The rice leaf folder C. medinalis has become a major threat to rice production in many Asian countries. The inherent molecular structure of RLF determines its characteristics, such as leaf folding, prolificacy, and dispersal. Therefore, an exhaustive understanding of molecular mechanisms regulating RLF behavior and activities may allow us to control the migratory pest with more sustainable and environmentally friendly approaches.
Surprisingly, BLASTx annotation of RLF transcriptome sequences revealed the highest similarity with T. castaneum of the order Coleoptera, while demonstrating a lower identity match (8.85%) with B. mori, a representative of the order Lepidoptera, which also encompasses RLF. To explain this similarity, we determined the number of protein sequences from T. castaneum, N. vitripennis, and B. mori in the Nr database and found 27,273, 10,637, and 6,842 protein sequences, respectively. It is clear that the protein sequences available in the database are fewer for B. mori than for T. castaneum. Therefore, the results may be due to the fewer protein sequences available for B. mori; however, the relationship between these species requires further investigation.
To the best of our knowledge, our work represents the first analysis of the RLF transcriptome and DGE profile data that greatly complemented and enriched the RLF database potentially to facilitate the determination of novel genes, gene functional analysis, insecticidal targets, developmental molecular mechanisms, and sex differentiation of RLF. Five DGE libraries of various developmental stages were constructed to analyze RLF gene expression patterns. In the RLF holometabolous development, the number of differentially expressed genes between the pupa and the adult was smaller than that between the pupa and the 3rd
instar larva; the latter was almost as large as that between the 3rd
instar larva and the egg. When examining the 3rd
instar larva differential gene expression, the number of upregulated genes was slightly more than the downregulated genes compared to the egg. The majority of upregulated genes were involved in metabolic processes, suggesting that the 3rd
instar larvae have stronger metabolic activities. In the pupal stage, the number of downregulated genes was greater than the upregulated genes compared with the 3rd
instar larvae. There were fewer differentially expressed genes in the pupa compared to the adult and more differentially expressed genes in the pupa compared to the larva. This result most likely indicates that pupa development occurs at the time of histogenesis when the old tissues have been destroyed and the new adult tissues or organs are being formed. Each RLF developmental stage demonstrates a large number of specifically differentially expressed genes that are most likely associated with developmental differentiation (Table S13
). Insect metamorphosis and development are physiological behaviors that are modulated by the procedural and tissue-specific expression of a series of genes under hormonal control. Several genes, such as EcR
(juvenile hormone binding protein
(Kruppel homolog 1), and broad
, were detected in the transcriptome database (Table S2
); it is believed that these genes play key roles in holometabolous development of insects 
In China, RLF has been primarily controlled by chemical methods over the years; therefore, under long-term selection pressures, the insect has developed resistance to commonly used insecticides, such as chlorpyrifos, trichlorphon, and carbosulfan. However, little is known about the molecular mechanisms behind the resistance of RLF. Our transcriptome data provide an enormous amount of genetic information that would facilitate research on resistance monitoring, mechanisms, and management strategy of the pest. Insect resistance to insecticides typically involves increases in the metabolic capability of detoxificative enzymes, as well as decreases in target site sensitivity; the former could be a more flexible way for insects to cope with xenobiotics. P450s and their associated P450 reductases can mediate resistance to all classes of insecticides via the upregulation and point gene mutations 
. In this study, we have characterized an additional 10 RLF P450 genes that fall into six families. Another important group of metabolic enzymes are GSTs that can mediate resistance to organophosphate, organochlorines, and pyrethroids through gene amplification and overexpression 
. To date, none of the GST genes from RLF has been reported in GenBank. We identified 10 GST genes of RLF and grouped them into six classes, thus filling the research gap. CarEs are also a class of important metabolic enzymes that are implicated in the resistance of insects to organophosphate, carbamates, and pyrethroids through gene amplification, upregulation, and coding sequence mutations 
. Six CarE genes were identified in the RLF transcriptome, thus providing new genetic resources where previously no such gene information existed. In view of this finding, our research provides the foundation for understanding the molecular mechanisms underlying insecticide resistance and further implementation of environmentally sustainable pest management.
The insect olfactory system is a highly specific and sensitive chemical detector essential for feeding, mating, finding sites of oviposition and development, and withdrawing from hostile environments. In insects, the recognition of odorants occurs through a complex series of events that involve a variety of proteins, such as OBP, CSP (chemosensory protein), and OR (odorant receptor). Hydrophobic odorant molecules are combined with OBPs in the hydrophilic lymph and are transported to reach ORs on the olfactory neuron twig, leading to activation. This result evokes olfactory nerve excitation that passes to the nerve center, leading to the perception of odorants 
. It has been believed that OBPs have key functions in recognizing and delivering hydrophobic odorants to ORs. We identified 14 OBPs from C. medinalis
, which were classified into three groups: GOBP, PBP, and ABP. Additionally, 8 CSPs (e. g. unigene846 and unigene2263) were found in the RLF transcriptome data and were believed to perform similar roles with OBPs, despite their different structures 
. These data provide valuable new information for further research on molecular mechanisms of RLF olfactory behavior.
is a migratory rice pest that possesses sufficient flight capacity for dispersal. It is thought that RLF fails to survive the winter in mainland China and the early-summer adults are supposed to be overseas migrants. The Indochinese peninsula serves as the source of RLF population that migrates annually during April and May to the south of mainland China, where these moths reproduce three to four generations before subsequently expanding northward during June and July. Throughout late August to early September, RLF migrates back southward and leaves China by November 
. RLF Migration is a key factor that induces the population abundance and infestation. This behavior is regulated not only by environmental conditions but also by its own physiological factors, with molecular modulation playing an important role in this process. The DGE profile indicated that several genes pertaining to RLF flight muscle cytoskeleton, including flightin
, and kettin
, were expressed at significantly higher levels in adults (Table S11
). These genes directly determine the flight muscle structure and the flying ability of RLF. Muscle contractions in insects are under the simultaneous regulation by both myosin and actin 
. The troponin (Tpn) complex is composed of three closely interacting subunits, namely, Tpn C, Tpn T, and Tpn I, which promote contraction 
. The expression of myosin
plays a key role in RLF migration behavior. Additionally, several other proteins participate in the assembly and regulation of myofibrils, comprising paramyosin, actinin, myosin rod protein, flightin, and kettin 
. We therefore propose that targeting the structural flight muscle genes may inhibit their expression, thereby degrading the muscles to control RLF.
The DGE profile indicated that the 12 genes pertinent to sex differences exhibited differential expression between the male and female moths; Vg
, and Tra2
genes demonstrated upregulated expression levels, whereas PBP
, and CYP6CV1
genes showed downregulated expression levels in the female compared to the male moth. The results were further confirmed by qRT-PCR. We were surprised to find that TSD
gene was differentially expressed between the male and female because such temperature-dependent sex determination (TSD) primarily exists in reptiles 
. The small heat shock proteins are involved in the TSD molecular process 
. This result implies that RLF is likely to employ TSD in a similar manner as reptiles; however, this finding requires further investigation. Previous studies have shown that PBP
genes are differentially expressed between the male and female adults 
; however, our DGE data have indicated that the corresponding mRNA expression levels are higher in RLF male moths than in female moths. In addition to detoxification functions, the three enzymes (GST, CarE7, and CYP6CV1) may also function as odorant-degrading enzymes (ODEs) that inactivate pheromones and other odorants 
. In fact, there are numerous candidate genes with significant differential expression between males and females, thereby providing a valuable resource for further studies on sex determination and differentiation in RLF.
In summary, we have performed a comprehensive gene expression analysis in RLF. Although the biological functions of most RLF genes remain unclear, the transcriptome and DGE data provide worthwhile information for further research concerning development, sex differentiation, migratory flight, olfactory behavior, and insecticide resistance to assist in uncovering the underlying molecular mechanisms of this agricultural pest.