Chemicals with ester functional groups such as species-specific pheromones, host plant odorants, and xenobiotic insecticides are important for insect physiology, biochemistry, and ecology (Xu and Turlings, 2018; Levi-Zada and Byers, 2021; Mdeni et al., 2022). Carboxylesterases (CXEs) belong to a superfamily that includes diverse functional enzymes (Oakeshott et al., 1999; Oakeshott et al., 2005). They hydrolyze esters to their corresponding acids and alcohols and are thus thought to be involved in xenobiotic detoxification, odorant degradation, and developmental regulations (Richmond et al., 1980; Leal, 2013; Godoy et al., 2021).

Insect CXEs possess highly conserved catalytic triads (Ser-Glu-His) and pentapeptide motifs (Gly-X-Ser-X-Gly). They are expressed in the midgut, fat body, and other detoxifying organs which metabolize pesticide organophosphates, pyrethroids, and carbamates in various insect species such as moths Helicoverpa armigera (Bai et al., 2019), Grapholita molesta (Li et al., 2023), Plutella xylostella (L.) (Li et al., 2021), and Spodoptera frugiperda (Carvalho et al., 2013); the aphid Aphis gossypii (Chang et al., 2010); and flies Lucilia cuprina (Heidari et al., 2005) and Drosophila melanogaster (Birner-Gruenberger et al., 2012). The overexpression of CXE genes has been reported as an important mechanism of insecticide detoxification. For instance, overexpressed CXE genes have been shown to be involved in indoxacarb resistance in Spodoptera litura (Shi et al., 2022); an overexpressed CXE (PxαE14) in the detoxification of multiple insecticides in P. xylostella (Li et al., 2022); and two overexpressed a-esterase genes mediated metabolic resistance to malathion in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Wang et al., 2015). Additionally, the alternative splicing and site mutation of CXEs are considered another mechanism in insecticide detoxification. The alternative splicing of TcCCE23 with different variants enhances fenpropathrin tolerance in Tetranychus cinnabarinus (Wei et al., 2023). Two different amino acid substitutions in the ali-esterase E3 confer alternative types of organophosphorus insecticide resistance in the sheep blowfly, Lucilia cuprin (Campbell et al., 1998).

In addition to xenobiotic detoxification, some CXEs have been demonstrated to be highly expressed at the antennae, the main insect olfactory organs, and function as odorant degrading enzymes (ODEs) that play crucial roles in olfactory cue degradation. The first insect ODE (ApolPDE) was identified from the silk moth Antheraea polyphemus; it displayed male antennae-specific expression and could rapidly degrade A. polyphemus major acetate sex pheromone, (E, Z)-6, 11-hexadecadienyl acetate [E6Z11–16Ac] (Vogt and Riddiford, 1981; Ishida and Leal, 2005). Another male antennae-specific CXE gene, the encoded protein (PjapPDE) of which was identified in the Japanese beetle, Popillia japonica, could degrade the sex pheromone component (R, Z)-5-(-)-(1-decenyl) oxacyclopentan-2-one [(R)-japonilure] (Ishida and Leal, 2008). In addition, an extracellular CXE, named esterase-6 (Est-6), in D. melanogaster was reported to be highly expressed in olfactory sensilla and involved in maintaining proper temporal dynamics of cis-vaccenyl acetate (cVA) detection (Chertemps et al., 2012). Subsequently, an increasing number of insect CXEs have been identified and functionally reportedly to be involved in degradation of both plant odorants and sex pheromones (Durand et al., 2010a; Durand et al., 2010b; Durand et al., 2011; Zhang et al., 2016; He et al., 2020).

Ectropis obliqua Prout and Ectropis grisescens Warren are two closely related tea geometrid species which have frequent outbreaks in tea gardens and result in serious damages to tea production (Zhang et al., 2014). At present, chemical control is still the main method against these two sibling species, which accelerates the risk of resistance development. A better understanding of the mechanism of insecticide detoxification and screening of the target genes involved in xenobiotic detoxification and odorant degradation is crucial for development of environmental friendly pest management strategies. The CXEs present potential targets due to their important roles in detoxifying various insecticides and degrading olfactory cues.

Previously, 35 EoblCXE genes have been identified in E. obliqua chemosensory organs and they displayed multiple tissues expression profiles (Sun et al., 2017). Whether these EoblCXEs in E. obliqua have their orthologs in the sibling species E. grisescens and the differences in their expression profiles are still unknown. Ectropis obliqua differs from E. grisescens in geographical distribution, sex pheromone composition, and symbiotic bacteria Wolbachia abundance (Luo et al., 2017; Li et al., 2019; Wang et al., 2020). Whether the differences in Wolbachia abundance are associated with CXE expression patterns should be investigated.

In this study, we focused on EoblCXE14, which showed a non-chemosensory organs-biased expression at the abdomen of E. obliqua adults (Sun et al., 2017). First, we cloned its orthologous gene EgriCXE14 in E. grisescens and analyzed its sequence characteristics; then we compared the expression profiles between the sibling species E. obliqua and E. grisescens and evaluated the potential effect of Wolbachia on their expression pattern. The results will help further investigation of the potential roles of CXE14 in xenobiotic detoxification.

Insect CXEs have been widely demonstrated to be involved in pesticide detoxification and pheromone and plant odorant degradation. Therefore, they represent a major target for development of eco-friendly pest management strategies. This study reports a pair of CXE orthologous genes, EoblCXE14 and EgriCXE14, which share a conserved motif and phylogenetic relationship but have different expression profiles in two closely related tea geometrid species, E. obliqua and E. grisescens.

Insect CXEs have been identified in most of the lepidopteran moth species, such as Spodoptera littoralis (Durand et al., 2010b), S. litura (Zhang et al., 2016), Carposina sasakii (Li and Zhang, 2022), Hyphantria cunea (Ye et al., 2021), and P. xylostella (Xie et al., 2017; Li et al., 2021; Wang et al., 2021), which represent important pests of agricultural food crops, cotton, fruit trees, and vegetables. By contrast, the identification of CXEs in tea plant pest, especially in the sibling species E. obliqua and E. grisescens, have not been well explored, although we have previously identified and characterized 35 CXEs in tea geometrid moth E. obliqua (Sun et al., 2017). In this study, we cloned a CXE gene from E. grisescens named EgriCXE14 which is the orthologs of EoblCXE14. Although it was not the full-length sequence, EgriCXE14 contained the main functional domain of insect CXEs including the highly conserved pentapeptide residues (GXSXA) and the Ser-His-Glu catalytic triad residues. In addition, some amino acid residues such as Arg, Phe, and His, which are considered key residues in the binding pocket associated with insecticide binding of H. armigera HarmCarE001g (Bai et al., 2019) or L. cuprina LcaE7 (Jackson et al., 2013), are also conserved in both EoblCXE14 and EgriCXE14 (Figure 1), indicating potential roles of these two CXE genes in detoxifying insecticides.

The phylogenetic analyses and larval tissue-specific expression patterns observed in this study support the above-mentioned speculation. EgriCXE14 and EoblCXE14 were found to cluster with HarmCarE001g, a carboxylesterase which has been demonstrated to detoxify pyrethroids in H. armigera (Bai et al., 2019) (Figure 1). Furthermore, qRT-PCR results showed that both EgriCXE14 and EoblCXE14 were highly expressed in the midgut, a detoxification metabolic organ of E. obliqua and E. grisescens larvae (Figure 3). Indeed, many CXEs of different moth species are highly detected in the midgut and play crucial roles in insecticide detoxification. The CXEs genes (CarE) in G. molesta have higher expression in the detoxification metabolic organs such as the midgut and fat body but lower expression in the head and cuticle, and are involved in the tolerance of insecticides (Li et al., 2023). In the diamondback moth, P. xylostella, PxEst-6 is highly expressed in the midgut and cuticles of the third instar larvae and is associated with pyrethroid insecticides metabolization (Li et al., 2021), whereas PxαE14 is predominantly expressed in the midgut and Malpighian tubule of larvae and contributes to detoxification of multiple insecticides (Li et al., 2022). CXE genes (SlituCOE) of S. litura are expressed in the midgut, Malpighian tubule, and fat body, and are related to insecticide indoxacarb resistance (Shi et al., 2022). Further functional studies of enzyme activities and RNAi knockdown are needed to test this hypothesis in two closely related tea geometrid Ectropis spp.

Despite the predominant expression in the larval midgut, the expression profiles of EoblCXE14 and EgriCXE14 are significantly different. The investigation of expression profiles at different developmental stages showed that EoblCXE14 was larvae-enriched in E. obliqua but EgriCXE14 was ubiquitously expressed at different developmental stages of E. grisescens (Figure 2). These results indicate that EoblCXE14 likely plays roles in E. obliqua larvae; however, EgriCXE14 probably functions in E. grisescens at multiple developmental stages. A possible role of CXE14 in degrading ester odorants cannot be excluded, because several CXEs in insect species reportedly contribute to degradation of insect pheromones and plant volatiles (Ishida and Leal, 2005; Durand et al., 2011; Chertemps et al., 2012; He et al., 2020).

The expressional comparison in the larval midgut of the two Ectropis spp. showed that the expression level of EoblCXE14 was significant higher than that of EgriCXE14 (Figure 4). Previous studies proposed that E. obliqua differs from E. grisescens in symbiotic bacteria composition, particularly the Wolbachia (Zhou et al., 2016; Wang et al., 2020); specifically, E. grisescens has a significant higher abundance of Wolbachia than E. obliqua. Wolbachia has been shown to affect the expression of genes in insect (Pfarr et al., 2008; Cai et al., 2021; Sun et al., 2022). Therefore, in this study, we tested whether Wolbachia was correlated with different expression levels of CXE14 in the midgut of the two Ectropis spp. The qRT-PCR results showed that EgriCXE14 in E. grisescens mutant, in which Wolbachia was removed using tetracycline, was also highly expressed in the midgut (Figure 5), and no significant differences in EgriCXE14 expression levels between E. grisescens and E. grisescens mutant were found (Figure 6). These results suggest that Wolbachia may not be related to EgriCXE14 expression in the E. grisescens midgut, and may not contribute to the different expression level in the midgut of these two sibling species. These findings are not consistent with our previous reports that Wolbachia could be correlated with the expression of a chemosensory gene CSP8 in E. obliqua and E. grisescens (Yan et al., 2022). Notably, E. grisescens mutant collected from the Yizheng, Jiangsu province, differs from the E. grisescens from Yueqing, Zhejiang province; therefore, we cannot rule out the possible effect of geographical population on the aforementioned findings. Further expression variation of target genes in different geographical population, and between wild (with Wolbachia) and mutant (without Wolbachia) E. grisescens from the same geographical region need to be evaluated.

In conclusion, this study characterized a pair of CXEs orthologs, EoblCXE14 and EgriCXE14, in two sibling geometrid mode species, E. obliqua and E. grisescens. The results revealed that EoblCXE14 and EgriCXE14 share a conserved motif and phylogenetic relationship but differ in expression profiles at different developmental stages and expression levels in the larval midgut. EoblCXE14 was predominately expressed in the E. obliqua larvae, whereas EgriCXE14 was abundant in E. grisescens at multiple developmental stages. The expression level of EoblCXE14 in the E. obliqua midgut was significantly higher than that of EgriCXE14 in the E. grisescens midgut. In addition, potential effects of Wolbachia on the CXE14 expression levels were examined. This study provides the first report on comparative expression profiles of orthologous CXE genes in two sibling geometrid moth species and could help identify potential targets for controlling tea geometrid.