Chitin is well known as an essential structure component of the exoskeletons (Yu et al., 2016; Noh et al., 2018; Qu et al., 2022a; Long et al., 2023), the wings (Xu et al., 2020), the peritrophic membrane of the midgut (Liu et al., 2012; Qu et al., 2022b), the intestinal lining of foregut (Zhang et al., 2021; Yu et al., 2024) and hindgut (Zhang et al., 2021), the taenidia of trachea (Yu et al., 2016; Qu et al., 2022a), as well as in the lumen of salivary glands (De Giorgio et al., 2023). It protects insects from various damages present in their living environment, such as chemical attacks, physical wear and tear, and pathogen invasion (Merzendorfer & Zimoch., 2003; Muthukrishnan et al., 2012; Zhu et al., 2016). Meanwhile, the rigid structure of chitin also restricts the growth of insects, so chitin is periodically synthesized and degraded during molting (Tetreau et al., 2015; Zhu et al., 2016). The enzymes that participate in the turnover of chitin are crucial for insect development, thus they are potential targets for designing eco-friendly pesticides (Shi et al., 2016; Chen & Yang., 2020; Chen et al., 2020; Liu et al., 2024).
The physiological significance of many enzymes associated with chitin turnover in insects has been extensively studied, such as chitin synthases (CHS) (Qu & Yang., 2011; Qu & Yang., 2012; Long et al., 2023) that are essential for the final step of chitin biosynthesis, chitin deacetylases (CDA) (Arakane et al., 2009; Yu et al., 2016) that participate in chitin modification, Lytic polysaccharide monooxygenases (LPMO) (Qu et al., 2022a; Qu et al., 2022b), chitinases (Cht) (Kramer et al., 1997; Li et al., 2015; Zhang et al., 2018; Zhu et al., 2019; Qu et al., 2021; Zhang et al., 2022; Li et al., 2024) and beta-N-acetylglucosaminidases (Hex) (Rong et al., 2013) that are in charge of chitin degradation. Insects usually encode multiple genes for each enzyme to fulfill their functions in different tissues. Take Locusta migratoria as an example, it encodes two CHSes (CHS1 and CHS2, also known as CHSA and CHSB), four CDAs, three LPMOs, fourteen chitinases, and four Hexes. Some of these genes possess alternative spliced variants to further accomplish their functions in different tissues. LmCHS1 contains two spliced variants LmCHS1A and LmCHS1B. LmCHS1A is mainly expressed in the integument while LmCHS1B is mainly expressed in the trachea. Both variants were proved to be essential for molting during the development of L. migratoria (Zhang et al., 2010). This alternative splicing is highly conserved in other insect species such as Tribolium castaneum (Arakane et al., 2004), Anopheles gambiae (Zhang et al., 2013), Sogatella furcifera (Wang et al., 2019). In lepidopteran CHSAs such as OfCHSA from Ostrinia furnacalis, there is an additional splicing site at the 5' region (OfCHSA-2a and OfCHSA-2b), which enables OfCHSA to produce four different transcripts (Qu & Yang., 2011). These transcripts were proved to be differently regulated during development (Qu & Yang., 2012). The BmCHSA-2b from Bombyx mori was proved to be specifically required for pupal wing development (Xu et al., 2017). LmCDA2 also contains two splice variants LmCDA2a and LmCDA2b. The variant LmCDA2a is essential for molting, whereas LmCDA2b seems to be dispensable for survival, although the expression patterns of LmCDA2a and LmCDA2b are similar (Yu et al., 2016). TcCDA2 from T. castaneum also has two selectively spliced transcripts, TcCDA2a and TcCDA2b, which appear to have different functions. TcCDA2a is needed for the establishment of the soft fermoraltibial joint cuticle, while TcCDA2b is involved in the formation of the hard elytra (Arakane et al.,2009). This splicing site is conserved in other insect CDA2 genes. Alternative splicing of chitinases has not been extensively investigated in insects. It is only reported in group IV chitinases from Locusta migratoria (Zhang et al., 2022), Bombyx mori (Abdel-Banat et al., 2002), and Lutzomyia longipalpis (Ortigão-Farias et al., 2018).
Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper-dependent enzymes that oxidatively cleave glycosidic bonds in polysaccharides such as cellulose, chitin, starch, xylan, and pectin (Vaaje-Kolstad et al., 2010; Couturier et al., 2018; Forsberg et al., 2019; Sabbadin et al., 2021). They provide additional attackable sites for glycosyl hydrolases (Couturier et al., 2018; Forsberg et al., 2019; Sabbadin et al., 2021), thus promoting the degradation of polysaccharides (Tandrup et al., 2018; Jagadeeswaran et al, 2021). They are categorized as members of the auxiliary activities (AA) family members in the Carbohydrate-Active enZymes (CAZy) database, belonging to AA9- AA11 and AA13–AA17. In the realm of insects, LPMOs are specifically classified within the AA15 (LPMO15) family (Sabbadin et al., 2018). They have been identified in Thermobia domestica (Sabbadin et al., 2018), Drosophila melanogaster (Zhu et al., 2008), Coptotermes gestroi (Franco Cairo et al., 2020), Tribolium castaneum (Qu et al., 2022a), L. migratoria (Qu et al., 2022a; Qu et al., 2022b), and Bombyx mori (Dong et al, 2016). Phylogenetically, insect LPMO15s can be categorized into four distinct groups, encompassing group Ⅰ through group Ⅳ. So far, the physiological significances of insect LPMO15s have only been investigated in L. migratoria and T. castaneum. In L. migratoria, three LmLPMO15s have been classified into group Ⅰ (LmLPMO15-1), group Ⅱ (LmLPMO15-2), and group Ⅲ (LmLPMO15-3) respectively (Qu et al., 2022a; Qu et al., 2022b). LmLPMO15-1 exhibits predominant expression within the trachea and epidermis. Its deficiency induced by RNAi leads to developmental stagnation and molting disruption in L migratoria. Transmission electron microscopy (TEM) analysis further elucidated its indispensable role in the degradation of the old cuticle during molting. LmLPMO15-3 has been proven to be mainly expressed in the midgut and participates in the degradation of the peritrophic matrix (Qu et al., 2022b). In T. castaneum, TcLPMO15-1 has been revealed to be pivotal in the degradation of chitin within the old cuticle and trachea during molting (Qu et al., 2022a). However, the knowledge about whether and how LPMOs participate in the turnover of chitin in other tissues is still insufficient.
In this study, alternative splicing of LmLPMO15-1 was discovered from the orthopteran pest Locusta migratoria, which generates two alternative spliced variants namely LmLPMO15-1a and LmLPMO15-1b. LmLPMO15-1a and LmLPMO15-1b were highly expressed in the trachea and foregut, respectively. They were proved to possess critical functions with tissue specificity by RNAi in combination with ultrastructure analysis. This work not only deepens our knowledge about the physiological functions of LPMO15s in multiple tissues during insect development but also imparts potential targets of great promise for future pesticide design.