Abstract
Background
Plant allelochemicals act as toxins, inhibitors of digestion, and
deterrents that affect the fecundity of insects. These compounds have
attracted significant research attention in recent decades, and much is
known about the effects of these xenobiotic plant secondary metabolites
on insect development. To date, although ecological interactions
between xenobiotic plant secondary chemicals that retard insect growth
have been observed in many species, it remains unclear how particular
allelochemicals influence insect development in a life stage-dependent
manner.
Results
We found that 2-tridecanone can affect insect development; this effect
appears similar to the symptoms induced by the physiological imbalance
between juvenile and molting hormones in cotton bollworm. We later
detected that a decrease in the concentration of 20-hydroxyecdysone
occurred alongside the observed symptoms. We next identified the
transcriptome of Helicoverpa armigera and eightdigital gene expression
libraries for shading light on how 2-tridecanone retarded the
development of cotton bollworm. The expression of CYP314A1, CYP315A1,
CYP18A1, CYP307A1, and CYP306A1 (unigenes 16487, 15409, 40026, 41217,
35643, 16953, 8199, 13311, and 13036) were found to be induced by
2-tridecanone; these are known to be related to the biosynthesis or
metabolism of 20-hydroxyecdysone. Expression analysis and RNA
interference studies established that the retardant effect of
2-tridecanone on the development of cotton bollworm is mediated by P450
genes.
Conclusions
The candidate P450 gene approach described and exploited here is useful
for identifying potential causal genes for the influence of plant
allelochemicals on insect development.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-016-3277-y)
contains supplementary material, which is available to authorized
users.
Keywords: Helicoverpa armigera, Cytochrome P450, Plant allelochemicals,
Insect development, Transcriptome
Background
Co-evolution strategies are a common phenomenon in herbivorous
insect-plant interactions [[33]1, [34]2]. Insects employ various
strategies to increase their performance and fitness, while plants also
develop efficient strategies to defend against particular insects
[[35]3]. Host plants can produce various allelochemicals to defend
against the damage of herbivorous insects [[36]4–[37]6]. Plant
allelochemicals possess beneficial or detrimental effects on the target
pests; allelochemicals with negative allelopathic effects are an
important part of plant defense against herbivory [[38]7, [39]8]. These
compounds can influence the growth, survival, and reproduction of other
organisms. For example, the phenolic aldehyde gossypol can retard the
developmental of the cotton bollworm, Helicoverpa armigera (H.
armigera) [[40]6]. The resistance of wild tomato (Lycopersicon hirsutum
f. glabratum) to several arthropods has been shown to be related to the
presence of high contencentrations of 2-tridecanone (2-TD) in leaves
[[41]5, [42]9]. 2-TD can stimulate ecdysone 20-monooxygenase activity
in Spodoptera frugiperda [[43]10]. 2-TD in wild tomato can defense
Manduca sexta and also plays an important role in the plant resistance
to Leptinotarsa decemlineate. 2-TD is also known to induce an enhanced
level of tolerance to the carbamate insecticide carbaryl in Heliothis
zea [[44]9]. Up to now, although the phenomena of plant allelochemicals
retarding the development of insects has been found in many species,
details remain unclear about the life-stage dependent manner and
pathway of allelochemicals to influence the insect development.
Insect pests have evolved various strategies with which to respond to
allelochemicals from host plants [[45]6]. Cytochrome P450 enzymes are a
major source of adaptation to plant defense mechanisms in insects
[[46]11, [47]12]. Plant allelochemicals are known to induce the
expression of various cytochrome P450 genes in insects. The cotton
bollworm is one of the most polyphagous and cosmopolitan pest species
in the world. Many studies have demonstrated that the expression levels
of cotton bollworm cytochrome P450 genes can be induced by plant
allelochemicals [[48]12–[49]14]. The expression of CYP9A subfamily and
CYP6AE14 genes can be induced by gossypol [[50]12, [51]15]. The
expression of CYP6AE, CYP9A, and CYP6B subfamily transcripts can be
induced by xanthotoxin [[52]16, [53]17]. 2-TD can significantly induce
the expression of CYP6B6 [[54]14].
High levels of P450 gene expression are typically thought to coincide
with an increased ability to metabolize exogenous compounds. Many
studies have focused on detoxification enzymes that can metabolize
plant natural products [[55]18–[56]21]. Cytochrome P450 enzymes not
only act as xenobiotic detoxification agents, but also play pivotal
roles in various physiological processes including the biosynthesis and
metabolism of 20-hydroxyecdysone (20E) and juvenile hormone (JH), which
are the major modulators of developmental processes that result in
molting and metamorphosis [[57]22].
We found that 2-TD can affect insect development, and this type of
effect was similar to the symptoms induced by the physiological
imbalance between juvenile and molting hormones in cotton bollworm. We
later discovered that a decrease in the concentration of 20E occurred
alongside the observed symptoms. We then profiled the transcriptome of
Helicoverpa armigera and used eight digital gene expression (DGE)
libraries for shading light on how 2-TD retarded the development of
cotton bollworm. These results should help to deepen our understanding
of how plant allelochemicals influence insect development.
Results
Effect of 2-TD on the development of H. armigera
6^th instar larvae were fed an artificial diet containing 2-TD
(10 mg/g, W:W) to evaluate the effects of 2-TD on development. 10 mg/g
2-TD is a sublethal dosage that was selected based on our studies
(Additional file [58]1). The pupation time of the treated group
(8.4d ± 2.01) was obviously longer than that of the control
(6.1d ± 1.67) (Table [59]1). The larval weight on the 1^st day of
treatment with 2-TD decreased significantly compared to the control
group, and the pupae weight at treatment day 10 was significantly lower
than that of the control group (Table [60]1). The pupation rate and the
adult emergence rate was significantly lower in the 2-TD treated group
as compared to the untreated group (Table [61]1). The 20E titer in
larvae was measured at 24 h. The 20E titer after treatment was
suppressed to a level that was only 57% of the control level
(Table [62]1). The adult emergence rate in the treatment group was
significantly lower than that of the control (Table [63]1).
Table 1.
Effect of 2-TD on larval development
Treatment group Pupation time (d) Pupae weight (g) *Weight gain rate
(%) **20E titers gain rate after treated for 24 h (%) Pupation rate
Adult emergence
Control 6.1d ± 1.67^b 0.243 ± 0.056^a 4.112 ± 1.578^a −0.75% ± 0.151 ^a
83.33% 68.0%
2-TD 8.4d ± 2.01^a 0.192 ± 0.049^b 1.980 ± 1.619^b −32.58% ± 0.21 ^b
45% 22.2%
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*Weight gain rate, the larval weight gain rate 24 h post-treatment
**20E titer gain rate = [(the concentration of 20E after 2-TD
treatment– the concentration of 20E before 2-TD treatment) / the
concentration of 20E before 2-TD treatment] × 100%. Each column sharing
the same superscript letter (a or b) for both treatment groups was not
significantly different at P > 0.05
Sequencing and sequence assembly of the H. armigera transcriptome
The effects of 2-TD on larval development are likely complicated and
may involve several pathways and related genes. We constructed a
transcriptome library of H. armigera with Illumina sequencing
technology. The library contained 43,756,144 clean reads
(101 bp + 101 bp) with an accumulated length of 4,419,370,544
nucleotides (nt) (Q20 = 98.07%), de novo assembly generated a total of
93,896 non-redundant transcripts, with a median N50 length of 597 bp,
and finally the total number of assembled unigenes is 42,463, the
median N50 length of these unigenes is 695 bp (Additional file [65]2).
All unigenes were compared with the nonredundant (nr) NCBI protein
database for functional annotation using BLASTX software with an
e-value cutoff of 1e^−5. A total of 19,382 (45.6% of all unigenes)
distinct sequences matched known genes, the species distribution of
unigene BLASTX matches against the nr protein database show in
Additional file [66]3. For further quantitative assessment of the
assembly and annotation completeness, we applied the software tool
BUSCO (Benchmarking Universal Single-Copy Orthologs), which is based on
evolutionarily informed expectations of gene content, with default
settings. Out of 2675 single copy orthologs for arthropods our assembly
is 27% complete (663 Complete and single-copy BUSCOs, 49 Complete and
duplicated BUSCOs ), while 19% of contigs are fragmented (510 BUSCOs)
and 54% are missing (1453 BUSCOs), the BUSCO analysis results show in
Additional file [67]4. Assignments of clusters of orthologous groups
(COG) were used to predict and classify the possible functions of the
unigenes (Additional file [68]5. Among the 25 COG categories, the
cluster for ‘General function prediction’ represented the largest group
(1421, 19.1%) followed by ‘Translation, ribosomal structure and
biogenesis’ (724, 9.76%) and ‘Replication, recombination and repair’
(683, 9.21%) (Additional file [69]5). Common gene ontology (GO)
annotation was used to classify the putative functions of the H.
armigera unigenes (Additional file [70]6). Pathway analysis of the
unigenes was conducted using the Kyoto Encyclopedia of Genes and
Genomes (KEGG) annotation system.
P450 sequence alignment and phylogenetic analyses
In our study, 153 putative P450 unigene sequences were annotated by
searching the nr NCBI protein database using BLASTX. 94 long P450
unigene sequences within 153 putative P450 unigene sequences and 47
full-length P450 sequences from B. mori were used to construct a
phylogenetic tree (Additional file [71]7). The annotated P450 unigenes
in the tree belonged to the CYP2, CYP3 (including CYP6 and CYP9), CYP4,
and the mitochondrial CYP (mito.CYP) clans [[72]23]. Among the 153
predicted P450 unigenes in H. armigera, 11, 11, 80, and 48 unigenes
were classified into the mito.CYP, CYP2, CYP3, and CYP4 clans,
respectively.
Comparison of P450 gene expression profiles at different developmental stages
In order to determine the P450 genes involved with the effects of 2-TD
on larval development, eight DGE libraries were constructed to identify
unigene expression profiles for shading light on how 2-tridecanone
inhibited retarded the development of cotton bollworm. After removing
low-quality reads, each library generated approximately eight million
clean reads. Among these clean reads, 5.8–7.3 million reads were mapped
to unigenes in transcriptome libraries. The Q20 ranged from 85.57 to
97.56% (Additional file [73]8). qPCR was used to confirm 34 unigenes
expression profile results. Compared with DGE libraries results, the
accuracy of these P450 unigenes expression detected by qPCR is up to
94% (Additional file [74]9, Additional file [75]10).
Figure [76]1a and b show the summed expression of P450 unigenes and the
numbers of P450 unigenes (including those of the CYP2, CYP3, CYP4 and
moti. CYP clans) in the different development stages. The total
expression amount and the numbers of P450 unigenes was higher in larvae
than in the egg and adult samples, with the highest total expression
level of P450s occurring in 3^rd instar larvae. During the
transformation from eggs to larvae, the percentage of expressed CYP4
clan unigenes sharply increased from 6.4 to 71.8%, while the CYP3,
CYP2, and moti. CYP clan unigenes decreased significantly from 82.4,
2.4, and 8.8% to 24.2, 1.0, and 3.0% respectively. During the
transformation from larvae to pupae, the percentage of expressed
annotated CYP4, CYP2 and moti. CYP clan unigenes increased dramatically
from 47.3, 1.1, and 3.2% to 57, 3.7, and 9.5%, respectively; the
percentage of expressed CYP3 clan unigenes decreased from 48.4 to 29.8%
during this transition (Fig. [77]2) During the transformation from
pupae to adults, the percentage of expressed annotated CYP4, CYP2 and
moti. CYP clan unigenes decreased dramatically from 57, 3.7, and 9.5%
to 24, 1.9, and 5.1%, respectively, while the percentage of expressed
CYP3 clan unigenes increased from 29.8 to 69.1% (Fig. [78]2). All the
expression of the 153 P450 unigenes in different DGE library were
listed in Additional file [79]11.
Fig. 1.
Fig. 1
[80]Open in a new tab
The DGE library data for the expression of P450 unigenes in different
development stages. a Sum expression of P450s in larvae at different
developmental stages. b The numbers of P450 unigenes at different
development stages. 1: 1^st instar larvae; 3: 3^rd instar larvae; 6:
6^th instar larvae
Fig. 2.
Fig. 2
[81]Open in a new tab
Percentage of each CYP clan expressed in different developmental
stages. RPKM < 0.1 was used as the criterion to judge the unigenes were
not expressed during a given developmental stage. The adults samples
consisted of equal numbers of female and male individuals
Among the 153 annotated P450 unigene sequences, the expression of 150
unigenes (98.1%) were detected in at least one DGE library. Three P450
unigenes were not detected in any DGE library; either these were not
expressed in the particular life stages that we examined or these were
possibly pseudogenes. Among the expressed P450 unigenes, 33% unigenes
(55 sequences) were expressed in all life stages (Fig. [82]3). Some
P450 unigenes were specifically expressed at a particular developmental
stage: eight P450 genes were specifically expressed in larvae, all of
these belonged to the CYP3 or CYP4 clans. We found one specific P450
unigene that was only expressed in eggs. Likewise, a single unigene was
expressed specifically in the pupae stage. Three P450 unigenes were
expressed only in females. All these specifically expressed P450
unigenes belonged to the CYP3 or CYP4 clans (Table [83]2).
Fig. 3.
Fig. 3
[84]Open in a new tab
Numbers of P450 unigenes expressed in a developmental stage-specific
manner. RPKM < 0.1 was used as the criterion to judge the unigenes were
not expressed in a given developmental stage. E: eggs; L: larvae; P:
pupae; M: males; F: females
Table 2.
P450 genes expressed at specific developmental stages
Family Stage Gene number Clan Homologous genes % similarity, organisms
RPKM^b
P450s^a Egg 32583 CYP3 6AX1.6B3 82%, N. vitripennis 0.72
Larval 13679 CYP3 6B2, 6B6, 6B7 89%, H. armigera 0.36
22278 CYP3 321B1 Spodoptera litura 1.58
15388 CYP 3 6AE14 100%, H. armigera 19.16
19180 CYP 3 9A18 74%, H. armigera 7.63
12812 CYP4 4 L6 85%, B. Mori 1.11
10466 CYP 4 340AA1 75%, S. littoralis 29.13
10601 CYP 4 341A2 88%, B. Mori 3.84
14820 CYP 4 341B1 88%, B. mori 3.57
Pupa 28770 CYP 3 9A14 83%, H. zea 4.72
Female 29309 CYP 3 6CV2 Plutella xylostella 17.73
29201 CYP 3 6B1 Papilio polyxenes 16.33
22936 CYP 4 402C1 78 %, Bemisia tabaci 14.07
[85]Open in a new tab
^aThe cytochrome P450 clan schema used here follows the system proposed
by Feyereisen et al. (2006)
^bRPKM < 0.1 was used as the criterion to judge the unigenes were not
expressed during a given developmental stage
Effect of 2-TD on the expression of P450 genes
Figure [86]4 shows the total expression levels of P450 unigenes and the
numbers of P450 unigenes observed in the DGE libraries of 6^th instar
larvae treated by 2-TD for 24 h compared with the control. The total
expression levels of P450 unigenes in the larvae treated by 2-TD was
2.6 fold higher than the control group (Fig. [87]4a). There were more
P450 unigenes expressed in the 2-TD-treated group than in the control
larvae; the additional two P450 unigenes belonged to the CYP3 clan
(Fig. [88]4b). The percentages of expressed CYP3 and CYP2 clan unigenes
were obviously higher in the 2-TD-treated group (64.2 and 2.8%) than in
the control (48.4 and 1.1%, respectively), while the percentages of
CYP4 and mito.CYP clan unigenes were higher in the control (47.3 and
3.2%) than in the 2-TD-treated group (31.3 and 1.7%) (Fig. [89]4c and
[90]d).
Fig. 4.
Fig. 4
[91]Open in a new tab
2-TD affects the expression of P450. a The sum of the expression of
P450 unigenes in 2-TD treated and untreated groups. b Numbers of P450
unigenes in 2-TD treated and untreated groups. c The percentage of each
CYP clan expressed in 6^th larvae. d The percentage of each CYP clan
expressed in 6^th larvae treated with 2-TD. Control: 6^th larvae
un-treated with 2-TD; 2-tridecanone: 6^th larvae treated with 2-TD for
24 h
An absolute value of Log[2] Ratio ≥ 1 were used as thresholds to judge
the differences of gene expression levels. 49 annotated P450 unigenes
were up-regulated, and 22 P450 unigenes were down-regulated in larva
treated by 2-TD, as compared to the control group. 7 of these annotated
P450 unigenes belonged to the CYP2 clan and 7 of these P450 unigenes
were classified into the mito.CYP clan; none of these 14 unigenes were
uniquely expressed in a particular developmental stage. However, 15
unigenes from the CYP3 and CYP4 clans were specifically expressed in a
particular stage of H. armigera development (Table [92]3).
Table 3.
Up- and down-regulated P450 genes in cotton bollworm in response to
2-TD treatment
Family Classification Homologous genes % similarity, organisms
Transcripts number Log[2]Ratio^a Expression stage 2-TD induced
P450s^b Mitochondrial 314A1 86%, B. mori 16487 2.55 All, 3 > 1 > 6 Up
314A1 83%, S. littoralis 15409 −2.29 All, 3 > 1 > 6 Down
333B3 69%, S. littoralis 12317 −1.12 All, 1 > 6 > 3 Down
333A3 75%, S. littoralis 25319 1.44 All, 3 > 1 > 6 Up
315A1 73%, S. littoralis 40026 −1.07 All, 3 > 1 > 6 Down
Clan 2 18A1 72%, S. littoralis 41217 4.12 All, 6 = 3 = 1 Up
18A1 81%, S. littoralis 35643 5.06 3 > 1, pupa, female Up
18A1 76%, S. littoralis 16953 4.73 3 > 1 Up
15C 71%, B. mori 14800 1.53 1, female Up
306A1 82%, S. littoralis 13036 −3.32 All, 3 > 1 > 6 Down
307A1 99%, H. armigera 8199 −4.55 3 > 6 > 1, pupa Down
307A1 99%, H. armigera 13311 −4.92 Egg, 3 > 6, pupa Down
Clan 3 (include CYP6 and CYP9) 6B2,6B6,6B7 96%, H. armigera 18705 3.08
Egg, female Up
6B2, 6B6, 6B7 94%, H. armigera 40306 2.23 All, 3 > 1 > 6 Up
321A2 98%, H. zea 17923 7.79 No expression Up
6B2 96%, H. armigera 2844 3.03 All, 6 > 3 > 1 Up
6B2, 6B6, 6B7 97%, H. armigera 41374 4.08 3 > 1 > 6, male Up
6B31 77%, S. littoralis 2950 1.67 Male Up
6AB31 74%, S. littoralis 5881 2.39 1 > 3 > 6, pupa, Adults Up
6B6 99%, H. armigera 39825 2.09 6 > 3 > 1 Up
6B2, 6B6, 6B7 89%, H. armigera 13679 4.38 3 Up
6AN4 72%, S. littoralis 12093 1.96 Egg,female,1 > 3 > 6 Up
6AN4 78%, S. littoralis 1833 1.07 1 < 3 < 6 Up
6AB4 74%, B. mori 42286 5.24 Male Up
6AB14 68%, S. littoralis 15424 −1.39 3 > 6 Down
6AE12 72%, H. armigera 41540 2.64 Female, male Up
6AE12 90%, H. armigera 3564 2.35 3 > 6 > 1, female Up
6AE14 99%, H. armigera 9094 −1.55 1 > 3 > 6 Down
6AE14 78%, H. armigera 4567 1.68 1 > 3 > 6, female, male Up
6AE14 100%, H. armigera 15388 1.97 All Up
6AE14 92%, H. armigera 6041 1.16 3 > 6 > 1 Up
6AE47 75%, S. littoralis 39097 2.37 6 > 1 > 3, female Up
6AE14 72%, H. armigera 30146 3.25 egg, 3 > 1 > 6, female Up
324A6 72%, S. littoralis 40289 2.84 3, pupa Up
337B3 99%, H. armigera 2773 2.64 Egg, female Up
337B3v1 85%, H. armigera 12899 1.29 All, 3 > 6 > 1 Up
35D18 97%, H. armigera 17285 −3.63 6 > 3 > 1 Down
35D18 91%, H. armigera 11744 −4.67 6 > 3 > 1 Down
324A1 77%, S. littoralis 36658 2.03 Female, male Up
321A1 96%, H. zea 38041 4.82 Egg, 1 < 3 < 6 Up
321A1 95%, H. zea 6465 6.56 3 > 6 > 1, male Up
321A2 91%, H. zea 9454 2.62 Egg, female Up
321A2 89%, H. zea 2372 1.53 Egg, pupa, female Up
321B1 90%, S. littoralis 40435 1.84 egg, female Up
9A18 88%, H. armigera 35600 −3.47 6 > 1 > 3, pupa Down
9A18 99%, H. armigera 40298 −3.49 All, 6 > 3 > 1 Down
9A18 99%, H. armigera 6517 −4.46 6 > 1 > 3 Down
9A12 96%, H. armigera 16315 1.511 3 > 1 > 6, female, male Up
9A14 94%, H. zea 13079 1.65 Egg, female Up
337B3v7 96%, H. zea 33786 1.50 Egg, female, 3 > 1 > 6 Up
321A2 92%, H. zea 12163 7.21 6 > 3 > 1, male Up
CYP4 4 V2 88%, Mamestra brassicae 17185 2.32 All, 3 > 1 > 6 Up
340 K4 69%, S. littoralis 8795 −1.95 3 > 1 > 6 Down
4 M7 96%, H. zea 32914 3.18 3 > 6 > 1, pupa Up
4 M7 97%, H. zea 34657 1.79 3 > 6 > 1, pupa Up
4 L12 74%, S. littoralis 41937 5.61 All, 1 > 3 > 6 Up
340AA1 66%, S. littoralis 18087 2.03 Egg, 3 Up
340AA1 70%, S. littoralis 23572 1.03 3, male Up
4M14V1 76%, S. litura 22567 1.50 Pupa Up
4C1 71%, Blaberus discoidalis 26692 1.77 All Up
4S1 96%, H. armigera 1070 −1.29 6 > 1 > 3,female,pupa Down
367B6 73%, S. littoralis 15813 2.03 3, pupa, female Up
340AA1 70%, S. littoralis 21273 2.03 3 Up
4G74 83%, S. littoralis 4194 −1.91 3 > 6 > 1,pupa, female Down
4G15 D. melanogaster 2239 −3.21 1 = 3 < 6, female Down
4G74 86%, S. littoralis 14395 −3.56 3 > 6 > 1, pupa, female Down
341B1 76%, B. mori 3370 −1.92 6 > 3 > 1 Down
341B1 67%, B. mori 40986 −1.19 Pupa Down
341B3 78%, S. littoralis 7936 −1.14 6 > 3 > 1 Down
341A13 83%, S. littoralis 26038 −1.55 3, female Up
4C1 88%, B. mori 22272 −2.43 6 Down
[93]Open in a new tab
^aRatio: RPKM of 2-TD treated samples/RPKM of untreated samples. RPKM:
Reads per kilo bases per million reads. RPKM < 0.1 was used as the
criterion to judge the unigenes were not expressed during a given
developmental stage. Absolute value of Log[2]Ratio ≥ 1 were used as
thresholds for ‘differential expression’. The P450 genes reported to be
involved in insect hormone biosynthesis and metabolism are shown in
bold. 1: 1^st instar larvae; 3: 3^rd instar larvae; 6: 6^th instar
larvae
^bThe cytochrome P450 clan schema used here follows the system proposed
by Feyereisen et al. (2006)
2-TD-induced P450 genes related to hormone biosynthesis and metabolism
Pathway analysis of the unigenes was conducted using the Kyoto
Encyclopedia of Genes and Genomes (KEGG) annotation system. To confirm
the unigenes expression profile results, the expression of P450
unigenes induced by 2-TD that related to hormone metabolism was
analyzed with Real-Time qPCR (Additional file [94]9). Figure [95]4
illustrates 2-TD affects the biosynthesis and metabolism of insect
hormones (JH and molting hormone). The expression of four P450 are
suppressed by 2-TD treatment: CYP307A1 (unigenes 8199, 13311), CYP306A1
(unigenes 13036), CYP314A1 (unigenes 15409), and CYP315A1 (unigenes
40026), these down-regulated genes are shown with solid blue lines in
Fig. [96]4. The expression of two P450 are significantly increased by
2-TD treatment: CYP18A1 (unigenes 41217, 35643, 16953), CYP314A1
(unigenes 16487), these up-regulated genes are shown with solid red
lines in Fig. [97]4. The dashed blue and red lines indicate the down-
and up-regulated products, respectively, and Fig. [98]5 shown that 20E
titers in the 2-TD treated group were higher than in the control group.
The expression of three hormone metabolism related unigenes were not
affected by 2-TD treatment (Additional file [99]9, Fig. [100]5). The
Real-Time qPCR results of the other 2-TD-induced 22 P450 unigenes were
consistent with the DGE gene expression profiles, suggesting that the
DGE results were reliable (Additional file [101]10).
Fig. 5.
Fig. 5
[102]Open in a new tab
2-TD affects the biosynthesis and metabolism of insect hormones (JH and
molting hormone). Genes down-regulated following treatment with 2-TD
are shown with solid blue lines, up-regulated genes are shown with
solid red lines. The dashed blue and red lines indicate the down- and
up-regulated products, respectively. The portion with a blue background
shows the biosynthetic pathway of 20E; portions with red backgrounds
show the metabolic pathways of insect hormones [[103]25, [104]29,
[105]33, [106]34]
RNA interference (RNAi) insect hormones-related P450 genes
CYP307A1 (unigenes 8199, 13311), an insect hormone-related P450 gene
that was down-regulated by 2-TD in H. armigera, was selected for RNAi
knockdown studies. The CYP307A1 dsRNA-treated larvae showed significant
reduction of CYP307A1 expression as compared to the larvae treated with
GFP dsRNA (Fig. [107]6a). Compared to the control, 90 and 85% of
CYP307A1 expression was suppressed at 12 h and 24 h after feeding
larvae artificial diet with 35 μg/g (w:w) CYP307A1 dsRNA, respectively.
However, no significant retardation of transcription was observed at 36
or 48 h after feeding (Fig. [108]6a).
Fig. 6.
Fig. 6
[109]Open in a new tab
CYP307A1 RNAi. a The dsRNA-mediated depletion of CYP307A1 transcripts
in larvae fed with CYP307A1 dsRNA. b RNAi CYP307A1 effects on the
development of H.armigera. Second-instar larvae were fed on a diet
containing 5 μg/g or 35 μg/g (w:w) dsRNA, and samples were collected at
12, 24, 36, and 48 h after feeding. GFP dsRNA was used as a control, at
the same concentrations. In the each diagram, bars sharing the same
letter for each time point group are not significantly different at the
P >0.05 level
The effect of the RNAi-based knockdown of CYP307A1 on larval survival
rates was evaluated in second instar larvae by feeding artificial diet
mixed with 35 μg/g (w:w) CYP307A1 dsRNA and 2-TD (0.1 mg/g) for 1, 3,
and 5 days. Compared to treated with ds CYP307A1 larvae, the survival
rate dramatically decreased in larvae treated the mixture of CYP307A1
with 2-TD. The survival rate was 72% for the treatment with ds CYP307A1
and 61% for the mixture of CYP307A1 with 2-TD, through continuous
feeding for 5 days (Fig. [110]6b).
Discussion
Our experimental results showed that the plant allelochemical 2-TD
affects insect development (Table [111]1), and we observed that a
decrease in the concentration of 20E occurred along with the growth
retardation symptoms following 2-TD treatment (Table [112]1). 2-TD
treatment induced the expression of P450 detoxification enzyme genes.
Insect P450 enzymes are classified into four major clans, namely the
CYP2, CYP3 (including CYP6 and CYP9), CYP4, and the mito.CYP clan
[[113]23]. The mito.CYP, CYP2, and CYP4 clans contain a variety of
single-member CYP families that are known to play important roles in
diverse physiological processes [[114]24–[115]31]. The CYP3 clan in
insects can be further subdivided into the CYP6 and CYP9 families,
which participate primarily in the metabolism of xenobiotic compounds
[[116]19, [117]32].
20E is a polyhydroxylated steroid hormone that controls molting and
thereby affects the growth of arthropods. Studies using D. melanogaster
have revealed that the Halloween P450 genes (CYP307A1/A2, CYP306A1,
CYP302A1, CYP315A1, and CYP314A1) are essential for each of the steps
in 20E biosynthesis [[118]25, [119]33, [120]34]. CYP18A1 belongs to the
CYP2 clan and takes part in 20E inactivation, converting 20E to 20,
26-dihydroxyecdysone [[121]29]. In B. mori, CYP18A1 not only has
temporal- and tissue-specific expression profiles, but also exhibits a
distinct expression pattern that closely coincided with the peak of
ecdysteroid accumulation in the hemolymph of B. mori, a finding that
further suggests that orthologous CYP18A1 in insects is closely related
to ecdysteroid homeostasis [[122]35]. Interestingly, we also observed
that 2-TD treatment dramatically increased the expression of CYP18A1
(41217, 35643, 16953) (Table [123]3, Fig. [124]5), the increasing
CYP18A1 will lead a lower concentration of 20E. Our results clearly
show that treatment of larvae with 2-TD decreased 20E concentrations
(Table [125]1) and suppressed larval growth. CYP306A1 (13036), CYP307A1
(8199, 13311), CYP314A1 (16487, 15409), and CYP315A1 (40026) may be
also essential for 20E biosynthesis. Treatment with 2-TD decreased the
expression levels of these unigenes. We used RNAi methods to confirm
the function of CYP307A1 in H. armigera. Larvae treated with CYP307A1
dsRNA had dramatically decreased survival rates compared to the GFP
dsRNA control, this symptom was similar with RNAi CYP307A1 in D.
melanogaster [[126]36]. Compared to treated with ds CYP307A1 larvae,
the survival rate dramatically decreased in larvae treated the mixture
of CYP307A1 with 2-TD (Fig. [127]6b), these results proved that the
retardant effect of 2-TD is mediated by CYP307A1 on development of
cotton bollworm. Some unigenes were annotated as the same P450 gene,
but these unigenes have different expression profiles in one sample,
these phenomenon maybe caused by these unigenes are not full-length
P450 genes or they have allele genes.
CYP15A1 encodes an enzyme that catalyzes the last step in JH
biosynthesis, catalyzing the epoxidation of methyl farnesoate into JH
III in D. punctate [[128]37]. CYP4C7, expressed in a heterologous
system, was able to metabolize JH III and JH precursors into
12-transhydroxy [[129]30]. Although CYP4C7 and CYP15A1 were not found
to be regulated by 2-TD, the percentage of expressed CYP4 genes
decreased following 2-TD treatment in H. armigera (Table [130]3), and
the percentage of expressed CYP4 unigenes suddenly increased during the
transformation from eggs to larvae; the percentage of expressed CYP4
decreased during the transformation from larvae to pupae (Fig. [131]1).
48 P450 unigenes of the CYP4 clan were found in our study. CYP4C15,
initially cloned from the steroidogenic glands (Y-organs) of crayfish,
has been suggested to be involved in ecdysteroid biosynthesis
[[132]38]. In Diploptera punctata, CYP4C7 is expressed selectively in
the corpora allata and metabolizes JH and its precursors into new
metabolites [[133]10, [134]30]. CYP4 unigenes in H. armigera homologous
to CYP15A1 and CYP4C7 may be involved in JH biosynthesis and
metabolism. In our study, the expression of these genes in larvae was
higher than in eggs, and was induced by 2-TD treatment (Table [135]3).
Four CYP4 unigenes (12812, 10466, 10601, 14820) were specifically
expressed in larvae, and one CYP4 unigene (22936) was solely expressed
in females. These expressed P450 unigenes seem likely to play important
roles during these specific stages (Table [136]1). 2-TD treatment
strongly induced the expression of CYP4 unigenes in 6^th instar larvae
(Fig. [137]4a), a finding consistent with previous research in other
insects [[138]23]. These imply that the increased expression of CYP4
transcripts induced by 2-TD treatment would likely also affect JH
biosynthesis and metabolism.
Our DGE analysis found that CYP333B3 (12317) and CYP333A3 (25319),
which belong to the mito.CYP clan, were also regulated by 2-TD
(Table [139]3). Other 2-TD-regulated mito.CYP genes are related to the
metabolism of molting hormone, but there have been no reports to prove
that these two unigenes are involved in the biosynthesis or metabolism
of molting hormone. Both the up- and down-regulation of these two P450
unigenes may be of critical importance in the development and
metamorphosis of insects. As many of these genes are conserved among
many insect species, our study provides a foundation for the functional
characterization of the roles of these two P450 unigenes in insect
development and metamorphosis.
About 80 P450 unigenes of the CYP3 clan were identified in our study.
Within the genus Papilio (Lepidoptera: Papilionidae), CYP6 family
members are known to detoxify furanocoumarins, secondary metabolites
characteristic of the host plant families consumed by these insects
[[140]14, [141]39–[142]45]. In our study, four P450 unigenes (13679,
22278, 15388, 19180) shared homology with CYP6B2, CYP321B1, CYP6AE14,
and CYP9A18. These unigenes all belong to the CYP3 clan are known to be
specifically expressed in larvae, and are thought to participate
primarily in the metabolism of plant allelochemicals [[143]12,
[144]14]. Two CYP3 unigenes were expressed only in adult females. One
CYP3 unigene was specifically expressed in egg and pupa, respectively
(Table [145]2). The ability of insects to metabolize xenobiotic
compounds at different development stages may be related to these CYP3
clan P450 unigenes.
Conclusions
In conclusion, we found that 2-TD can retard the development of cotton
bollworm, and a decrease of the concentration of 20E occurred alongside
the retardant symptoms (Table [146]1). In order to further illuminate
the relationship between 2-TD and its function in retarding the
development of insects, the transcriptome of H. armigera was sequenced
and digital gene expression libraries were constructed in the present
study. The expression of CYP314A1, CYP315A1, CYP18A1, CYP307A1, and
CYP306A1 was found to be induced by 2-TD, and these genes were also
related to the biosynthesis or metabolism of 20E. Expression analysis
and RNAi studies proved that the retardant effect of 2-TD is mediated
by P450 genes on development of cotton bollworm.
Methods
Insect samples
The cotton bollworm population used in this study (a laboratory
population) was initially collected from the Handan region of Hebei
Province, China, in 1998, and reared on an artificial diet in a growth
room maintained at 26 ± 1 °C, 70–80% relative humidity, with a
photoperiod of 16:8 (L:D). The population was never exposed to any
pesticides. The composition of the artificial diet was as follows: corn
flour 300 g, soybean powder 100 g, yeast extract powder 100 g, citric
acid 2.5 g, vitamin C 10 g, sorbic acid 1.5 g, vitamin B 1.5 g,
erythromycin 0.05 g, propionic acid 5 mL, vitamin E 1.5 g, water 2.5 L.
Adults were held under the same conditions and supplied with a 10%
sugar solution. Females were induced to oviposit into gauze. Eggs were
collected from this gauze. All specimens at all life stages were
pesticide-free and were reared in a growth chamber set to the
aforementioned environmental conditions. The newly molted 6^th instar
larvae, after molted for 1 day, were treated by 12 h of starvation
treatment, then the larvae were exposed to the artificial diet mixed
with 2-TD (Sigma-Aldrich, MO, USA) (99% purity) 10 mg/g (w:w) for 24 h
(ethyl alcohol as negative control). Each treatment contained twenty
five larvae, and these experiments were repeated four times.
Quantification of Ecdysteroids
Total 20Ewere quantified by enzyme immunoassay (EIA). Newly molted
sixth instar larvae treated with 2-TD (twenty five larvae/tube with
four replicates) were homogenized and extracted as described previously
[[147]46]. The extracts were evaporated, redissolved, and subjected to
ecdysteroid enzyme-linked immunosorbent assay (ELISA). The ELISA was
performed in a competitive assay format using anti-20E rabbit antiserum
(Cayman Chemical, Michigan, USA), acetylcholinesterase-conjugated 20E
(Cayman Chemical, Michigan, USA), and standard 20E (Sigma-Aldrich, St.
Louis, MO, USA). The acetylcholinesterase activity was quantified by
Ellman’s Reagent (Cayman Chemical, Michigan, USA), and the absorbance
at 415 nm was detected with a Benchmark microplate reader (Bio-Rad
Laboratories, Hercules, USA).
RNA isolation
Total RNA was isolated from specimens at the following developmental
stages: eggs collected within 24 h of post-oviposition; first-instar
larvae; third-instar larvae; sixth-instar larvae not treated with
2-tridecane; pupae; mating adults (females and males, within 6 days of
eclosion); and sixth-instar larvae treated with 2-TD. For each sample,
approximately 800 mg of insect material was homogenized with liquid
nitrogen in a mortar in order to reduce the effect of error. RNA was
extracted using an RNeasy plus Micro Kit (Qiagen GmbH, Germany)
following the manufacturer’s instructions. RNA was quantified by
measuring the absorbance at 260 nm using a NanoDrop® 1000A
spectrophotometer (GE Healthcare, Uppsala, Sweden). The purity of all
RNA samples was assessed at an absorbance ratio of OD[260/280] and OD
[260/230], and the integrity of RNA was confirmed by electrophoresis on
1% agarose gels.
Construction of the cDNA library and Illumina sequencing for transcriptome
analysis
Briefly, 12 mg total RNA (a mixture of RNA from eggs, 1^st instar
larvae, 3^rd instar larvae, 6^th instar larvae, pupae, adult females
and males, all at equal proportions) was used to construct a cDNA
library of transcriptome. Poly (A) mRNA was purified from total RNA
using oligo (dT) magnetic beads. These short fragments were then used
as templates for the synthesis of first-strand cDNA. Second-strand cDNA
was synthesized using DNA polymerase I, and the samples were treated
with RNaseH. Short fragments were purified using a QiaQuick PCR
extraction kit (Qiagen GmbH, Hilden, Germany). These fragments were
subsequently washed with elution buffer for end reparation poly (A)
addition and then ligated to sequencing adapters. Suitable fragments,
as determined by agarose gel electrophoresis, were selected for use as
templates for PCR amplification. The cDNA library was sequenced using
the Illumina Solexa platform.
Assembly and functional annotation of the transcriptome
Using Trinity program to assembly transcripts, all of the raw sequences
were filtered to remove low quality and adaptor sequences [[148]47].
Open reading frame (ORF) of the unigenes were predicted using the ORF
finder tool ([149]https://www.ncbi.nlm.nih.gov/orffinder/). All
unigenes were queried against the NCBI Nr protein database with an
e-value cutoff of 1e^−5 for functional annotation. The BLASTN algorithm
was also used to query the unigenes against the NCBI Nt nucleotide
databases (Nt; e-value < 10^-5). For quantitative assessment of the
assembly and annotation completeness, in comparison with the arthropod
profile in OrthoDB v8 [[150]48], we applied the software tool BUSCO
[[151]49], which is based on evolutionarily informed expectations of
gene content, with default settings. Then, the BLAST results were used
to do a tentative functional annotation of the unigenes with GO, KEGG
and COG databases (e-value < 10^-5). The clean reads and
computationally assembled sequences from this study were submitted to
the Sequence Read Archive (SRA) database (Accession number: SRX374716).
Selection of cytochrome P450 sequences and phylogenetic analysis
Sequences encoding genes related to cytochrome P450s were identified by
BLASTX analysis against the NCBI nr database, with a cut-off value of
e-value < 10^-5. Sequences that returned redundant BLASTX results, or
those that showed high homology with each other as determined by the
alignment results, were eliminated as likely allelic variants or
different portions of the same gene. MEGA 6.0 software was used to
analyze the phylogenetic relationships between the P450 unigenes of H.
armigera and the published P450 sequences from Bombyx mori (B. mori).
The amino acid sequences for each predicted protein were aligned using
MAFFT 7.110 [[152]50]. Neighbor-joining trees were produced using MEGA
6.0 with Poisson correction of distances [[153]51], and 1000
neighbor-joining bootstrap replicates.
Preparation and sequencing of the DGE library
RNA was extracted separately from eggs, 1^st instar larvae, 3^rd instar
larvae, 6^th instar larvae, pupae, adults (females and males), and
sixth-instar larvae treated with 2-TD. The extractions were performed
using an RNeasy plus Micro kit (Qiagen GmbH, Hilden, Germany) according
to the manufacturer’s instructions. Approximately 10 μg RNA from each
sample was used for the construction of DGE libraries. mRNA was treated
as described in the cDNA library construction methods, above. The
fragments were purified by agarose gel electrophoresis and enriched by
PCR amplification. The library products were then sequenced with the
Illumina Solexa platform. The raw data (tag sequences and counts) were
deposited in the NCBI SRA database, under accession number: SRX684363.
Bioinformatics pipeline and analysis of DGE libraries
Sequencing raw data were transformed by base calling into raw sequence
data. Clean tags were obtained after the raw sequences were filtered to
remove adaptor sequences, empty tags, low quality tags, tags that were
too short (<200 bp), and tags with a copy number of 1. All clean tags
were mapped to the transcriptome of H. armigera with a stringency
allowing no more than 1 nucleotide mismatch. The number of unambiguous,
clean tags for each gene was calculated, and then normalized to RPKM
(Reads Per Kilo bases per Million reads), using the following equation:
[MATH: RPKM=10
6/CNL/103 :MATH]
in which C is the number of reads uniquely mapped to a given gene, N is
the number of reads uniquely mapped to all genes, and L is the total
length of the exons in the given gene. For genes with more than one
alternative transcript, the longest transcript was selected to
calculate the RPKM. The RPKM method eliminates the influences of
different gene lengths and sequencing discrepancies on gene expression
calculations. Therefore, RPKM values can be used directly for comparing
differences in gene expression among samples. [[154]52]. RPKM <0.1 was
used as the criterion to judge if a given unigene was not expressed in
one specimen.
For gene expression profiling analysis, unigenes were assigned GO terms
using the Blast2GO and canonical pathways tools of the KEGG pathway
enrichment analysis. Analysis of the differentially expressed genes was
performed based on the GOstat algorithm [[155]53]. To identify the
differentially expressed genes among different development libraries
(egg, 1^st instar larvae, 3^rd instar larvae, 6^th instar larvae,
pupae, adult females and males libraries), each library compared with
egg library, and the fold change Log[2] Ratio ≥ 1 values were used as
threshold criteria to judge the differences in gene expression
[[156]54]. Compared with 6^th instar larvae library, the differentially
expressed genes among 6^th instar larvae library and 2-TD treated
library were also identified by Log[2] Ratio ≥ 1 values. The percentage
of each CYP clan (mitochondrial, clan 2, clan3 and clan4) expressed in
each DGE library was calculated according to the following formula:
(Sum RPKM of each CYP clan)/(Sum RPKM of P450) × 100%.
Validation of P450 gene expression profiles by Real-Time PCR
To confirm the gene expression profile results from the DGE libraries,
the expression of 35 P450 unigenes (including 12 hormone-related P450
unigenes) were analyzed with Real-Time qPCR. Specific primers were
designed using Primer 5.0 software, and are listed in Additional file
[157]12. EF-α was used as an internal control. Three biological
replicates were performed for qPCR assay. The efficiency of each set
primer was about 100% (Additional file [158]12). RNA isolation was
performed using TRIzol reagent, according to the manufacturer’s
instructions (Invitrogen, Carlsbad, CA, USA). Samples were treated with
RNase-free DNase I (Takara Biotechnology Dalian Co., Ltd., Dalian,
China). First-strand cDNA synthesis was performed with 1 μg of total
RNA by using a Transcriptor First Strand cDNA Synthesis Kit (Takara
Biotechnology Dalian Co., Ltd., Dalian, China). cDNA was amplified
using an Applied Biosystems7500 qPCR System (Applied Biosystems, Foster
City, USA) with a Real Master Mix SYBR Green PCR kit (Invitrogen
Carlsbad, CA, USA). Amplification conditions consisted of an initial
pre-incubation at 95 °C for 5 min, followed by amplification of the
target DNA for 40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for
30s and 95 °C for 5 min. The melting curves of the amplicons were
measured by taking continuous fluorescence readings whilst increasing
the temperature from 58 to 95 °C, with 0.5 °C incremental increases
every 10 s. geNorm version 3.5 [[159]55] and Normfinder version 0.953
[[160]56] software were used to evaluated the raw CT values of the
selected reference genes as described in their manuals. Candidate gene
with the lowest M value should be the most stably expressed reference
gene, and EF-1a was chose as the reference gene (Additional file
[161]13). For each gene, a standard curve was generated for each set of
primers, and the efficiency of each reaction was determined.
Statistical analyses of Real-Time qPCR results were performed using
GraphPad Prism 5.0 software (GraphPad prism, Prism 5 for Windows).
Statistical significance was determined by using a Student’s t-test,
and a p value less than 0.05 was considered to indicate statistical
significance.
RNAi insect hormone-related P450 genes
Based on the CYP307A1 gene sequence (Gene bank number: [162]KM016704.1)
and predicted possible interference sites obtained using online
prediction software ([163]http://www.dkfz.de/signaling/e-rnai3/), we
designed specific primers using DNAMAN 6.0 software. A 494-bp fragment
of CYP307A1 (position 730–1310) was amplified and cloned into the
pMD-18simple-T vector (Takara, Dalian, China), using the
dsRNAi-CYP307A1-1 and dsRNAi-CYP307A2-2 primer pair (Additional file
[164]12), which contained additional T7 promoter sequences. Purified
plasmids served as templates for RNA synthesis using a MEGAscript T7
transcription kit (Ambion, Austin, TX, USA). GFP dsRNA, which was used
as the control, was synthesized with the same procedures as above,
using the dsGFP-F and dsGFP-R primers (Additional file [165]12). dsRNA
from GFP and CYP307A1 were derived by using the MEGAscript T7
transcription kit with an extended transcription time of 5 h at 37 °C.
The resulting dsRNA was digested by DNase I and RNase to remove DNA and
any single-stranded RNA, and finally dissolved in DEPC water.
Second-instar larvae, after being starved for 12 h, were exposed to
artificial diet containing CYP307A1 dsRNA (15 μg/g or 35 μg/g, w/w) mix
or not mix with 2-TD (0.1 mg/g, w/w) for 12, 24, and 36 h; GFP dsRNA
was used as a control. Thirty larvae were used in each treatment, and
three replications were performed. The dsRNA-mediated depletion of
CYP307A1 transcripts was experimentally evaluated with qPCR by using
the qCYP307A1 -F and qCYP307A1 -R primers (Additional file [166]12).
Acknowledgments