Distinct Roles of Two UDP-N-Acetylglucosamine Pyrophosphorylase Genes in Chitin Biosynthesis and Molting of Oedaleus asiaticus (Orthoptera: Acrididae)

TL;DR

This study identifies two genes in grasshoppers that are essential for chitin production and molting, offering a potential target for safer pest control methods.

Contribution

The study reveals distinct roles of OaUAP1 and OaUAP2 genes in chitin biosynthesis and molting in Oedaleus asiaticus, providing new insights for biological pest control.

Findings

OaUAP1 is primarily expressed in the epidermis, while OaUAP2 is mainly expressed in the fat body.

Silencing either OaUAP1 or OaUAP2 disrupts molting and leads to nymph death.

20-hydroxyecdysone increases UAP gene activity, while validamycin decreases it.

Abstract

The Asian grasshopper (Oedaleus asiaticus) is a serious pest in dry and semi-dry grasslands of Inner Mongolia, China, where outbreaks destroy crops and pastures, harming farming, animal husbandry, and the environment. Current control depends mostly on chemical pesticides, but these cause insect resistance, pollution, and health risks. This study identified two genes, OaUAP1 and OaUAP2, that help produce chitin, the hard material forming the insect’s outer covering. The genes work in different body parts: OaUAP1 mainly in the epidermis and OaUAP2 in the fat body. Silencing either gene prevented proper molting, causing body shrinkage, cracked shells, and death. A natural molting hormone boosted their activity, while a substance that blocks chitin production reduced it. These results show that the genes have distinct but vital roles in insect growth and molting. The findings offer new knowledge for creating safe, targeted biological insecticides that avoid chemical harm, supporting sustainable pest control and the protection of grasslands and agriculture.

UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the insect chitin biosynthesis pathway; however, little is known regarding its molecular functions in Oedaleus asiaticus Bey-Bienko (Orthoptera: Acrididae). Here, two UAP genes, OaUAP1 and OaUAP2, were identified and characterized in O. asiaticus. The effects of exogenous treatments, including the molting hormone 20-hydroxyecdysone (20E) and the chitin biosynthesis inhibitor validamycin (VA), were assessed on chitin synthesis. Sequence analyses have shown that the cDNA and deduced amino acid sequences of O. asiaticus share over 90% identity with UAPs in Locusta migratoria. OaUAP1 and OaUAP2 are widely expressed in many tissues and developmental stages but exhibit different expression patterns: OaUAP1 shows higher expression in the epidermis and fifth-instar nymphs, while OaUAP2 is primarily expressed in the fat body and in the fifth-instar nymphs and adults. The functional analysis of two OaUAPs revealed that OaUAP2 was crucial in molting; moreover, its implication also exists in other biosynthetic processes since nymphs maintained normal growth and development. Both OaUAP expressions were upregulated by 20E and downregulated by VA in the chitin biosynthesis pathway. Our findings provide a vital molecular insight into the chitin biosynthesis pathway of O. asiaticus and lay a solid foundation for developing environmentally safe biological insecticides.

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1. Introduction

The second most abundant natural polymer in living organisms after cellulose, chitin is a cellulose-derived biopolymer and a linear homopolymer of N-acetylglucosamine (GlcNAc) linked by β-1,4 glycosidic bonds [1,2,3,4]. It is so widely distributed among diverse taxa including fungi, sponges, nematodes, mollusks, arthropods, fishes, amphibians, and some certain algae that it indicates an important general biological role [5,6,7,8]. The importance of chitin in insects includes the formation of exoskeletal structures such as those of the cuticle, linings of tracheae, and the peritrophic matrix of the midgut, whereas, physiologically, it supports important processes like growth, metamorphosis, and actual survival, serving thereby to maintain physical resilience for developmental success [2,9,10]. That might actually jeopardize the whole developmental and adult life of an insect, thus giving the chitin a place in the concept of pest management [11,12,13,14,15,16].

Chitin biosynthesis gives rise to a potent and pest-selective pest control methodology. Indeed, it is possible to cause impairment to the formation of exoskeleton structures and gut integrity in insects [5,6,13,14,15,17,18]. For example, insects fail to molt properly, or the peritrophic matrix in the midgut may weaken or thin, with all these leading to stunted growth or death, as demonstrated to occur in a few selective cases where the target was enzymes such as chitin synthase or UAP [9,19,20,21,22]. RNA interference-induced suppression of essential chitin metabolism enzymes has proven effective as a promising green method of insect growth and development control with little environmental toxicity for use either in the open or in any other recommended approach [23,24,25,26]. Insect growth, development, and molting depend on a delicate dynamic equilibrium between chitin synthesis and degradation, controlled by a coordinated activity of chitin-metabolizing enzymes [9,27,28,29]. From trehalose, chitin biosynthesis occurs via a chain of enzyme-catalyzed processes which include hexokinase, glucose-6-phosphate isomerase, glutamine-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate N-acetyltransferase, phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase (UAP), and chitin synthase, with UAP supplying the active precursor UDP-N-acetylglucosamine [30,31,32,33]. Although UAP is evolutionarily highly conserved, the structural divergence between prokaryotic and eukaryotic homologs shows a special functional adaptation of UAP in higher organisms [27,34,35]. UAP is essential for the epidermis and the peritrophic matrix (PM) of the larval midgut in insects. Disruption of UAP activity compromises chitin biosynthesis, thereby causing defective molting and reduced survival [9,19,22,36].

The rapid growth of genomic and transcriptomic resources in insects has allowed the identification and functional characterization of UAP orthologs in various taxa, thereby providing new avenues in the regulation of chitin metabolism [37,38,39].

Different species of insects show many variations in the number of UAP genes. Among the various species studied, UAP genes are identified only in Drosophila melanogaster Meigen, Aedes aegypti L., and Bombyx mori L. [14,35,40,41]. On the other hand, two separate UAP gene sequences have been identified in the species Tribolium castaneum Herbst, Locusta migratoria L., and Leptinotarsa decemlineata Say, suggesting the evolutionary diversification and possible functional specialization of UAPs among insects [5,15,42,43,44]. UAPs during insect development clearly play vital roles in mediating certain processes that are essential for the growth and morphogenesis of the organism. In D. melanogaster, mutations in DmUAP produce severe defects in cuticle formation, tracheal tube morphogenesis, and CNS fasciculation, underscoring the critical nature of this enzyme for overall developmental integrity [12,42,45]. In T. castaneum, the RNAi-induced silencing of both TcUAPl and TcUAP2 disrupts the sequence of all developmental transitions: larval–larval, larval–pupal, and pupal–adult eclosion, whereas the specific amendment of TcUAP2 declines the growth of larvae severely [46,47,48]. In L. decemlineata, LdUAP1 and LdUAP2 show spatially distinct functions associated with the roles in the cuticle layer synthesis and the deposition of midgut chitin, respectively [15,49]. Likewise, in L. migratoria, LmUAP1 is required for the biosynthesis of chitin, in both the epidermis and midgut, whereas LmUAP2 appears to be an unessential gene with respect to nymphal development, thus pointing out species-specific changes in the functionalities of genes within this family [42,50]. Locusts reproduce very quickly, are voracious in appetite, and can migrate over long distances; this is a combination that seriously jeopardizes agricultural and livestock production [42,51]. The adverse effects of locusts on direct damage to crops and pastures go far beyond such damage: they also have major ecological and socio-economic impacts, among which we find grassland degradation, desertification, and disruption of ecosystem services [52,53]. An annual invasion of up to 100,000 hectares by O. asiaticus in Inner Mongolia, which is prone to semi-arid types of grazing, can be very detrimental to agricultural production, animal husbandry, and local ecological balances [54,55,56,57]. Furthermore, O. asiaticus is also a bioindicator of grassland degradation and hence reflects the health of the ecosystem at the larger scale [54,58,59].

Despite ongoing efforts to diversify management strategies, control of O. asiaticus remains heavily reliant on chemical pesticides, with only limited integration of biological measures [60,61,62]. The long-term and extensive application of chemical pesticides has led to environmental residues, accelerated development of pest resistance, ultimately resulting in reduced control efficacy and threats to ecological safety [63,64,65,66]. Whereas chemical pesticides used for a long time have promoted resistance and environmental residues, genetic and molecular approaches to managing O. asiaticus remain largely unexplored, highlighting a critical gap in sustainable pest management strategies. Two UDP-N-acetylglucosamine pyrophosphorylase genes (OaUAP1 and OaUAP2) have been identified and characterized in the current study on O. asiaticus, thus giving molecular insights for sustainable locust control strategies.

2. Materials and Methods

2.1. Insect Rearing and Sample Collection

Oedaleus asiaticus eggs were collected from the wild grasslands of Siziwang Banner, Inner Mongolia, China (41°22′ N, 111°21′ E), and brought to Hohhot, where they were set up under controlled conditions for incubation. After hatching out, nymphs were fed with fresh wheat seedlings with added bran at 25 ± 1 °C, 40 ± 5% RH, and a photoperiod L/D 14:10 h. In order to keep the consistency of treatment across experiments, individual specimens were synchronized so that all individuals reached the same developmental stage and physiological age simultaneously. Samples were healthy, spreading over all developmental stages, including eggs, first- to fifth-instar nymphs, and adult males and females. For tissue specificity analysis, higher fourth-instar nymph organs were dissected and included fat body, Malpighian tubules, foregut, midgut, hindgut, salivary glands, trachea, and epidermis. All dissected tissues were snap-frozen in liquid nitrogen immediately and stored at −80 °C until the time of molecular analysis.

2.2. Transcriptome-Based Identification of UAP Genes

UAP genes in O. asiaticus were identified by exhaustively interrogating the published transcriptome database of the species. For this purpose, pooled mRNA samples from synchronized nymphal and adult stages were subjected to interrogation using specific keywords pertaining to UAP and UDP-N-acetylglucosamine pyrophosphorylase. By employing the Blastx algorithm for homology search, two distinct cDNAs, OaUAP1 and OaUAP2, were revealed corresponding to two divergent UDP-N-acetylglucosamine pyrophosphorylases. These findings provided a basis for downstream molecular cloning, expression profile studies, and functional characterization.

2.3. Molecular Cloning of UAP Genes

For the purpose of validating and obtaining full-length sequences of the predicted UAP genes, cDNA was synthesized from total RNA extracted from whole-body samples of fourth-instar nymphs using Eastep^®^ Super Total RNA Extraction Kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. First-strand cDNA synthesis was conducted using GoScript^®^ Reverse Transcription Mix (Promega, Madison, WI, USA) and Oligo(dT) kit (Promega). Gene-specific primers were designed with Premier 5.0 software (Table 1) and used in reverse transcription quantitative PCR (RT-qPCR) to amplify target cDNA sequences. The PCR products were then analyzed on 1% agarose gels, purified, and cloned into the pMD18-T vector for sequencing by Hooseen Biotechnology Co., Ltd. (Beijing, China). This study followed all standard procedures for molecular cloning to guarantee sequence accuracy.

2.4. Bioinformatics and Phylogenetic Analysis

Both cDNA sequences of OaUAP1 and OaUAP2 were translated into amino acid sequences using the ExPASy server (http://cn.expasy.org, accessed on 16 September 2025). Key physicochemical properties such as molecular weight, theoretical isoelectric point, and open reading frames were computed via NCBI’s Blastp tool (http://www.ncbi.nlm.nih.gov, accessed on 17 September 2025). Amino acid sequences were compared with homologous UAPs from other insect species for sequence conservation. Pairwise alignments were created between the deduced amino acid sequences of OaUAP1 and OaUAP2 and their corresponding homologs LmUAP1 and LmUAP2 from L. migratoria using DNAMAN 6.0 (Lynnon Biosoft, San Ramon, CA, USA) to evaluate sequence homology [42]. Out of these 21 representative protein sequences retrieved from the NCBI database, they were later used to elicit evolutionary relationships among the insect UAPs. Their phylogenetic reconstruction employed the neighbor-joining (NJ) method with 1000 bootstrap replicates in MEGA 5, which gave really solid support for the obtained inferred evolutionary linkages [67,68]. Therefore, this phylogeny can provide a basis through which the divergence and conservation of OaUAP sequences with respect to other insect UAPs can be interpreted.

2.5. Expression Profiling of UAP Genes

OaUAP1 and OaUAP2 gene expressions were studied across developmental stages and in sampled tissues within fourth-instar nymphs such as the fat body, Malpighian tubules, foregut, midgut, hindgut, salivary glands, trachea, and epidermis. Quantitative PCR (qRT-PCR) was performed using GoTaq^®^ qPCR Master Mix (Promega, Madison, WI, USA) in a 20 μL reaction volume, consisting of 1 μL template cDNA, 0.4 μL for each primer, 0.2 μL ROX reference dye, 10 μL 2× Master Mix, and nuclease-free water. The thermal cycling was started by an initial denaturation of 2 min at 95 °C followed by 40 cycles of 3 s at 95 °C and 30 s at 60 °C. Dissociation curve analysis was performed to further confirm the specificity of the amplification. Relative transcript abundance was normalized to that of β-actin, serving as an internal reference. Data were analyzed statistically by one-way nested ANOVA followed by Duncan’s multiple range test (IBM SPSS Statistics v25.0) to determine significant differences among developmental stages and tissues.

2.6. Functional Analysis of UAP Genes

Functional studies of OaUAP1 and OaUAP2 were performed through RNA interference (RNAi). A double-stranded RNA (dsRNA) specific for each gene was injected into the fourth-instar nymphs by microinjection 3 days post-molt (5 μL per individual, containing 5 μg dsRNA at a concentration of 1 μg/μL). Each treatment was independently replicated three times (20 nymphs per group). The locusts were then sampled after injection at 12, 24, 48, and 72 h post-treatment for evaluating the efficiency of gene silencing through RT-qPCR and checking the phenotypic effects under controlled rearing conditions. The RNAi was performed for knockdown of OaUAP1 and OaUAP2 to assess their individual contributions toward chitin synthesis and locust development.

2.7. Hormone and Inhibitor Treatments

In the regulatory effect studies on OaUAP expression, healthy fourth-instar nymphs were firstly injected with 20E or VA with different concentration programs at 3 days post-molting (20E from Sigma-Aldrich, St. Louis, MO, USA, dissolved in DMSO as solvent control: DMSO alone; VA from Huaxia Chemical Reagent, Chengdu, China, dissolved in ddH_2_O as solvent control: ddH_2_O alone). Gene expression was quantified at 12, 24, 48, and 72 h after 20E treatment and at 12, 24, and 48 h after VA treatment in nymphs subjected to RNAi-mediated gene knockdown. This allowed assessment of transcriptional regulation of OaUAP1 and OaUAP2 through both hormonal induction and chemical inhibition.

2.8. Statistical Analysis

All measurements were assessed by applying a one-way nested analysis of variance (ANOVA) followed by Tukey’s post hoc test (IBM SPSS Statistics v 25.0).

3. Results

3.1. Molecular Characterization and Phylogeny of UAP Genes

Annotational data identified two chitin biosynthesis pathway genes in O. asiaticus called OaUAP1 and OaUAP2, with nucleotide sizes of 2157 bp and 3646 bp, respectively (Figure 1). The open reading frames (ORFs) of OaUAP1 and OaUAP2 translate into proteins with lengths of 484 and 493 amino acids, respectively. Their calculated molecular weights were 55.08 kDa and 55.88 kDa, while the isoelectric points were 6.05 and 6.25. Functional domain analysis found that neither of the two proteins possess any signal peptides. The OaUPA1 amino acid sequence from O. asiaticus was very similar to that of the L. migratoria LmUAP1 homolog for the first two thirds of the sequence, in that they showed >90% identity with each other in those regions. However, OaUPA1 contained many more amino acids: 1127 for OaUPA1 compared to 699 for LmUAP1 (Figure 1). The OaUPA2 amino acid sequence had only slightly more amino acids than LmUAP2 and was >90% identical overall (Figure 1). Phylogenetic analysis of the full-length amino acid sequences of 21 insect species showed that OaUAP1 and OaUAP2 clustered closely with LmUAP1 and LmUAP2 but were found in different branches (Figure 2).

3.2. Expression Patterns of UAP Genes

The spatiotemporal expression analysis indicates clear differences between OaUAP1 and OaUAP2 with respect to time and tissue (Figure 3A,B and Figure 4A,B). The highest expression of OaUAP1 was found in the epidermis, whereas expression in the other tissues was considerably lower. Instead, OaUAP2 appeared to be highly expressed in the fat body. OaUAP1 peaked in the fifth-instar nymphs, whereas expression was low in all other stages. In comparison, OaUAP2 was more highly expressed in both the fifth-instar nymphs and adult stages.

3.3. Functional Roles of UAP Genes

RNA interference (RNAi) was adopted for studying the functions of OaUAP1 and OaUAP2 in the development and growth of O. asiaticus. The dsRNAs specific to both genes were injected into fourth-instar nymphs three days post-molt. The expression levels of OaUAP1 and OaUAP2 were significantly reduced post-injection, with OaUAP1 showing 77.78% knockdown at 48 h and OaUAP2 showing 41.10% knockdown at 72 h (Figure 5A). Nymphal mortality reached about 100% after four days of dsRNA treatment (Figure 5B). Phenotypic observations showed that dsOaUAP1-injected nymphs revealed shrinkage all over the body and a lack of ecdysis particularly for the cuticle of the abdomen and hind legs and they could not move normally until death. In contrast, dsOaUAP2-injected nymphs showed partial normal development, with dorsal plate cracks, exudation of green fluid, and wing eversion. The proportion of representative lethal phenotypes at 48 h post-injection was 76% for dsOaUAP1 and 83% for dsOaUAP2 (Figure 5C). The green morph of O. asiaticus represents solitary individuals with weaker resistance, while the brown morph is gregarious with stronger resistance; in this experiment, the proportion of green individuals was 35%.

3.4. Regulation of UAP Gene Expression

The foreign hormones affect and control the developmental transition in the insects. The 20E injection resulted in very high up-regulation of chitin biosynthesis pathway genes. Compared with the ethanol control (10%), 20E increased the transcript level for OaUAP. At 2.5 μg/μL, OaUAP1 increased by 9 at 12 h and by 19.9 at 24 h (Figure 6A), whereas OaUAP2 expression rose by 10.4 at 24 h (Figure 6B). In contrast, VA treatment resulted in a dose-dependent decrease in OaUAP transcript levels, indicating that this compound exerts an inhibitory effect on chitin synthesis genes.VA inhibited OaUAP1 transcripts by almost 86.6% at 20 μg/μL within a period of 12 h (Figure 6C). Last, but not least, the highest inhibition for the same concentration occurs at 48 h with a reduction in transcript levels of about 81.7% for OaUAP2 (Figure 6D).

4. Discussion

UAP genes are key regulators of chitin biosynthesis in insects in the formation of the exoskeleton, tracheal linings, and other chitin-containing structures while also being involved in protein glycosylation [40,45,69,70]. Historically, only one single UAP gene was reported in insect genomes [15,46,71] but increasing genomic and transcriptomic resources have revealed the presence of additional paralogs in species like T. castaneum [44,46], L. migratoria [42,72], and L. decemlineata [15,73], indicating species-specific duplications which may suggest functional specialization. The present study has successfully identified two UAP genes, namely OaUAP1 and OaUAP2, from the O. asiaticus transcriptome. The nucleotide lengths were found to be 2157 and 3646 bp, respectively (Figure 1). The ORFs produce 484 and 493 amino acids, and their molecular weights are 55.08 kDa and 55.88 kDa, respectively. The isoelectric points are 6.05 and 6.25, respectively (Figure 1). Phylogenetic analysis positioned OaUAP1 and OaUAP2 next to LmUAP1 and LmUAP2 in terms of relationships but on different branches, indicating both conserved enzymatic functions and possible divergence (Figure 2). Spatiotemporal expression profiling revealed that OaUAP1 is predominantly expressed in the epidermis, with the highest peaks occurring in fifth instars, whereas OaUAP2 is mainly expressed in the fat body, with increased levels in fifth instars and adults (Figure 3 and Figure 4). The differential expression strongly indicates functional divergence following gene duplication, thus allowing tissue- and stage-specific control of chitin biosynthesis and the associated metabolism [9,74]. These patterns are consistent with previous findings in L. migratoria, where LmUAP1 is enriched in epidermal tissues and LmUAP2 is preferentially expressed in the fat body [42,72], and in L. decemlineata, where LdUAP1 and LdUAP2 are specialized in the cuticle and peritrophic matrix, respectively [75].

RNA interference (RNAi)-based functional validation established the indispensable roles of OaUAP1 and OaUAP2 in molting and survival. The knockdown of either gene led to failed molting, which included incomplete ecdysis and lethal phenotypes (Figure 5), thus confirming their very vital role in chitin biosynthesis and exoskeleton formation. Essentially, OaUAP1-dsRNA-injected nymphs underwent shrinkage of the whole body, failure of cuticle shedding from the abdominal region, and locomotion, whereas OaUAP2-dsRNA-injected nymphs showed cracks in the dorsal plates, exudation of greenish fluid, and everted wings (Figure 5C). The high proportions of lethal phenotypes (76% for dsOaUAP1 and 83% for dsOaUAP2 at 48 h) underscore the critical dependency on these genes, with the green morph (35% in our experiment) showing potentially higher susceptibility due to its solitary and weaker resistance traits compared to the gregarious brown morph. The phenotypes are consistent with previous observations in L. migratoria and L. decemlineata [15,36,42,75], indicating UAP paralogs that, while differing in their expression patterns, function in a convergent manner for the maintenance of molting integrity [42,46]. However, notable differences were observed when compared to L. migratoria. In L. migratoria, the RNAi-mediated knockdown of LmUAP1 in the early seconds and fifth-instar nymphs led to a rapid cessation of feeding, loss of responsiveness to physical stimuli, and 100% mortality within approximately two days, whereas the knockdown of LmUAP2 had no significant effect on molting, development, or survival [42]. In contrast, in O. asiaticus, the silencing of both OaUAP1 and OaUAP2 resulted in severe molting defects and high mortality rates, although the phenotypes were distinct between the two genes (e.g., whole-body shrinkage and complete ecdysis failure for OaUAP1, versus partial development with dorsal cracks and wing eversion for OaUAP2). These contrasting outcomes highlight potential species-specific functional divergence in the roles of UAP paralogs within orthopteran chitin biosynthesis pathways.

Moreover, hormonal regulation elucidated the dynamic roles of OaUAPs. When exposed to 20E, OaUAP1 and OaUAP2 were significantly upregulated (Figure 6A,B) as expected, in agreement with the conserved role of ecdysteroids in molting and remodeling cuticles. On the contrary, VA considerably suppressed OaUAP expression in terms of doses as well as time (Figure 6C,D), thereby linking energy metabolism to chitin biosynthesis. These results were identified in Spodoptera exigua (Hübner) and Nilaparvata lugens (Stål) as well as soybean aphid and Panonychus citri (McGregor) [76,77,78]; however, recent work [46,79] adds to the evidence of the hormonal modulation of UAP expression across insect taxa. In developmental profiling, OaUAP1 expression was detected early in nymphal development at stages N1 and N2, peaked in fifth-instar nymphs, and afterward decreased in adults (Figure 3A and Figure 4A), suggesting a key role in the final deposition of the nymphal cuticle [15,36].

The short molting cycles for very early instars may explain the low expression levels seen during this period, illustrating an adaptive form of chitin metabolism regulation according to developmental demands [80,81].

5. Conclusions

OaUAP1 and OaUAP2 are identified as important molecules involved in chitin biosynthesis, molting, and hormonal regulation in O. asiaticus. By phylogenetics, spatiotemporal expression, RNAi functional assays, and hormonal treatments, this study forms a comprehensive framework for UAP function in orthopteran insects. These findings do not just add to basic knowledge but also provide a basis for RNAi-based pest management targeting UAP genes towards the environmentally sustainable control of grasshopper populations.