Abstract

   Cnaphalocrocis medinalis (Guenée) is one of the important insect pests
   in rice field. Bt agents were recommended in the C. medinalis control
   and Bt rice is bred as a tactic to control this insect. However, the
   tolerance or resistance of insect to Bt protein is a main threat to the
   application of Bt protein. In order to investigate the response of C.
   medinalis transcriptome in defending a Cry1C toxin, high-through
   RNA-sequencing was carried in the C. medinalis larvae treated with and
   without Cry1C toxin. A total of 35,586 high-quality unigenes was
   annotated in the transcriptome of C. medinalis midgut. The comparative
   analysis identified 6,966 differently expressed unigenes (DEGs) between
   the two treatments. GO analysis showed that these genes involved in
   proteolysis and extracellular region. Among these DEGs,
   carboxylesterase, glutathione S-transferase and P450 were differently
   expressed in the treated C. medinalis midgut. Furthermore, trypsin,
   chymotrypsin, and carboxypeptidase were identified in DEGs, and most of
   them up-regulated. In addition, thirteen ABC transporters were
   downregulated and three upregulated in Cry1C-treated C. medinalis
   midgut. Based on the pathway analysis, antigen processing and
   presentation pathway, and chronic myeloid leukemia pathway were
   significant in C. medinalis treated with Cry1C toxin. These results
   indicated that serine protease, detoxification enzymes and ABC
   transporter, antigen processing and presentation pathway, and chronic
   myeloid leukemia pathway may involved in the response of C. medinalis
   to Cry1C toxin. This study provides a transcriptomal foundation for the
   identification and functional characterization of genes involved in the
   toxicity of Bt Cry protein against C. medinalis, and provides potential
   clues to the studies on the tolerance or resistance of an
   agriculturally important insect pest C. medinalis to Cry1C toxin.

Introduction

   The rice leaffolder, Cnaphalocrocis medinalis (Guenée), is one of the
   important migratory insect pests of rice in China [[32]1–[33]2]. It
   widely distributes in humid tropical and temperate regions of Oceania,
   Africa, and Asia [[34]3–[35]4]. It scrapes folds and feeds the rice
   leaves causing the reduction of photosynthetic activity [[36]1,[37]5].
   Heavy occurrence often happens in main rice-growing areas like China,
   Japan, Korea, Vietnam, Philippines, and so on [[38]3], and frequently
   results in great rice yield losses [[39]2]. The damage area by C.
   medinalis were more than 20 million hm^2 in China in the most of years
   ranged from 2003 to 2010 [[40]2]. A report from the Ministry of
   Agriculture, China showed that the damage area by C. medinalis was up
   to 15.5 million hm^2 in 2015 [[41]4].

   So far, the control of C. medinalis mainly relied on the chemicals.
   However, the misuse and overuse of chemicals resulted in many negative
   issue such as insect resistance, resurgence, environmental pollution,
   human health concerned by public [[42]6]. Environment-friendly methods
   are recommended in the pest control. Bacillus thuringiensis, as one of
   the important biological agents has been applied in the pest control
   for more than 60 years due to its crystal protein with insecticidal
   activity against insect pests [[43]7–[44]9]. Cry genes derived from B.
   thuringiensis were successfully transferred into plants like cotton,
   rice and these plants harbored resistance to Lepidopteran insect pests
   [[45]10–[46]13]. In China, Bt agent was recommended as a friendly
   bio-agent to use in the control of C. medinalis and Bt rice in China
   was approved to release for production test in limited area in 2009,
   but not commercially planted[[47]11]. The application of Bt protein and
   Bt crop could greatly reduce the use of chemical insecticides. However,
   insect resistance is a big threat to the application of Bt protein and
   Bt crop. So far, at least eight insect pests evolved resistance against
   Bt crop or Bt protein [[48]14–[49]15]. Coincidentally, many insect
   pests evolved resistance to Bt proteins under selection with Bt protein
   in the laboratory condition [[50]8,[51]16]. Considering the sustainable
   use of the Bt protein or Bt rice, the potential of resistance evolution
   of C. medinalis against Bt protein will become an inescapable issue
   [[52]17].

   Midgut, one of the important organs in the insect body, not only is a
   place for food ingestion and utilization, but also a place for
   detoxification to xenobiotics [[53]18]. Interaction of Bt protein and
   insect mainly occurred in midgut. Several reports revealed that
   resistance of insect against Bt protein were correlated with the
   variation of genes in midgut [[54]14,[55]19–[56]21]. Next generation
   sequencing (NGS) is a strong technology which could generate large
   volumes of sequence information [[57]22]. NGS provides us with
   unprecedented high-throughput and low-cost sequencing platforms applied
   in a variety of manners, including de novo whole-genome sequencing,
   resequencing of genomes to identify variations, de novo transcriptome
   and gene expression profiling, and detecting methylation patterns
   [[58]23]. Now, the Illumina/Solexa sequencing technology is dominated
   in the NGS market, featuring high data accuracy and a broad range of
   applications.

   In this study, transcriptome of C. medinalis midgut was sequenced and
   de novo assembled using Illumina sequencing technology. The gene
   expression patterns in the midgut were compared in C. medinalis larvae
   treated with and without Bt Cry1C protein. Moreover, the genes of
   digestion and detoxification system were given greater attention. The
   results provide clues on the roles of midgut in the response to Bt
   proteins, information for further study candidate genes involved in
   interaction of Bt protein and C. medinalis and promote the
   understanding of potential resistance or tolerance of C. medinalis to
   Bt proteins.

Materials and methods

Insect rearing

   Adults of C. medinalis were collected with sweep net from paddy fields
   in suburb of Hangzhou, Zhejiang, China in August, 2015. The moths were
   placed in plastic cup covered with nylon mesh and fed with 10% honey
   solution. Eggs laid on the mesh were removed and transferred to a box
   with detached leaf of 45 d-old Taichung Native 1 (TN1). The larvae that
   hatched from eggs were cultured in the detached leaf then used in the
   experiment. All insect cultures were kept at 27±1°C with 70–80% RH and
   a photoperiod of 14: 10 (L: D) h. Insect collection is from our
   experimental field, and C. medinalis is not endangered or protected
   species.

Treatment of C. medinalis larvae with Cry1C toxin

   Fifth instar larvae of C. medinalis were selected for the feeding
   experiments. The larvae were reared on rice leaves dipped with Cry1C
   activated toxin (MP, Cavey, CWRU, US) solution which resulted in a
   growth inhibition but did not cause visible death of the larvae for 48
   hours. Larvae treated with PBS buffer were used as negative control.
   All treated larvae were incubated for 48 h at 27±1°C with 70–80% RH and
   a photoperiod of 14: 10 (L: D) h. Approximately 150 larvae were used
   for each treatment.

RNA isolation, cDNA library construction, transcriptomic sequencing

   Total RNA was isolated from the midgut of C. medinalis larvae using
   TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the
   manufacturer's protocol. The total RNA quantity and purity were
   analyzed with Bioanalyzer 2100 and RNA 6000 Nano Lab Chip Kit
   (Agi-lent, CA, USA) with RIN number >7.0.

   Approximately 10 μg of total RNA was subjected to isolate Poly (A) mRNA
   with poly-T oligo attached magnetic beads (Invitrogen). Following
   purification, the mRNA is fragmented into small pieces using divalent
   cations under elevated temperature, and the cleaved RNA fragments were
   reverse-transcribed to create the final cDNA library in accordance with
   the protocol for the mRNA-Seq sample preparation kit (Illumina, San
   Diego, USA). The cDNA library was sequenced run with Illumina
   HiSeqTM2000 sequence platform at LC Sceiences (USA) following the
   vendor's recommended protocols. Two or three biological replicates for
   each treatment were prepared.

De novo RNA-seq assembly and annotation

   After sequencing, the raw data were filtered against low-quantity reads
   and adaptor contamination using Trinity. Clean reads were de novo
   assembled using Trinity with K-mer = 25 [[59]24]. Annotations of all
   the unigenes were performed by a BLASTx search against the Nr,
   Swiss-Prot, KOG, KEGG databases and Pfam with a cut-off E-value of
   ≤1e-5. We obtained the Gene Ontology terms
   ([60]http://www.geneontology.org) of each C. medinalis unigene with the
   software Blast2GO ([61]http://www.blast2go.org) using the default
   parameters [[62]25]. Gene expression level was normalized by RSEM-based
   lgorithm to get RPKM value [[63]26–[64]27]. Based on the expression
   levels, thresholds of FDR<0.05 and log2 fold-change (log2FC) ≥1 were
   set for identifying significant differential expressed unigenes (DEGs)
   between control and Cry1C treated larvae.

Enrichment analysis

   All the DEGs were mapped to terms in KEGG databases to identify
   significantly enriched metabolic pathway or signal transduction
   pathways in DEGs. Pathway enrichment analysis provides all terms that
   significantly enriched in differentially expressed genes in comparison
   to the control C. medinalis. The P-value was calculated for each
   pathway by Fisher's exact test with a Q value threshold<0.05.

Quantitative real-time PCR (qRT-PCR) validation

   To conform the data, a subset of DEGs was validated by qRT-PCR. qRT-PCR
   was performed in 20 μL reaction mixtures composed of 2.5 μL of template
   cDNA,10 μL of 2×SYBR Green PCR Master Mix (Fermentas,Waltham, MA, USA),
   and 0.25 mM each primer on the CFX96 Real-Time System (Bio-Rad,
   Hercules, CA, USA). The selected genes were verified with the following
   cycling conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 30
   s, 60°C for 35 s. The melting curve analysis was used to analyze the
   specificity of the qPCR product. The sequences of the primers used are
   listed in [65]S1 Table. A ubiquinol-cytochrome c reductase served as an
   internal control. The relative gene expression values were calculated
   using the 2^−ΔΔCt method [[66]28].

Results

Assembly and annotation of the C. medinalis transcriptome

   The transcriptomes of the midgut of C. medinalis larvae treated with
   and without Cry1C protein were sequenced and compared for each
   treatment. A total of 37.33G bases of clean reads were obtained from
   the cDNA libraries, and 35,586 high-quality unigenes and 65,016
   transcripts were assembled ([67]Table 1). The average gene and
   transcript length were 784 and 895 base pair (bp), respectively
   ([68]Table 1). Mean GC% of gene and transcript were 40.66% and 39.18%,
   respectively. N50 (the shortest sequence length at 50% of the
   transcriptome) of gene and transcript were 1,211 and 1,378 bp,
   respectively. The size distributions of all the unigenes studied were
   as follows: 18,720 unigenes (17.8%) exhibited lengths of more than
   2,000 bp; 17,474 unigenes (16.7%) showed lengths between 500 and 1000
   bp; 26,983 unigenes (25.7%) demonstrated lengths between 300 and 500
   bp; and 41,789 unigenes (39.8%) were shorter than 300 bp ([69]S1 Fig).
   The size distributions of all the transcripts studied were as follows:
   18,720 unigenes (17.8%) exhibited lengths of more than 2,000 bp; 17,474
   unigenes (16.7%) showed lengths between 500 and 1,000 bp; 26,983
   unigenes (25.7%) demonstrated lengths between 300 and 500 bp; and
   41,789 unigenes (39.8%) were shorter than 300 bp ([70]S2 Fig).

Table 1. Summary statistics for the transcriptome of larval midgut of
Cnaphalocrocis medinalis.

   Statistics            gene       transcript
   Total number          35,586     65,016
   Total assembled bases 27,902,176 58,253,881
   Min length (bp)       201        201
   Max length (bp)       17,428     17,428
   length > 2 kb         2,725      6,530
   Average length (bp)   784        895
   Median length (bp)    471        572
   N50 length (bp)       1,211      1,378
   Mean (G + C)s (%)     40.66      39.18
   Median (G + C)s (%)   38.60      36.60
   [71]Open in a new tab

   To annotate these unigenes, All unigene sequences were against the
   protein databases (Nr, SwissProt, KEGG, COG, Pfam, GO) using BLASTx
   (E-value<e-5). Only 16,741 unigenes (47.04% of all unigenes) displayed
   Nr annotations. 9,789 unigenes (27.51% of all unigenes) displayed
   swiss-prot annotations. 12,317 unigenes (34.61% of all unigenes)
   displayed Pfam annotations. 7,401unigenes (20.80% of all unigenes)
   displayed KEGG annotations. 11,010 unigenes (30.94% of all unigenes)
   displayed KOG annotations. 9,003 unigenes (25.30% of all unigenes)
   displayed KEGG annotations. 11,010 unigenes (30.94% of all unigenes)
   displayed GO annotations. Only small partial genes were annotated to
   the databases because of the short nucleotide lengths and the lack of
   C. medinalis genomic information.

   Species-specific distribution revealed that 37.2% of the C. medinalis
   sequences matched with the silkworm, Bombyx mori. In addition, 27.2% of
   the C. medinalis sequences resembled those of Danaus plexippus,
   followed by those of Papilio xuthus (2.8%), Tribolium castaneum (1.3%),
   Zootermopsis nevadensis (1.2%), Papilio polytes (0.9%) ([72]Fig 1A).

Fig 1. Overview of genes annotations from Cnaphalocrocis medinalis midgut.

   [73]Fig 1
   [74]Open in a new tab

   (A) Species distribution of the BLASTX results. (B) GO categories of
   all unigenes and DEGs. (C) euKaryotic orthologous Groups (KOG)
   classification.

Classifications for C. medinalis unigenes

   GO assignments (level 2) were used to predict the functions of the C.
   medinalis unigenes. A total of 12,653 unigenes were assigned GO terms
   of three categories (biological process, cellular component, and
   molecular function) based on sequence homology. The dominant items of
   the three categories were nucleus, ATP-binding, cytoplasm, integral to
   membrane, transcription DNA-dependent. ([75]Fig 1B).

   The C. medinalis unigenes were also annotated with COG classifications.
   Among the 11,010 unigenes with COG annotations, 21% (2,063) belonged to
   the cluster of general function; followed by 10% (1,079) under the
   cluster of Signal transduction mechanisms; and 10% (986) under the
   cluster of Posttranslational modification, protein turnover,
   chaperones. By contrast, Cytoskeleton corresponded to the smallest
   clusters ([76]Fig 1C).

   The C. medinalis unigenes were also annotated with KEGG
   classifications. Among the 9,003 unigenes with KEGG annotations, 21%
   (2,145) belonged to the cluster of Metabolism; followed by 10% (1,014)
   under the cluster of Organismal Systems; and 10% (991) under the
   cluster of Human Diseases. Genetic Information Processing, Cellular
   Processes, Environmental Information Processing.

Overview on complex response of C. medinalis to Cry1C toxin

   The number of DEGs in the midgut of C. medinalis treated with Cry1C was
   6,966. Of that, 4,621 genes were down-regulated, and 2,345 genes were
   up-regulated ([77]Fig 2A). The changed ratios of most DEGs accumulated
   between 2^−15 and 2^13 ([78]Fig 2B). For the top 20 upregulated
   unigenes in the midgut of C. medinalis treated with Cry1C, three
   unigenes had explicit annotations, i.e., adenosinetriphosphatase,
   nucleoprotein TPR, and heat shock 70kDa protein 1/8 ([79]S2 Table).
   Meanwhile, for the top 20 downregulated unigenes in the midgut of C.
   medinalis treated with Cry1C, 10 unigenes had explicit annotations,
   i.e., encoded NADH dehydrogenase I subunit 4, beta-2-microglobulin,
   glypican 5, CD74 antigen, tyrosine 3-monooxygenase/tryptophan
   5-monooxygenase activation protein, valyl-tRNA synthetase, tyrosine
   3-monooxygenase/tryptophan 5-monooxygenase activation protein,
   valyl-tRNA synthetase, protein transport protein SEC61 subunit alpha,
   saposin ([80]S3 Table).

Fig 2. Differentially expressed unigenes (DEGs) in the midgut of
Cnaphalocrocis medinalis larvae fed with Cry1C toxin.

   [81]Fig 2
   [82]Open in a new tab

   (A) Number of DEGs. (B) Volcano plot to show the fold change and error
   rates. The non-DEGs are indicated by black dots, the DEGs up-regulated
   are indicated by red dots, and the DEGs down-regulated are indicated by
   blue dots.

   From the statistic of GO enrichment, most of DEGs involved in
   transcription DNA-dependent, regulation of transcription, and
   proteolysis under biological process; most of DEGs involved in
   nucleusm, cytoplasm, and integral to membrane under cellular component
   function item; most of DEGs involved in ATP-binding, metal ion binding,
   and DNA binding under molecular function ([83]Fig 3A). According to the
   GO enrichment scatterplot ([84]Fig 3B), large amount of genes involved
   in proteolysis, extracellular region, extracellular space, and
   serine-type endopeptidase activity were differently expressed in larval
   midgut of C. medinalis treat with and without Cry1C toxin.

Fig 3. Statistic of GO enrichments of DGEs.

   [85]Fig 3
   [86]Open in a new tab

   (A) Histogram. (B) Scatter plot.

   To validate the transcriptome data, qRT-PCR analysis was performed for
   DGEs ([87]S1 Table). The qRT-PCR validation results were presented as
   fold-changes normalized to the ubiquinol-cytochrome c reductase gene. A
   comparison of the mean values of qRT-PCR results and the DEGs is
   summarized in [88]S3 Fig. The consistent expression trend in qRT-PCR
   results and the original DEGs indicates the reliability of gene
   expression profiling from transcriptome analysis.

Interest genes and Bt related genes in the transcriptome of C. medinalis
midgut

   Here, we investigated DEGs potentially involved in insecticidal
   tolerance/resistance in C. medinalis, including metabolic and
   Bt-related genes. DEGs sequences encoding enzymes function ingestion
   and detoxification and receptors of the insecticide and toxins were
   extracted from the RNA-seq database from the C. medinalis midgut. Here,
   alkaline phosphatase, cytochrome P450, glutathione S-transferase (GST),
   carboxylesterase, ABC transporter, and serine protease including
   transmembrane protease, trypsin and chymotrypsin were (FDR < 0.05 and
   the absolute value of log2FC ≥ 1) were found significant different in
   the midgut of C. medinalis larvae treated with Cry1C protein ([89]Table
   2). A total of thirteen DEGs encoding carboxylesterases were
   differentially expressed between treated and untreated C. medinalis, of
   which 11 of these unigenes were up-regulated and two were
   down-regulated in the Cry1C-treated C. medinalis ([90]S4 Table). A
   total of 99 serine protease genes were differently expressed including
   trypsin, chymotrypsin, carboxypeptidase, aminopeptidase, elastase,
   cathepsin and so on ([91]Fig 4). Fourty-one unigenes encoding trypsin
   were differentially expressed between treated and untreated C.
   medinalis, of which thirty-four unigenes were up-regulated and seven
   were down-regulated in the Cry1C-treated C. medinalis ([92]Table 3).
   Eight chymotrypsin were 1.24- to 3.69- fold upregulated. Twenty
   carboxypeptidase genes were differently expressed with 17 genes
   upregulated and 3 downregulated in the Cry1C-treated C. medinalis
   ([93]Table 4). Four aminopeptidase N were upregulated and two
   downregulated, and two alkaline phosphatase were upregulated in the
   midgut of C. medinalis larvae treated with Cry1C. One cadherin in Cry1C
   treated C. medinalis larvae were differently downregulated by 1.36-fold
   compared with those of the untreated larvae. The DEGs were annotated to
   the ABC transporter including five subfamilies (ABCB, ABCC, ABCE, ABCF
   and ABCG), and three upregulated and 13 down regulated ([94]Fig 5).

Table 2. Differently expressed unigenes potentially involved in
Cnaphalocrocis medinalis response to Cry1C toxin.

   Genes Treated/Control [95]^a
   up down Total
   Metabolic insecticide resistance and insecticide targets
   Cytochrome P450 monooxygenase 3 3 6
   Carboxylesterase 11 2 13
   Glutathione S-transferase 2 1 3
   Acetylcholinesterase 0 0 0
   Nicotinic acetylcholine receptor 0 0 0
   GABA receptor 0 1 1
   Neuropeptide receptor 0 2 2
   Glutamate receptor 0 1 1
   G-protein coupled receptor 0 0 0
   Ryanodine receptor 0 2 2
   Lipophorin receptor 0 0 0
   Sodium channel 0 0 0
   Chloride channel 0 1 1
   NADH dehydrogenase 0 9 9
   NADH oxidoreductase 0 0 0
   Catalase 0 1 3
   Peroxidase 2 9 11
   Superoxide dismutase 1 3 4
   Bt resistance
   Cadherin 0 1 1
   Aminopeptidases N 4 2 6
   Alkaline phosphatase 2 0 2
   Glycolipid 0 0 0
   ABC transporter 3 13 16
   carboxypeptidase 17 3 20
   trypsin 34 7 41
   chymotrypsin 8 0 8
   cathepsin 2 5 7
   [96]Open in a new tab

   ^a Differentially expressed genes listed here have a FDR of <0.05 and
   the absolute value of log2ratio ≥ 1 between C. medinalis larvae treated
   with and without Cry1C toxin.

Fig 4. Serine proteases differently expressed in Cnaphalocrocis medinalis
larvae treated with Cry1C toxin.

   [97]Fig 4
   [98]Open in a new tab

Table 3. Trypsin genes of Cnaphalocrocis medinalis midgut in response to the
ingestion of Cry1C toxin.

   Gene ID      Annotation Treated vs Control[99]^a
                           Log2FC FDR       regulated
   comp57649_c2 trypsin     2.72  2.19E-132 up
   comp53936_c0 trypsin     2.89  2.53E-126 up
   comp58709_c0 trypsin     2.57  8.96E-87  up
   comp62679_c0 trypsin     2.61  1.72E-82  up
   comp52245_c0 trypsin     2.40  2.94E-81  up
   comp52333_c0 trypsin     3.05  3.82E-79  up
   comp59607_c0 trypsin     3.02  4.98E-79  up
   comp53824_c0 trypsin     2.57  1.17E-77  up
   comp55688_c0 trypsin     2.55  2.58E-56  up
   comp50084_c0 trypsin     3.37  6.08E-38  up
   comp58071_c0 trypsin     1.81  1.19E-36  up
   comp59543_c0 trypsin     2.28  4.09E-34  up
   comp55740_c0 trypsin     1.92  6.33E-33  up
   comp59573_c0 trypsin     1.48  1.99E-29  up
   comp51336_c0 trypsin     3.79  5.20E-27  up
   comp54289_c0 trypsin     1.79  2.94E-26  up
   comp59285_c0 trypsin     2.24  2.88E-21  up
   comp50389_c0 trypsin     4.20  2.35E-18  up
   comp53422_c0 trypsin     2.46  1.35E-16  up
   comp55884_c0 trypsin     2.28  3.08E-16  up
   comp51937_c0 trypsin     2.80  4.03E-16  up
   comp46739_c0 trypsin     3.24  2.46E-12  up
   comp43712_c0 trypsin     3.81  2.48E-09  up
   comp53870_c1 trypsin     1.61  6.66E-09  up
   comp61336_c0 trypsin     1.15  7.34E-07  up
   comp47248_c0 trypsin     2.57  3.22E-06  up
   comp53870_c0 trypsin     2.61  7.99E-05  up
   comp46414_c0 trypsin     2.62  2.28E-03  up
   comp51898_c0 trypsin     1.67  4.34E-03  up
   comp40870_c1 trypsin     2.36  5.12E-03  up
   comp53728_c0 trypsin     1.44  1.33E-02  up
   comp40870_c0 trypsin     2.95  1.70E-02  up
   comp56917_c0 trypsin     1.18  4.07E-02  up
   comp47218_c0 trypsin     1.72  4.76E-02  up
   comp62257_c0 trypsin    -1.05  6.09E-15  down
   comp62572_c0 trypsin    -1.40  1.11E-09  down
   comp50375_c0 trypsin    -4.12  1.04E-05  down
   comp59978_c0 trypsin    -1.05  2.51E-04  down
   comp59413_c0 trypsin    -1.73  1.03E-03  down
   comp46878_c0 trypsin    -10.52 1.57E-02  down
   comp58010_c0 trypsin    -1.24  4.26E-02  down
   [100]Open in a new tab

   ^a Differentially expressed genes listed here have a FDR of <0.05 and
   the absolute value of log2ratio ≥ 1 between C. medinalis larvae treated
   with and without Cry1C toxin.

Table 4. Carboxypeptidase genes of Cnaphalocrocis medinalis midgut in
response to the ingestion of Cry1C toxin.

   Gene ID Annotation Treated vs Control[101]^a
   Log2FC FDR regulated
   comp58247_c0 carboxypeptidase 1.42 1.13E-05 up
   comp62619_c0 carboxypeptidase A 2.36 3.14E-51 up
   comp61191_c0 carboxypeptidase A 2.19 2.05E-18 up
   comp55473_c0 carboxypeptidase A 2.38 4.30E-12 up
   comp58294_c0 carboxypeptidase A 1.23 2.09E-11 up
   comp60636_c0 carboxypeptidase A 1.51 6.93E-10 up
   comp57980_c0 carboxypeptidase A 2.07 4.13E-08 up
   comp61570_c0 carboxypeptidase A 1.08 2.82E-07 up
   comp58731_c0 carboxypeptidase A 1.54 1.08E-05 up
   comp57828_c2 carboxypeptidase A 1.08 4.94 E-04 up
   comp51659_c1 carboxypeptidase A 1.99 6.15 E-03 up
   comp57845_c0 carboxypeptidase A 1.18 2.81E-02 up
   comp55631_c0 carboxypeptidase A2 3.13 2.12E-54 up
   comp60005_c0 carboxypeptidase A2 2.68 3.70E-31 up
   comp59859_c0 carboxypeptidase A2 3.06 1.52E-14 up
   comp58033_c0 carboxypeptidase A4 5.14 1.32E-244 up
   comp61876_c0 lysosomal Pro-X carboxypeptidase 1.63 5.50E-13 up
   comp54790_c0 carboxypeptidase A -3.54 4.03E-14 down
   comp45043_c0 carboxypeptidase A -4.51 1.57E-03 down
   comp62420_c0 lysosomal Pro-X carboxypeptidase -1.87 1.02E-07 down
   [102]Open in a new tab

   ^a Differentially expressed genes listed here have a FDR of <0.05 and
   the absolute value of log2ratio ≥ 1 between C. medinalis larvae treated
   with and without Cry1C toxin.

Fig 5. ABC transporters differently expressed in Cnaphalocrocis medinalis
larvae treated with Cry1C toxin.

   [103]Fig 5
   [104]Open in a new tab

Significant enriched pathways in the C. medinalis larvae treated with Cry1C
toxin

   Metabolic pathway enrichment analysis demonstrated that 770 DEGs
   involved in 232 pathways ([105]S5 Table) might play a role in the
   response of C. medinalis to Cry1C toxin. DEGs were significantly
   enriched in two functional categories (antigen processing and
   presentation, and chronic myeloid leukemia) in C. medinalis treated
   with Cry1C (Q-value < 0.05) ([106]Table 5). Twenty-five DGEs were found
   in antigen processing and presentation pathway, and twelve DGEs were
   found in chronic myeloid leukemia pathway.

Table 5. Enriched pathway of DEGs in Cnaphalocrocis medinalis response to
Cry1C toxin (Q-value<0.05).

   Pathway_ID            Pathway Name             Gene number Q-value
    ko04612   Antigen processing and presentation     25      0.0105
    ko05220        Chronic myeloid leukemia           12      0.0487
   [107]Open in a new tab

Discussion

   Despite C. medinalis is one of the important insect pests in rice field
   of Asia, and also the target insect of Bt agent and Bt rice
   [[108]3,[109]11]. But the interaction between this insect pest and Bt
   toxin is unclear. The response of C. medinalis to Bt toxin related with
   the tolerance and resistance to Bt toxin. In this study, larval midgut
   transcriptome was sequenced and analyzed, 35,586 high-quality unigenes
   were obtained, and 6,966 DEGs were found between C. medinalis larvae
   fed with and without Cry1C toxin. In Plutella xylostella midgut
   transcriptome, 2,925 and 2,967 unigenes were differently expressed in
   two resistant strains compared with susceptible strains [[110]29]. In a
   pink bollworm (Pectinophora gossipiella) larval midgut transcriptome,
   39,874 unigenes were obtained from18,623,508 assembled reads [[111]30].
   The number of bases in C. medinalis midugt transcriptome in this study
   was smaller than those in P. xylostella and P. gossipiella larval
   midgut transcriptome. In the transcriptome of the Lymantria dispar
   (gypsy moth) larval midgut in response to infection by B.
   thuringiensis, the DEGs primarily associated with digestive function
   (eg. a-amylase, lipase and carboxypeptidase), immune response (eg.
   C-type lectin 4), developmental genes (eg. arylphorin) as well as a
   variety of binding proteins including cellular retinoic acid binding
   protein (lipid-binding), insulin-related peptide binding protein
   (protein binding) and ovary C/EBPg transcription factor (nucleic
   acid-binding) [[112]31]. Many DGEs involved in the metabolic pathway,
   ABC transporter pathway, aminopeptidases and cadherins were found
   between Cry1Ac-resistant and susceptible strains of P. xylostella
   [[113]29]. Here we focused on the interest genes involved insecticides
   and Bt resistance or tolerance. These findings may facilitate the
   understanding of interaction of insect and Bt protein.

Detoxification enzymes

   Carboxylesterase, cytochrome P450, and glutathione S-transferase (GST)
   are important enzymes in insect system and function on the metabolic
   and detoxification of the xenobiotics. GST in C. medinalis could
   involved in the detoxification of chlorpyrifos [[114]32]. Chen et al.
   [[115]33] discovered that two P450 genes CYP6CV1 and CYP9A38 may be
   involved in detoxification of rice phytochemicals. Veegala and Vemuri
   [[116]34] reported carboxylesterase and GST mediated in the resistance
   of C. medinalis to insecticide. In this study, 6 cytochrome P450, 3
   GSTs, 13 carboxylesterase were significant different expression in C.
   medinalis treated with Cry1C, suggesting that detoxification enzymes
   may be involved the early response of C. medinalis larvae to Cry1C
   toxin. Similarly, Guo et al. [[117]35] discovered that carboxylesterase
   were increased in S. exigua as the increased time of exposure to Bt
   cotton and considered that the allocation of carboxylesterase in body
   is a metabolic tolerance of herbivorous insects response to a toxic
   protein.

Protect enzymes

   Catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) are
   the important protection enzymes in insect [[118]36]. Here, we found 1
   CAT, 11 PODs and 4 SODs were found. In midgut of C. medinalis treated
   with Cry1C, 1 CAT, 9 PODs and 3 SODs were down regulated, and 2 other
   PODs and one SOD were upregulated. These results showed Cry1C impose
   environmental stress to C. medinalis larvae, anyhow the upregulated
   PODs indicated that the body of C. medinalis adjust the physiological
   activity to reply to Cry1C. Many reports indicated that the increase of
   POD is the emergence measures of the insect reply to Bt toxin
   [[119]35,[120]37].

Serine protease

   Serine proteases always function on the digestion and utilization of
   food, and the detoxification of exogenous proteins. Trypsin and
   chymotrypsin are two kinds of serine proteases and involved in the
   activation and degradation of Bt toxin [[121]38–[122]40]. The changes
   of serine proteases may be correlated with the resistance of the insect
   to Bt toxins. Tetreau et al. [[123]41] reported an increase in larval
   gut proteolytic activities in a Bt resistance strain of the Dengue
   fever mosquito. Elleuch et al. [[124]42] found that early degradation
   of Cry toxins by proteases in mosquito larvae midgut may be one of the
   reasons why Bt protein toxicity decreased. In our study, 34 trypsin and
   8 chymotrypsin were upregulated and 7 other trypsin down-regulated. In
   addition, twenty carboxypeptidases were differently expressed with 17
   genes up-regulated and 3 genes down-regulated. These results indicated
   that the trypsin, chymotrypsin and carboxypeptidases could play an
   important role on the tolerance of C. medinalis to Cry1C through
   degradation.

Bt receptors

   In the Bt action, Bt receptors are important on the formation of
   toxicity [[125]19]. So far, at least cadherin, alkaline phosphatase,
   and aminopeptidase N (APN) were putative to be the receptor of Bt
   toxins in insects [[126]15,[127]19]. However, there are no reports on
   these proteins as Bt receptors in C. medinalis. Unigenes on these
   proteins could provide invaluable information for the further study to
   prove these proteins as Bt receptors in C. medinalis. A previous study
   in the cabbage looper (Trichoplusia ni) indicated that the Cry1Ac
   resistance was correlated with the concurrent up-regulation of APN6 and
   down-regulation of APN1 [[128]43]. In our study, four APN were
   up-regulated (1.68- to 2.54-fold) and two APNs downregulated (2.76- and
   5.57- fold) in the C. medinalis treated with Cry1C toxin. The detailed
   role of these APNs on the tolerance or resistance of C. medinalis to
   Cry1C remained to be determined. Alkaline phosphatases have also been
   identified as Bt receptors in many insects [[129]19,[130]44–[131]46].
   Chen et al. [[132]47] found a toxin-binding alkaline phosphatase
   fragment could synergize Bt toxin Cry1Ac against susceptible and
   resistant Helicoverpa armigera. Here, two alkaline phosphatases were
   upregulated in C. medinalis larvae treated with Cry1C. According to the
   role of alkaline phosphatase in Bt action, alkaline phosphatases in
   tolerant or resistant insects to Bt toxin may do not increase, the
   results in our studies may indicate these two alkaline phosphatases may
   be not the receptor of the Cry1C or the results may be unexpected.
   Flores-Escobar et al. [[133]48] found alkaline phosphatase is more
   important than APN for Cry1Ab toxicity, while Cry1Ac relied principally
   on APN1 through RNAi experiment. Cadherin is an important Bt receptor
   and its mutation was correlated with the insect resistance to Bt toxins
   [[134]14,[135]19,[136]49]. Here, one unigene annotated as cadherin was
   downregulated in C. medinalis larvae treated with Cry1C. However, the
   upregulated cadherin belongs to flamingo-cadherin which is different
   with the cadherin in other insects related with Bt action.
   Additionally, the other cadherin in C. medinalis was not differently
   expressed. Whether this flamingo-cadherin correlated with Cry1C action
   remains to be determined.

ABC transporters

   ATP-binding cassette (ABC) transporters is a super family involved in
   various functions including the transport of lipids, inorganic ions and
   especially the detoxification of xenobiotics [[137]50]. ABC transporter
   plays important role in drug resistance [[138]51]. And in insect ABC
   transporters were shown functions in glucoside sequestration [[139]52],
   the transport of eye color pigments [[140]53] and insecticidal
   resistance [[141]54]. In this study, sixteen ABC transporters
   attributed to 5 subfamilies were regulated in C. medinalis treated with
   Cry1C and 3 upregulated and 13 down regulated. Previous studies have
   linked ABC transporters with Cry1 resistance in seven lepidopterans
   including Heliothis virescens, H. armigera, P. xylostella, T. ni,
   Ostrinia nubilalis, H. punctigera and B. mori
   [[142]29,[143]55–[144]59]. In the transcriptome of P. xylostella
   Cry1Ac-susceptible and resistant strains, many DGEs of ABC transporters
   were observed [[145]29]. The role of the ABC transporters found in this
   study on tolerance or resistance of C. medinalis to Cry1C warrants
   further investigation.

Enriched pathway

   Pathway analysis indicated that two pathways were significant enriched
   in the C. medinalis treated with Cry1C. The antigen processing and
   presentation pathway had 25 DEGs in the C. medinalis treated with
   Cry1C. Antigen processing is an immunological process that prepares
   antigens for presentation to special cells of the immune system called
   T lymphocytes [[146]60]. Subsequent presentation of the antigens on
   class I or class II major histocompatibility complex (MHC) molecules is
   dependent on the pathway, and MHC I and MHC II antigen presentation
   typically involves the endogenous and exogenous pathway of antigen
   processing, respectively [[147]60]. The transporter associated with
   antigen processing (TAP) represents a key machinery in MHC class I
   antigen presentation by translocating proteasomal degradation products
   into the lumen of the endoplasmic reticulum (ER) for loading onto MHC
   class I molecules, and TAP is a member of the ABC transporters
   [[148]61]. The ABC transporter pathway was an important enriched one
   due to previous report that ABC transporter was correlated with Bt
   resistance in insects [[149]62–[150]63]. Chronic myeloid leukemia (CML)
   pathway correlated with the apoptosis, and twelve DGEs were found in
   this pathway. ABC transporter also was important in the CML pathway
   [[151]64]. In addition, a mechanism of Bt action was reported to be
   related to apoptosis [[152]65–[153]67]. These two pathways may provide
   more information on the Bt action and insect response to Bt protein.

   In conclusion, multiple aspects were involved in the C. medinalis
   response to Cry1C protein from the transcriptomal data. Serine
   protease, detoxification enzymes, ABC transporter, antigen processing
   and presentation pathway, and chronic myeloid leukemia pathway may be
   main elements involved in the response of C. medinalis to Cry1C toxin.

Supporting information

   S1 Fig. Distribution of unigenes from the midgut transcriptome of
   Cnaphalocrocis medinalis.

   (TIF)
   [154]Click here for additional data file.^ (36.6MB, tif)
   S2 Fig. Distribution of transcripts from the midgut transcriptome of
   Cnaphalocrocis medinalis.

   (TIF)
   [155]Click here for additional data file.^ (36.1MB, tif)
   S3 Fig. Validation of transcriptome data by qRT-PCR.

   The means of at least three biological replicates are presented as
   log[2]FC ± SE.

   (TIF)
   [156]Click here for additional data file.^ (29.8MB, TIF)
   S1 Table. List of primers of genes for the validation of transcriptome
   data by qRT-PCR.

   (DOCX)
   [157]Click here for additional data file.^ (20.5KB, docx)
   S2 Table. Top 20 downregulated unigenes in the midgut of Cnaphalocrocis
   medinalis treated with Cry1C toxin.

   (DOCX)
   [158]Click here for additional data file.^ (27.8KB, docx)
   S3 Table. Top 20 upregulated unigenes in the midgut of Cnaphalocrocis
   medinalis treated with Cry1C toxin.

   (DOCX)
   [159]Click here for additional data file.^ (30KB, docx)
   S4 Table. Carboxylesterase genes differently expressed in
   Cnaphalocrocis medinalis larvae treated with Cry1C toxin.

   (DOCX)
   [160]Click here for additional data file.^ (28.3KB, docx)
   S5 Table. KEGG summary of Cnaphalocrocis medinalis response to Cry1C
   toxin.

   (XLSX)
   [161]Click here for additional data file.^ (19.4KB, xlsx)

Data Availability

   All relevant data are within the paper and its Supporting Information
   files.

Funding Statement

   This work was funded by National Natural Science Foundation of China
   (31501669), the earmarked fund for China Agriculture Research System
   (CARS-01-36), National Special Key Project of China (2016ZX08001-001),
   and Zhejiang Provincial Natural Science Foundation of China
   (LY15C140003).

References