Journal of Insect Science, (2019) 19(5): 16; 1–9 doi: 10.1093/jisesa/iez092 Molecular Entomological Genetics Regulation of Hormone-Related Genes in Ericerus pela (Hemiptera: Coccidae) for Dimorphic Metamorphosis Liu PengFei,1,2 Wang Weiwei,1 Ling Xiaofei,1 Lu Qin,1 Zhang Jinwen,1 He Rui,1,3 and Chen Hang1,3,4 1Research Institute of Resources Insect, Chinese Academy of Forestry, Kunming 650224, China, 2NanJing Forestry University, Nanjing 210000, China, 3The Key Laboratory of Cultivating and Utilization of Resources Insects, State Forestry Administration, Kunming 650224, China, and 4Corresponding author, e-mail: [email protected] Subject Editor: Bill Bendena Received 13 July 2019; Editorial decision 14 August 2019 Abstract Insect hormones regulate metamorphosis including that leading to sexual dimorphism. Using RNA-Seq, we discovered that the second-instar male larva (SM) of the white wax insect, Ericerus pela, have 5,968 and 8,620 differentially expressed transcripts compared with the second-instar female larva (SF) and the first-instar male larva (FM), respectively. The expression levels of genes involved in the apoptosis of old tissues and the reconstruction of new ones in the SM significantly enhanced, while the SF mainly has enhanced expression levels of anabolic genes such as chitin. We predicted that the second-instar larvae are the developmental origin of sexual dimorphic metamorphosis. Meanwhile, in the juvenile hormone (JH) metabolic pathway, CYP15A1 and JH esterase (JHE) are differentially expressed; and in the 20-hydroxyecdysone (20E) metabolic pathway, CYP307A1, CYP314A1, and CYP18A1 are differentially expressed. In the SM, the expression levels of CYP307A1 and CYP314A1 are significantly increased, whereas the expression level of CYP18A1 is significantly decreased; in the SF, the expression levels of the above genes are opposite to that of the SM. Expression trends of RNA-seq is consistent with the expression level of qRT–PCR, and seven of them are highly correlated (R ≥ 0.610) and four are moderately correlated (0.588 ≥ R ≥ 0.542). Key words: transcriptome, dimorphic, metamorphosis, hormone The white wax scale insect Ericerus pela (Chavannes) (Hemiptera: the pupation process under the wax layer. The adult male dies after Coccidae) is distributed in northern subtropical, subtropical and mating with the female on the 10th to 15th day (Chen 2011). temperate regions. It is a vital resource insect with over a thousand It was previously reported that the sexual dimorphism of years usage history in China. The second-instar male larvae secrete a E. pela was an ecological phenomenon of insect sex separation and large amount of white wax whose main component is ceryl cerotate, differentiation caused by differential mating processes or sexual a 26-acid and 26-lipid (C25H51COOC26H53) compound, with a high selection (Yang et al. 2015), and which was jointly regulated by ju- nutritional, health, economic, and ecological value. It is widely used venile hormone (JH) and molting hormone (20-hydroxyecdysone in printing, machinery, medicine, foodstuff, cosmetics, and related [20E]) (Röller et al. 2010). JH, a sesquiterpenoid hormone, has fields (Chen 2011). the functions of maintaining the growth characteristics of larvae: Ericerus pela shows a unique sexual dimorphic metamorphosis in regulating growth and development, promoting ovarian mat- its life cycle. The female develops from an egg (E), to a first-instar uration, and preventing larvae from prematurely entering the larvae (FF), a second-instar larvae (SF), an early female adult (EA), next instar (Qu et al. 2018). 20E, a steroid hormone, regulates through to a late female adult (LA) in a hemimetabolous development the metamorphosis process of the insect body, including molting, mode. In contrast, the male develops from an egg (E), to a first-instar tissue apoptosis, and reconstruction (Mai et al. 2017). When the larvae (FM), a second-instar larvae (SM), a pre-pupal stage (PP), a concentration of JH in the larvae is high, the insect larvae state pupal stage (P), through to an adult (MA) in holometabolous devel- is maintained, and the metamorphosis is prevented. At the end opment mode (Fig. 1a). The life cycles also differ—the female up to of each larvae instar, the concentration of JH decreases and 20E 1 yr, with a main purpose of laying eggs in the adult stage, while the increases, promoting pupation or metamorphosis into adulthood male life cycle is only about 5 mo. In males, the SM produce white (Rachinsky et al. 1990, Sakurai and Satake 1998, Truman and wax which they use to cover themselves and then they complete Riddiford 1999). JH and 20E play major regulatory roles in the © The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ 1 licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Journal of Insect Science, 2019, Vol. 19, No. 5 Fig. 1. (a) The life history of male and female worms of E. pela, in which E represents egg, FF represents first-instar female nymph, SF represents second-instar female nymph, EA represents early female adult, and LA represents late female adult. FM represents the first-instar male nymph, SM represents the second- instar male nymph, PP represents the pre-pupa, P represents the pupa stage, and MA represents the male adult stage. (b) Results of database annotation of the transcriptome of E. pela; x-axis is the database, right-y-axis is the number of transcripts annotated and left-y-axis is percentage. (c) VN diagram analysis of transcripts of the expressed between the six stages of E. pela. development of the unique sexual dimorphic metamorphosis in RNA-seq E. pela (hemimetabolous of the female and holometabolous of the Hundred milligrams of E. pela tissues of each developmental stage male) (Yang et al. 2015), but their regulatory mechanisms are not were used to isolate total RNA separately using an EZ-10 Total RNA completely understood. Mini-Preps Kit (Sangon Biotech, Shanghai, China) according to the We explore differential expression of transcripts using transcrip- manufacturer’s protocols. RNA purity and integrity were assessed tome data, with a particular emphasis on the second-instar larvae. using a RNA Nano 6000 Assay Kit (Agilent Technologies, CA). We use KEGG metabolic pathway to identify genes related to JH and To prepare for RNA-seq analysis, the mRNA was enriched with 20E metabolism, analyze the expression levels of ones, and predict Oligo (dT) magnetic beads, broken into short fragments with a frag- the key genes involved in regulating dimorphic metamorphosis. We mentation buffer, and the first strand cDNA was synthesized with also preliminarily illustrate the differences phenomenon in sexual random hexamer primers using mRNA as a template. The second dimorphic metamorphosis and establish the development patterns strand of cDNA was then synthesized by adding buffer, dNTPs, of sexual dimorphic of E. pela. DNA polymerase I, and RNase H. The double-stranded cDNA was purified with AMPure XP beads, the ends were repaired (the poly Materials and Methods A tail and the sequencing linker were ligated), and the fragment size was selected using AMPure XP beads. Finally, PCR amplification Sample Collection was performed and the PCR product was purified using AMPure XP Ericerus pela samples were collected from Ligustrum lucidum by the beads to obtain the final cDNA library. Research Institute of Resource Insect, Chinese Academy of Forestry, After the library was constructed, preliminary quantification was Kunming, China (25° 3′20′′N, 102° 45′ 16′′E, 1,950 m above sea performed using Qubit2.0, and the library was diluted to 1.5 ng/μl. level), and the eggs come from Zhaotong, Sichuan. Samples of FF, SF, The insert size of the library was detected using an Agilent 2100. EA, LA, FM, and SM were collected in three sets of each group, and As the insert size was as expected, the effective concentration of the were stored at −80°C. library was determined by qPCR; accurate quantification (effective Journal of Insect Science, 2019, Vol. 19, No. 5 3 library concentration > 2 nM) was performed to ensure the quality of obtained transcripts of the FF, SF, EA, and LA of the female, and the the library. The different libraries were then pooled according to the FM and SM of the male. After Trinity splicing, there were 528,309 effective concentration and the target data volume, and sequenced transcripts (Supp Fig. S1 [online only]). The longest cluster sequence with Illumina HiSeq. obtained by HCA was used in subsequent analysis, and we obtained 249,288 longest transcripts (unigenes) (Supp Fig. S2 [online only]). Nonparametric Transcriptome Assembly and The 249,288 transcripts were then annotated with seven major data- Annotation bases: NR, NT, KO, SwissProt, PFAM, GO, and KOG; there were RNA-seq reads were filtered to remove adaptors and low-quality 79,710, 56,789, 90,672, 97,375, 98,216, 59,154, and 16,273 tran- sequences to obtain clean reads. The clean reads were spliced using scripts annotations, respectively; and all the transcripts were suc- Trinity (Grabherr et al. 2011) to obtain transcript files stored in cessfully annotated (Fig. 1b and Supp Fig. S3 [online only]). In total, Fasta format. The transcripts were then hierarchically clustered 158,968 (63.76%) transcripts were expressed in the FM, 149,025 using the number of reads and expression patterns of the aligned (59.78%) transcripts in the SM, 162,651 (65.25%) transcripts in the transcripts on Corset’s official website at https://code.google.com/p/ FF, 182,538 (73.22%) transcripts in the SF, 120,901 (48.50%) tran- corset-project/ (Davidson and Oshlack 2014).