Juvenile hormone (JH) is a sesquiterpenoid hormone synthesized and secreted by the corpora allata that regulates the development, metamorphosis, reproduction, and other physiological processes of insects (Bernardo and Dubrovsky 2012, Bilen et al. 2013, Flatt et al. 2005, Jindra et al. 2013, Riddiford 2012). Its primary role is to maintain the status quo during larval development, in close cooperation with molting hormones (ecdysteroids) (Riddiford 2012). Although the mode of action of JH was described many years ago, the molecular mechanisms underlying JH were largely unknown until the candidate JH receptor, methoprene-tolerant (Met) protein, a basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) family of transcription factors (TFs), first described in Drosophila melanogaster Meigen (Diptera: Drosophilidae) (Ashok et al. 1998, Wilson and Fabian 1986), was identified. Met homologs have since been identified in other taxa (Kayukawa et al. 2012, Konopova et al. 2011, Lin et al. 2015, Zhu et al. 2010). JH first binds to Met and then recruits the heterodimer partner steroid receptor coactivator (also called Taiman or FISC) to form an active JH–receptor complex that regulates the expression of JH-responsive genes (Furness et al. 2007; Jindra et al. 2013, 2015a, 2015b; Li et al. 2011; Partch and Gardner 2010; Zhang et al. 2011).

The TF Krüppel homolog 1 (Kr-h1), first discovered in D. melanogaster and homologous with the segmentation gene Krüppel (Beck et al. 2004, Pecasse et al. 2000), is one such gene that acts as a key regulator suppressing metamorphosis in insects. Kr-h1 contains eight C2-H2-type Zinc fingers (DNA binding motif) and has been shown to be the direct target of Met and to regulate insect development, metamorphosis, and vitellogenesis. In the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), functional analysis of Kr-h1 has revealed that knockdown of both TcMet and TcKr-h1 causes precocious metamorphosis (Konopova and Jindra 2007, Minakuchi et al. 2009, Parthasarathy et al. 2008). In addition, RNA interference (RNAi) suppression of Kr-h1 in Pyrrhocoris apterus (L.) (Hemiptera: Pyrrhocoridae) causes precocious metamorphosis at the early fourthinstar nymphs (Konopova et al. 2011), whereas overexpression of Kr-h1 in Drosophila and Bombyx mori L. (Lepidoptera: Bombycidae) prevented pupation (Kayukawa et al. 2014, Minakuchi et al. 2008).

In addition to repressing metamorphosis, Kr-h1 has been found to play a role in female reproduction by regulating vitellogenin and oogenesis in the mosquito Aedes aegypti (L.) (Diptera: Culicidae) (Ojani et al. 2018, Shin et al. 2012, Zou et al. 2013) and migratory locust Locusta migratoria L. (Orthoptera: Acrididae) (Song et al. 2014). Although RNAi suppression of Kr-h1 did not reduce fecundity in bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), it reduced hatchability (Gujar and Palli 2016).

It is now evident that Kr-h1 plays a crucial role in both metamorphosis and reproduction. Recent studies have revealed that Kr-h1 prevents the activation of metamorphosis by suppressing the expression of the Broad-complex (BR-C) and Ecdysone induced protein 93F (E93) genes (Belles and Santos 2014, Jindra et al. 2013). Mechanistically, Kr-h1 binds directly to the consensus Kr-h1 binding site (KBS) in the promoter regions of target genes to suppress their transcription (Kayukawa et al. 2016, 2017). It has recently been discovered that Kr-h1 suppresses steroidogenic enzyme by binding to the KBS regions of steroidogenic enzyme gene promotors in D. melanogaster and B. mori, thereby suppressing 20-hydroxyecdysone (20E) synthesis (Liu et al. 2018, Zhang et al. 2018). Although the role of Kr-h1 in insect development and reproduction is relatively well understood, the potential TF genes that are regulated by Kr-h1 remain largely unknown.

In this study, we used the rice (Oryza sativa L.) pest Chilo suppressalis (Walker) (Lepidoptera: Crambidae) as a model species and used RNAi and high-throughput RNA sequencing to identify potential TFs that regulated by Kr-h1. The results indicate that several TFs may be regulated by Kr-h1 to mediate development and reproduction in C. suppressalis.

Materials and Methods

Insect rearing. Chilo suppressalis larvae were collected from the campus greenhouse of Hunan Agricultural University in April 2017 and reared on water bamboo Zizania latifolia (Griseb.) Turcz. Stapf. at 28 ± 1°C and 80 ± 10% relative humidity under a 16-h photoperiod. Adults were fed on 10% sucrose.

RNA isolation and synthesis of first-strand cDNA. Total RNA was isolated from samples by using RNAiso Reagent (TaKaRa, Dalian, China) according to the manufacturer's instructions. Its purity and concentration were quantified with a spectrophotometer (2.1 > OD 260/280 > 1.8; NanoDrop™ 1000, Thermo Fisher Scientific, Waltham, MA). Complementary DNA (cDNA) was synthesized using the PrimeScript RT reagent kit with gDNA eraser (TaKaRa) according to the manufacturer's recommendations.

RNAi. The target gene fragment was amplified from C. suppressalis pupal cDNA by using a PrimeSTAR HS DNA polymerase kit (TaKaRa) with a flanking T7 promoter and T7 terminator site. A segment of enhanced green fluorescent protein (EGFP; accession no. U55762) was the negative control for nonspecific effects in RNAi experiments. For double-stranded (ds)RNA synthesis, a T7 RiboMAX™ Express RNAi System (Promega, Madison, WI) was used to produce dsRNA against Kr-h1 and EGFP from approximately 1 mg of the DNA template. The integrity and concentration of the dsRNA obtained were verified by electrophoresis in 1% native agarose gel.

Five hundred nanoliters of dsKr-h1 solution (5 lg/ll) was injected into the abdomen of female pupae 24 and 48 h after pupation by using a nanoinjector (Drummond Scientific Co., Broomall, PA). The control group was injected with dsEGFP according to the same protocol. Four individuals were randomly selected for detection of RNAi efficiency and high-throughput RNA sequencing.

cDNA library construction and transcriptome analysis. Total RNA was extracted from individuals in the control and treatment groups with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA integrity was verified with gel electrophoresis, and its concentration was measured with a Qubit^® RNA Assay Kit in a Qubit^® 2.0 fluorometer (Life Technologies, Rockville, MD). We used 1.5 lg of total RNA to construct the cDNA library, which was sequenced using a HiSeq platform (Illumina, San Diego, CA).

A method adapted from Qiu et al. (2018) was used to produce clean reads, and a Trinity assembly was created using Trinity software based on the left.fq and right.fq (Grabherr et al. 2011). The putative function of unigenes was deduced by aligning them against Pfam (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Nr (National Center for Biotechnology Information [NCBI] nonredundant protein sequences), and Nt (NCBI nonredundant nucleotide sequences).

Differential gene expression. The fragments per kilobase of transcripts per million mapped reads method was used to measure the differential expression of genes in the treatment and control groups (Mortazavi et al. 2008). DESeq R package (Version 1.10.1) was used to analyze the differential expression of genes in each group. Benjamini–Hochberg approach was used to adjust P values to control the false discovery rate. Genes with an adjusted P value of <0.05 were considered differentially expressed.

Identification of TFs. TFs were verified by manually using BLASTX with an E-value cutoff of ≤10^–5 to check their amino acid sequences against the NCBI nonredundant protein database.

RT-qPCR. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to verify the expression of candidate TFs that were regulated by Kr-h1. Total RNA was isolated from the control and treatment (iEGFP and iKr-h1) groups, and cDNA was synthesized using a PrimeScript RT reagent kit with gDNA eraser (TaKaRa) as described above. Gene-specific primers (Table 2) were designed using the NCBI profile Server (http://www.ncbi.nlm.nih.gov/tools/primer-blast). The C. suppressalis elongation factor-1 was used as the internal reference gene for RT-qPCR (Hui et al. 2011, Zhu et al. 2016). Quantitative real-time PCR was conducted with a SYBR Premix ExTaq (Perfect Real Time) kit (TaKaRa) according to the manufacturer's instructions and a CFX 96 Touch System (Bio-Rad Laboratories, Hercules, CA). The efficiency of the RT-qPCR primers was assessed from a standard curve (cDNA concentration versus cycle threshold [Ct]) based on a fivefold dilution series of pupal cDNA corresponding to 1 mg of total RNA. The qPCR protocol used was that of Qiu et al. (2018).

Table 2 Specific primers used in real-time quantitative polymerase chain reaction (RT-qPCR) and RNA interference (RNAi).^a

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Table 2 Continued.

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Data analysis. Quantitative expression data were analyzed using the 2^–44Ct method (Pfaffl 2001), and the statistical significance of differences in treatment group means were assessed with a one-way analysis of variance (ANOVA) in the Statistical Package for the Social Sciences program for Windows (SPSS, Chicago, IL).

Results

RNAi depletion of Kr-h1 in C. suppressalis pupae. High-throughput sequencing combined with Kr-h1 RNAi was used to identify potential TFs that were regulated by Kr-h1. Injection of Kr-h1 dsRNA significantly reduced transcription of Kr-h1 by 77% compared with EGFP dsRNA (Fig. 1A). We identified 7,845 differentially expressed transcripts controlled by Kr-h1 in female pupae (Fig. 1B, C).

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Kr-h1 RNAi depletion revealed differential expression of TFs. Total RNA from dsRNA-treated pupae was subjected to high-throughput sequencing and screening to identify differentially expressed TFs. The TFs identified included Zinc finger-C2H2, Zinc finger-GATA, Zinc finger-LITAF (lipopolysaccharide-induced tumor necrosis factor–activating factor-like), Zinc finger-BED, ZBTB, THAP, PAX, MYB, HSF, Homeobox, HMG, CSD, bHLH, STAT, RHD, and MBD families (Table 1). Functional annotation identified that iKr-h1 downregulated 29 and upregulated 12 of the above-mentioned genes, suggesting that these 41 genes interact with Krh1 to regulate development and reproduction.

Table 1 Differential expression of candidate transcription factor genes after RNA interference knockdown of Kr-h1 in Chilo suppressalis.

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Table 1 Continued.

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Table 1 Continued.

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Table 1 Continued.

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Validation of high-throughput sequencing data with RT-qPCR. To verify the differential expression of candidate TFs, total RNA was isolated for analysis via RT-qPCR. Specific primers were designed based on the sequences of 14 randomly selected Kr-h1-regulated genes identifed by RNA high-throughput sequencing. For the bHLH families, two genes, CL2668.Contig2_All and CL238.Contig8_All, were more highly expressed in the treatment group than in the control group. This result is consistent with that of the transcriptome analysis and confirms that the mRNA levels of these two genes increased in pupae injected with Kr-h1 dsRNA. Expression levels of the other tested genes, CL290.Contig1_All and CL2209.Contig8_All, did not significantly differ between the treatment and control groups (Fig. 2A). With respect to the Zinc finger-C2H2 families, expression of CL569.Contig2_All was slightly higher in the treatment group but that of Unigene1796_All was not significantly different from that in the control group. These results are inconsistent with those from the high-throughput sequencing data (Fig. 2B). With respect to the HMG TFs, expression of CL1804.Contig1_All in the treatment group was not significantly different from that of the control group, whereas expression of CL2860.Contig1_All was lower in the treatment group than in the control and that of Unigene16623_All was higher (Fig. 2C). We also analyzed the expression levels of CL1287.Contig1_All, Unigene3678_All, and Unigene18554_All, which belong to the ZBTB, THAP, and Zinc finger-LITAF-like families, respectively. Expression of all three genes was lower in the treatment group than in the control (Fig. 2D, F, G). Contrary to the results of the transcriptome analysis, RT-qPCR indicated that expression of CL1640.Contig2_All (Zinc finger-GATA) and Unigene18466_All (Zinc finger-BED) differed between the treatment and control groups (Fig. 2E, H).

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Discussion

Kr-h1 has been shown to be induced by JH hormone and to be involved in preventing metamorphosis and promoting reproduction in both holometabolous and hemimetabolous insects (Konopova et al. 2011, Lozano and Belles 2011, Minakuchi et al. 2011, Parthasarathy et al. 2010, Zhu et al. 2010). Consequently, Kr-h1 continues to be one of the most important target molecules for understanding the JH signaling pathway mechanism (Lozano and Belles 2011).

Kr-h1 is not only induced by JH to repress metamorphosis but also is induced by ecdysone signaling (Beckstead et al. 2005, Pecasse et al. 2000). There is evidence to suggest that JH is not necessary to induce Kr-h1 expression in B. mori and, like BmKr-h1, D. melanogaster Kr-h1 can also be upregulated by 20E (Beckstead et al. 2005, Pecasse et al. 2000). Kr-h1 plays a key role in reproduction; however, previous studies have found that Kr-h1 does not directly induce Vg expression, implying that Kr-h1 activates other regulators of Vg expression via other TFs in the fat body (Song et al. 2014). We used RNAi and RNA high-throughput sequencing to identify the candidate TFs that regulated by Kr-h1 (Table 1) and RT-qPCR to confirm the expression of TF genes that were induced and suppressed by iKr-h1 (Fig. 2).

We screened Zinc finger TF families, including Zinc finger-C2H2, Zinc finger-GATA, Zinc finger-LITAF, and Zinc finger-BED. TFs (GATAs) have been demonstrated to be involved in the proliferation, differentiation, and development of eukaryotes (Patient and McGhee 2002). In some blood-feeding insects, GATA is synthesized after a blood meal and it, in turn, activates the expression of Vg (Attardo et al. 2005, Boldbaatar et al. 2010, Park et al. 2006, Sun et al. 2018). Functional analysis has shown that BmGATAβ4 binds directly to the GATA cisresponse element 1 (CRE1) and CRE2 in the promoter of BmVg to upregulate Vg expression (Liu et al. 2019). Although Kr-h1 may induce Vg expression via the JH signaling pathway, there is no evidence that it binds directly to the Vg promoter. Consequently, we speculate that Kr-h1 regulates the transcription of Vg in combination with GATAs or antagonized GATAs.

We postulate that Kr-h1 belongs to Zinc finger families that directly bind to the Krh1 binding site (KBS) to promote the pupal specifier gene Broad-Complex (BR-C) and the adult specifier gene Ecdysone induced protein (E93), thereby suppressing larval-to-pupal transition and adult metamorphosis, respectively (Belles and Santos 2014; Jindra et al. 2013; Kayukawa et al. 2016, 2017). Kr-h1 has also recently been found to bind directly to KBS in the promoter region of steroidogenic enzyme genes to downregulate ecdysone biosynthesis in Drosophila and Bombyx (Zhang et al. 2018). As part of this study, we confirmed the transcription levels of E93 and BR-C in C. suppressalis with qPCR and iKr-h1, and the results were consistent with those of previous studies (data not shown). Based on our qPCR data (Fig. 2), the expression of other candidate Zinc finger TF genes were up- or downregulated by RNAi; however, the mechanism regulating the interaction between Kr-h1 and these factors needs further study.

Our results identified bHLHs family genes as likely TFs, and transcripts of CL238.Contig8_All and CL2668.Contig2_All were higher in the treatment than in the control group, suggesting that this was due to the knockdown of Kr-h1. Met is a well-established JH receptor that belongs to the bHLH-PAS TF family. Previous studies have demonstrated that the bHLH domain is indispensable for inducing Krh1 transcription (Cui et al. 2014); hence, we suspect that bHLH proteins may mediate Kr-h1 expression either directly or indirectly by interacting with other bHLH factors like Met via JH signaling. Functional analysis suggests that Kr-h1 mediates the bHLH gene to regulate develoment and reproduction, but confirming this requires further gene identification and characterization.

In conclusion, a combination of RNAi, high-throughput sequencing, and qPCR identified candidate TFs that regulated by Kr-h1 in C. suppressalis, including members of the bHLH, Zinc finger, and other protein families. This the first study to comprehensively identify candidate TFs that regulated by Kr-h1 in C. suppressalis. Consequently, our results provide a useful foundation for future research on these proteins in both C. suppressalis and other insect pests.