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In Locusta migratoria a worldwide
In Locusta migratoria, a worldwide agricultural pest, we previously identified 51 CPRs containing eight endocuticle structural glycoproteins, which are homologs of SgAbd-1, SgAbd-2, SgAbd-3, SgAbd-4, SgAbd-5, SgAbd-6, SgAbd-8, and SgAbd-9 in the adult desert locust, Schistocerca gregaria [17,26]. Although their amino Apicidin sequences were initially analyzed, their biological functions in locusts remain unclear. In the present study, we analyzed the molecular characteristics, spatiotemporal expression, and response to 20E of the endocuticle protein gene LmAbd-9 identified according to transcriptome data of L. migratoria [27]. We analyzed its biological function using RNAi combined with hematoxylin and eosin staining and transmission electron microscopy.
Materials and methods
Results
Discussion
In S. gregaria, eight post-ecdysial cuticular proteins have been sequenced (SgAbd-1, SgAbd-2, SgAbd-3, SgAbd-4, SgAbd-5, SgAbd-6, SgAbd-8, and SgAbd-9), and they all contain an RR-1 consensus sequence [17]. We have previously identified eight post-ecdysial cuticular proteins based on the L. migratoria transcriptome [27], which have a high degree of sequence conservation with those of S. gregaria. In the present study, we characterized LmAbd-9, which has high similarity (95.3%) with SgAbd-9, belonging to the RR-1 subclass of CPR family in L. migratoria. The LmAbd-9 may contain two O-linked N-acetylhexosamine residues (Serine (S115) and Threonine (T137)) (Figs. 1 and 2), and may be the structural components of the extracellular matrix.
The insect cuticle consists of epicuticle and procuticle (exocuticle and endocuticle), and the exocuticle and endocuticle forms and deposits before ecdysis and after ecdysis, respectively [35]. Spatiotemporal profiling suggested that the LmAbd-9 protein might be involved in the formation of the endocuticle synthesized post-ecdysis. In addition, insect molting is controlled by 20E and the juvenile hormone [[36], [37], [38]]. 20E binds to its cognate nuclear receptor (EcR/USP) to activate the expression of 20E-response genes, which regulate the expression of cuticle protein genes and enable subsequent metamorphosis [[39], [40], [41]]. For example, the Drosophila EDG84A and EDG78E are both pupal cuticle protein genes that are regulated by a 20E pulse [42]. FTZ-F1 is a typical nuclear receptor transcription factor [43] that has been identified as a potential regulator of the transcription of fushi tarazu (ftz) in D. melanogaster [44], and is a critical transcriptional activator of EDG84A [45]. In B. mori, BmbetaFTZ-F1 was also demonstrated to regulate two CP genes (BmWCP2 and BmWCP5) via a 20E pulse [46,47]. The locust genome encodes two forms of the protein, betaFTZ-F1 and FTZ-F1beta, which are similar to other species insects including D. melanogaster. In the present study, the mRNA expression pattern of LmAbd-9 was inversely proportional to the 20E titer in nymphs [34]. We further confirmed that LmAbd-9 was negatively regulated by a 20E pulse, and that LmFTZ-F1beta is a negative regulator of LmAbd-9 (Fig. 4), but not LmbetaFTZ-F1 (Supplemental Fig. 1). However, whether LmFTZ-F1beta directly or indirectly regulates the expression of LmAbd-9 requires further study.
Cuticular proteins play roles in the formation of the cuticle during insect molting. For the CPR family, RNAi against CPR genes caused lethal phenotypes or led to a disorganization of the laminar architecture [16,21,22,48]. More recently, an ungrouped cuticular protein gene NlCP21.92 of N. lugens was studied, which can lead to abnormal or lethal morphological phenotypes after RNAi mediated by dsRNA, and is crucial for normal endocuticle formation [25]. In B. mori, a hypothetical cuticular protein, BmorCPH24, produced the abnormal body shape after knocking out its encoding gene and was observed to be involved in synthesis of the endocuticle [24]. However, so far, no RR-1 protein has been reported to be involved in the formation of the endocuticle from ultrastructural analysis. Although there were no visible abnormal phenotypes after injection dsRNA against LmAbd-9 in the fifth instar nymph, and even from 3rd and 4th instar nymphs continuously injected with dsLmAbd-9 (Supplemental Fig. 2), we found that the cuticle of nymphs treated with dsLmAbd-9 was thinner, and there were significantly fewer endocuticular lamellae than in the control, as shown from microscopic and ultrastructure by H&E staining and TEM analysis, respectively (Fig. 5). During insects molting, it is believed that multiple proteins and related factors are required for the formation and construction of the cuticular structure, and are cross-linked; however, the specific mechanism of the action remains unclear. Meanwhile, whether LmAbd-9 and other members of the Abd family of proteins have synergistic or complementary effects with each other remains to be determined.