (DOCX 18 kb)
[144]Additional file 2:^ (1.8MB, tif)
Unigene length distribution in G. pyloalis midgut with different
treatments. X axis represents the length of unigenes. Y axis represents
the number of unigenes. A: “Control” unigene length distribution. B:
“Heat shock” unigene length distribution. (TIFF 1807 kb)
[145]Additional file 3:^ (5MB, tif)
Distribution of annotated species. (TIFF 5077 kb)
[146]Additional file 4:^ (570.5KB, xlsx)
List of the significantly up- and down-regulated genes. (XLSX 570 kb)
[147]Additional file 5:^ (1.4MB, tif)
Pathway functional enrichment of DEGs. Based on the KEGG results, the
metabolic pathway was the most enriched KEGG pathway under heat stress.
(TIFF 1458 kb)
[148]Additional file 6:^ (61.9KB, tif)
qRT-PCR analysis of the expression levels of five randomly selected B.
mori genes. (TIFF 61 kb)
[149]Additional file 7:^ (15.6KB, docx)
Primers used for B. mori CYPs. (DOCX 15 kb)
[150]Additional file 8:^ (19.7KB, docx)
Primers used for G. pyloalis. (DOCX 19 kb)
Acknowledgements