Insect Chitin Research Articles

Unravelling the Role of Chitin and Chitosan in Prebiotic Activity and Correlation With Cancer: A Narrative Review.

This review describes the state of the art regarding the prebiotic role of chitin and the interactions of chitin and chitosan with cancer cells. Chitin is the second most abundant polysaccharide in nature and a constitutive component of crustacean shells and the exoskeleton of insects. Chitosan is the deacetylated form of chitin, which is obtained by chemical processing or the enzymatic activity of deacetylases found in microorganisms and insects. Edible insects have recently been introduced in Western countries, thus raising concerns regarding food safety and due to their chitin content and the release of chitosan during the digestive process. The roles of insect chitin and chitosan in the gastrointestinal tract, microbiome modulation, and cancer have been widely investigated. Several in vitro and in vivo studies have shown the possible microbiota modulation of chitin and its relevant communication with the immune system, thus confirming its prebiotic activity. No evidence has been provided on the cancerogenic activity of chitin; however, studies have suggested that chitin has a cytotoxic effect on cancer cell lines. Chitosan has been confirmed to exhibit apoptotic and cytotoxic activities on cancer cells in several in vitro studies on cancer cell lines and in vivo models. In conclusion, the literature does not show a direct connection between the presence of chitin or chitosan and the onset of cancer. However, cytotoxic and apoptotic activities in relation to cancerous lines have been demonstrated.

Knockdown of the glucosamine-6-phosphate N-acetyltransferase gene by RNA interference enhances the virulence of entomopathogenic fungi against rice leaffolder Cnaphalocrocis medinalis

Insect cuticle acts as a first line of defense and a physical protective barrier against entomopathogens. Chitin biosynthesis pathway plays a crucial role in chitin formation in the cuticle of insects. Glucosamine-6-phosphate N-acetyltransferase (GNA) is a key enzyme in insect chitin biosynthesis that regulate the chitin formation. However, how GNA-mediated cuticle metabolism influences virulence of entomopathogenic fungi is still unknown. In this study, CmGNA gene was cloned and characterized from the rice leaffolder Cnaphalocrocis medinalis. The CmGNA contains an open read frame (ORF) 600 nucleotides, encoding 199 amino acids with an isoelectric point of 8.65 and a molecular weight of 22.30 kDa. The expression profile showed that CmGNA was highly expressed in 4th instar larvae and in the cuticle. Here, we also reported the impact of CmGNA gene and entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, on expression pattern of chitin biosynthesis genes, feeding behavior, survival rate and average body weight of infected larvae, phenotypic deformities, rate of pupation, and adult emergence. Our results showed that knockdown of CmGNA and application of M. anisopliae and B. bassiana three days after RNA interference (RNAi) significantly decreased the expression of CmGNA and other associated genes, reduced feeding efficiency and survival rate, and caused loss of average body weight, less rate of pupation and adult emergence of infected larvae. Knockdown of CmGNA gene also increased the lethality of larvae caused by M. anisopliae and B. bassiana and resulted in significantly phenotypic deformities of infected larvae. Our findings illustrated that RNAi-mediated CmGNA knockdown disturbed the chitin synthesis genes that led to enhancing the virulence of M. anisopliae and B. bassiana, which can provide us new insights to develop novel biocontrol strategies against C. medinalis.

Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation have been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

Structure information analysis and relative content determination of protein and chitin from yellow mealworm larvae using Raman spectroscopy

Insect protein extract is one of the high-quality protein sources and is frequently viewed as a potential nutrition alternative. However, a more precise method for protein measurement is still needed due to protein overestimation by the Kjeldahl method due to the presence of a large amount of chitin in insects. Therefore, we demonstrated the monitoring of chitin and protein extracted from yellow mealworm larvae through the information on molecular vibration obtained using Raman spectroscopy and infrared (IR) spectroscopy. The NH vibration at 3475 cm−1 is the characteristic peak of chitin in defatted product observed in the Raman spectra. The nitrogen-to-protein conversion factor in protein extracted from larvae by the Raman method was determined based on the NH vibration and found to be 5.66 ± 0.01. We also compared these experimental data to theoretical Raman and IR spectra and determined the possible reasons for why nitrogen elements in chitin affect the determination of protein content. The method of sequentially removing fat and protein could provide more accurate quantification of protein and chitin. Raman spectroscopy is feasible for various types of insects with high chitin content. Compared with the Kjeldahl method, the Raman method is a faster and more accurate measurement method. Moreover, it provides the content of impurities, purity, and structural information.

Buprofezin delayed the molting of Pardosa pseudoannulata, a predatory enemy for insect pests, by suppressing chitin synthase 1 expression

Spiders, the major predatory enemies of insect pests in fields, are vulnerable to insecticides. In this study, we observed that the recommended dose of buprofezin delayed the molting of the pond wolf spider Pardosa pseudoannulata, although it had no lethal effect on the spiders. Since buprofezin is an insect chitin biosynthesis inhibitor, we identified two chitin synthase genes (PpCHS1 and PpCHS2) in P. pseudoannulata. Tissue-specific expression profiling showed that PpCHS1 was most highly expressed in cuticle. In contrast, PpCHS2 showed highest mRNA levels in the midgut and fat body. RNAi knockdown of PpCHS1 significantly delayed the molting of 12-days old spiderlings, whereas no significant effect on the molting was observed in the PpCHS2-silencing spiderlings. The expression of PpCHS1 was significantly suppressed in the spiderlings treated with buprofezin, but rescued by exogenous ecdysteroid ponasterone A (PA). Consistent with this result, the molting delay caused by buprofezin was also rescued by PA. The results revealed that buprofezin delayed the molting of spiders by suppressing PpCHS1 expression, which will benefit the protection of P. pseudoannulate and related spider species.

Insecticidal activity and underlying molecular mechanisms of a phytochemical plumbagin against Spodoptera frugiperda.

Plumbagin is an important phytochemical and has been reported to exhibit potent larvicidal activity against several insect pests, However, the insecticidal mechanism of plumbagin against pests is still poorly understood. This study aimed to investigate the insecticidal activities of plumbagin and the underlying molecular mechanisms against a devastating agricultural pest, the fall armyworm Spodoptera frugiperda. The effects of plumbagin on S. frugiperda larval development and the activities of two detoxification enzymes were initially examined. Next, transcriptomic changes in S. frugiperda after plumbagin treatment were investigated. Furthermore, RNA-seq results were validated by qPCR. Plumbagin exhibited a high larvicidal activity against the second and third instar larvae of S. frugiperda with 72h LC50 of 0.573 and 2.676mg/g, respectively. The activities of the two detoxification enzymes carboxylesterase and P450 were significantly increased after 1.5mg/g plumbagin treatment. Furthermore, RNA-seq analysis provided a comprehensive overview of complex transcriptomic changes in S. frugiperda larvae in response to 1.5mg/g plumbagin exposure, and revealed that plumbagin treatment led to aberrant expression of a large number of genes related to nutrient and energy metabolism, humoral immune response, insect cuticle protein, chitin-binding proteins, chitin synthesis and degradation, insect hormone, and xenobiotic detoxification. The qPCR results further validated the reproducibility and reliability of the transcriptomic data. Our findings provide a valuable insight into understanding the insecticidal mechanism of the phytochemical plumbagin.

Advances in understanding insect chitin biosynthesis

Chitin, a natural polymer of N-acetylglucosamine chains, is a principal component of the apical extracellular matrix in arthropods. Chitin microfibrils serve as structural components of natural biocomposites present in the extracellular matrix of a variety of invertebrates including sponges, molluscs, nematodes, fungi and arthropods. In this review, we summarize the frontier advances of insect chitin synthesis. More specifically, we focus on the chitin synthase (CHS), which catalyzes the key biosynthesis step. CHS is also known as an attractive insecticidal target in that this enzyme is absent in mammals, birds or plants. As no insect chitin synthase structure have been reported so far, we review recent studies on glycosyltransferase domain structures derived from fungi and oomycetes, which are conserved in CHS from all species containing chitin. Auxiliary proteins, which coordinate with CHS in chitin biosynthesis and assembly, are also discussed.

Physicochemical characterization of chitin extracted by different treatment sequences from an edible insect

Chitin present in the shell of edible insects is a potential source of chitin, lipids, and proteins, and it exerts various biological activities. Thus far, only a few studies have focused on the use of chitin as a source of high-protein-diet oligosaccharides. The use of insect chitin for the production of high-protein-diet oligosaccharides can lessen the reliance on diet crops. Moreover, although chitin composition in Tenebrio molitor larva, pupa, and adult has been extensively investigated, chitin extraction from T. molitor larval whole body and exuvium has received poor attention. The present study compared the effectiveness of two techniques for extracting high-protein-diet chitin oligosaccharide from an edible insect (T. molitor). Two different extraction sequences of chitin from the larval stage (molitor stage larvae) and adult stage (molitor stage adult) of edible T. molitor were investigated. Two processing steps were employed: (a) deproteinization (DEP) and (b) demineralization (DEM) treatments. Differences in the order, conditions, and period of their application resulted in two different chitin extraction procedures. The viscosity, degree of polymerization, and crystallinity index of the chitin extracted using the two procedures were measured, and its chemical components (chitin, ash, protein, fat, and moisture contents) were determined. T. molitor adults and larvae treated sequentially with DEM-DEP demonstrated the greatest yield of approximately 14.62 % ± 0.15 and 6.096 % ± 0.10 %, respectively. By contrast, when treated sequentially with DEP-DEM, the recorded yields were 10.96 % ± 0.18 and 5.31 % ± 0.38, respectively. Differences in the degree of deacetylation between both methods were observed. Additionally, Fourier transform infrared spectroscopy and X-ray diffractometry of the extracted chitin along with a commercial sample revealed consistent chain conformation, mean hydrogen bonding, and crystallinity index. In this way, residues produced by farmed edible insects can be recovered and used as a novel source of chitin.

Synthesis, DFT study, molecular docking and larvicidal activity of Chitin inhibitor alanine derived compounds

In order to obtain an effective larvicidal against A. aegypti the vectors for Dengue, Zika and Chikungunya virus, Sodium 2-{(E)-[(2-hydroxy-3-methoxyphenyl) methylidene] amino} propanoate (NaLH) ligand and its Cu(II) complexes derived from alanine amino acid with and without coligand (C1 &C2) have been synthesized. All three molecules were characterized by physicochemical techniques including FTIR, UV–Vis, proton NMR and Mass spectrum. The geometry optimization and frontier orbitals HOMO, LUMO and band gaps were calculated by using B3LYP functional and SVP basis sets for all three compounds (NaLH, complex C1 and C2). The values of HOMO/LUMO energies observed were −4.2/−1.7 eV for ligand, −5.2–3.5 eV for C1 and −5.0/−3.7 eV for C2 and corresponding values of energy gaps were 2.5 eV, 1.7 eV and 1.3 eV for NaLH, C1 and C2 respectively which increases in the following order NaLH > C1 > C2. Complex1 has been arranged in slightly distorted octahedral geometry while complex2 acquired a square pyramidal geometry. Molecular docking study of the reported larvicidal against insect acetylcholistenerase (PDB id: 6ARY) and Chitin acetylase (PDB id 5ZNT) revealed significant free energy of binding in the active site of target molecules with binding energy of −6.14 & −10.20Kcal/mol and −6.79 & −11.75Kcal/mol for C1 &C2 respectively. In addition, Larvicidal activity of complex 2 was observed with 100% mortality rate in just 2 hrs time using 1.0% concentration of larvicide. The LC50 value is 0.0015 µg/mL while LC90 value is 0.0018 µg/mL for C2. The complexes were observed to be lethal against Chitinous body layer of larvae of Aedes aegypti, the vectors for Dengue, Zika and Chikungunya virus.

FDP-Na-induced enhancement of glycolysis impacts larval growth and development and chitin biosynthesis in fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae)

Fructose 1, 6-diphosphate (FDP) is an endogenous intermediate in the glycolytic pathway, as well as an allosteric activator of phosphofructokinase (PFK). Based on the role in promoting glycolysis, FDP has been widely used as a therapeutic agent for mitigating the damage of endotoxemia and ischemia/reperfusion in clinical practice. However, the effect of exogenous FDP-induced glycolysis activation on insect carbohydrate metabolism and chitin synthesis remains largely unclear. Here, we investigated for the first time the effects of FDP-Na, an allosteric activator of PFK, on the growth and development of Hyphantria cunea larvae, a serious defoliator in agriculture and forestry, especially on glycolysis and chitin synthesis. The results showed that FDP-Na significantly restrained the growth and development of H. cunea larvae and resulted in larval lethality. After treatment with FDP-Na, hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) were significantly activated, and HcHK2, HcPFK, HcPK were dramatically upregulated, which suggested that FDP-Na enhanced glycolysis in H. cunea larvae. Meanwhile, FDP-Na also distinctly impacted chitin biosynthesis by disturbing transcriptions of genes in the chitin synthesis pathway, resulting in changes of chitin contents in the midgut and epidermis of H. cunea larvae. Therefore, we considered that FDP-Na caused the growth and development arrest, and impacted chitin biosynthesis, probably by disturbing in vivo glycolysis and carbohydrate metabolism in H. cunea larvae. The findings provide a new perspective on the mechanism by which glycolysis regulates insect growth and development, and lay the foundation for exploring the potential application of glycolysis activators in pest control as well.

Functional importance of groups I and II chitinases in cuticle chitin turnover during molting in a wood-boring beetle, Monochamus alternatus

Insects must periodically replace their old cuticle/exoskeleton with a new one in a process called molting or ecdysis to allow for continuous growth through sequential developmental stages. Many RNA interference (RNAi) studies have demonstrated that certain chitinases (CHTs) play roles in this vital physiological event because knockdown of these CHT genes resulted in developmental arrest during the ensuing molting period in several insect species. In this research we analyzed the functions of group I (MaCHT5) and group II (MaCHT10) CHT genes in molting of the Japanese pine sawyer, Monochamus alternatus, an important forest pest known as a major vector of the pinewood nematode. Real-time qPCR revealed that these two CHT genes differ in their expression patterns during late stages of development. Depletion of either MaCHT5 or MaCHT10 transcripts by RNAi resulted in lethal larval-pupal and pupal-adult molting defects depending on the double-stranded RNA (dsRNA) injection timing during development. The insects were unable to shed their old cuticle and died. Furthermore, transmission electron microscopic analysis revealed that, unlike dsEGFP-treated controls, dsMaCHT5- and dsMaCHT10-treated pharate adults exhibited a failure of degradation of the endocuticular layer of their old pupal cuticle, retaining nearly intact horizontal chitinous laminae and vertical pore canal fibers. Both enzymes were indispensable for complete turnover of the chitinous old endocuticle, which is critical for insect molting. The possible functions of two spliced variants of MaCHT10, namely, MaCHT10a and MaCHT10b, are also discussed. Our results add to the knowledge base for further functional studies of insect chitin catabolism by revealing the relative importance of both MaCHT5 and MaCHT10 in chitin turnover with subtle differences in their action. These essential genes and their encoded proteins are potential targets to manipulate for controlling populations of M. alternatus and other pest insects.

Chitin and omega-3 fatty acids in edible insects have underexplored benefits for the gut microbiome and human health.

A healthy gut microbiome is critical for nutrient metabolism, pathogen inhibition and immune regulation, and is highly influenced by diet. Edible insects are good sources of protein and micronutrients, but unlike other animal-derived foods, they also contain both dietary fibre and omega-3 fatty acids that can modulate gut microbiota. Here we explore the potential impacts of insect consumption on the microbiome. Laboratory, animal and human studies indicate that insect fibre in the form of chitin and its derivatives can modify gut microbiota with beneficial outcomes. Some insects also contain favourable omega-3/omega-6 ratios. We identify gaps in the literature-especially a dearth of human studies-that must be addressed to better understand health impacts of entomophagy. Insects, already eaten across the globe, can be farmed using fewer resources than conventional livestock. Widening the research scope offers an opportunity to advance use of edible insects to address interconnected environmental and health challenges.

Electrophoretic determination of chitin in insects

An electrophoretic method (on-line coupled capillary isotachophoresis with capillary zone electrophoresis with conductometric detection (cITP-CZE-COND)) for the determination of chitin in insects based on the analysis of glucosamine after acidic hydrolysis of the sample is described. Chitin is deacetylated and hydrolyzed to glucosamine by acidic hydrolysis (6 M sulfuric acid, 110 °C, 6 h). Under optimized electrophoresis conditions, glucosamine (GlcN) is separated from other sample components in cationic mode and detected with a conductometer within 15 min. The performance method characteristics of the GlcN assay, i.e., linearity (0.2–20 μmol), accuracy (103 ± 5%), repeatability (1.9%), reproducibility (3.4%), limits of detection (0.06 μmol/L) and quantification (0.2 μmol/L), were evaluated. On a series of 28 insect samples, it was proven that cITP-CZE-COND provides results of chitin content in insects comparable to the literature data. The important features of the developed cITP-CZE-COND method are easy sample treatment, high sensitivity and selectivity, and low running costs. It is clear from the above that the cITP-CZE-COND method is suitable for analysis of insect samples for chitin content.

Application of Chitosan-Based Hydrogel Obtained from Insects in Pine Planting.

Agrogels, a hydrogel applied in the soil that collects water during irrigation or rainfall and distributes moisture to plant roots during drought, are a solution to water shortage concerns. Extending the release of low molecular weight chemicals has the potential to minimise mineral fertiliser losses as well as water and soil pollution. Thus, the aim of the research is to obtain chitosan from insect chitin, to synthesize a hydrogel based on chitosan with included mineral and organic fertilisers, and to report on experiments with agrogels in the field. In this study, chitosan was obtained from the adult beetles Zophobas morio. IR spectroscopy was used to examine chitosan. The existence of absorption lines typical of primary amines was demonstrated. In one step, a technique for the manufacture of chitosan-based hydrogels containing embedded mineral fertilisers was established. Hydrogel has a swelling coefficient of 60 g/g. Agrogels were evaluated while planting spruce seedlings on "Semei Ormany" LLP experimental locations. The survival rate of seedlings was found to be 40% higher in the experimental group than in the control group.

Molecular and functional analysis of chitin synthase genes in Chilo suppressalis (Lepidoptera: Crambidae).

The rice stem borer, Chilo suppressalis, has developed a high level of resistance to many of the compounds currently used for control. There is therefore an urgent need to develop novel control methods for C.suppressalis. Insect chitin synthases (CHS) have attracted interest as a potential target for insect pest management. However, to date, CHS have not been characterized in C.suppressalis. Two CHS genes (CsCHS1 and CsCHS2) were identified and cloned from C.suppressalis. Two transcript variants were identified for CsCHS1, CsCHS1a and CsCHS1b. Spatiotemporal expression profiling showed that both transcripts of CsCHS1 are most highly expressed on the last day of each larval instar stage and show the highest expression levels in the integument. In contrast, CsCHS2 is predominantly expressed during the larval feeding stages and shows the highest expression levels in the midgut. Knockdown of CsCHS1 by RNA interference significantly inhibited the molting and pupation of C.suppressalis, and knockdown of CsCHS2 significantly affected growth during the larval stage, but had no significant effect on the pupation. Moreover, knockout of CsCHS1 by CRISPR/Cas9 genome editing severely lowered the hatching rate, larval survivorship, pupation rate, and eclosion rate, but only larval survivorship at the G0 generation was lowered after the knockout of CsCHS2. These results demonstrate that CsCHS1 and CsCHS2 play vital roles in the growth and development of C.suppressalis, and so have potential as insecticidal targets for the control of this highly damaging pest.

Physicochemical Characteristics of Biochar from Waste Cricket Chitin (Acheta domesticus).

The properties of biochar (BC) from crustacean chitin are relatively well understood, while there are few studies on BC from insect chitin. This study presents the characterization and phytotoxic assessment of BC produced from crickets and cricket chitin. Cricket powder (BCCR) and cricket chitin (BCCH) were pyrolyzed at 500 °C and 700 °C. Physicochemical characteristics, N ad-/desorption, FTIR, were examined. SEM images were also performed. Regardless of the pyrolysis temperature, biochars were characterized by a densely "packed" solid surface/monolithic type with a non-porous structure (0.05-0.22 m2/g) and high content of N (9.4-11.8%). BCCHs showed a higher pH (12.2-12.4) compared to BCCR (8.7-10.8). Based on the XRD analysis, BCs were characterized by an amorphous carbon turbostratic structure and a randomly oriented graphitic-like micro-crystallite structure. FTIR spectra of BCs confirmed the presence of various O2 and N-functional groups on the BC surface. BCCHs added to soil at rates from 0.5 to 1.5% significantly reduced the germination of Lepidium sativum. Stimulation of root elongation was also observed in the case of BCCR500 1.0% and BCCR700 1.5%. Thermal degradation of cricket powder and cricket chitin promotes the formation of organic N-containing heterocyclic rings, which lead to the production of N-doped carbons with potential uses in energy storage and the contaminations sorption.

Development of a Quantitative UPLC-ESI/MS Method for the Simultaneous Determination of the Chitin and Protein Content in Insects

In a context where the commercial and nutritional interest in insect chitin is always increasing, an accurate and precise method to quantify this biopolymer, especially in food/feed, is required. In addition, quantification of insect crude protein through nitrogen determination is normally overestimated due to the presence of chitin. In this work, for the first time, an RP-UPLC-ESI/MS method for the simultaneous quantification in insects of chitin, as glucosamine (GlcN), and protein, as total amino acids, is presented. The method is based on acid hydrolysis and derivatization of amino acids and GlcN with the AccQ Tag reagent. Method was optimized and validated in terms of linearity, LOD and LOQ, intraday and inter-day repeatability, and accuracy. A hydrolysed commercial chitin was selected as reference standard for calibration. The instrumental LOD and LOQ correspond respectively to a concentration of 0.00068 mM and 0.00204 mM. The intraday precision satisfied the Horwitz ratio. Data from inter-day precision showed the necessity to perform the analysis within 1 week utilizing standard calibration solutions freshly prepared. A matrix effect was observed, which suggested the necessity to use an internal calibration curve or to work in a particular concentration range of GlcN. The chitin and protein content in black soldier fly (Hermetia illucens) and lesser mealworm (Alphitobius diaperinus) were found in agreement with results obtained by independent methods. The optimized method was also tested on two different commercial food supplements, suggesting its applicability on a wide range of matrices. This newly developed method proved to be simple, more accurate, and faster if compared to methods which separately analyse chitin and protein content.

Fatty acid binding protein is required for chitin biosynthesis in the wing of Drosophila melanogaster

Chitin, the major structural polysaccharide in arthropods such as insects and mites, is a linear polymer of N-acetylglucosamine units. The growth and development of insects are intimately coupled with chitin biosynthesis. The membrane-bound β-glycosyltransferase chitin synthase is known to catalyze the key polymerization step of N-acetylglucosamine. However, the additional proteins that might assist chitin synthase during chitin biosynthesis are not well understood. Recently, fatty acid binding protein (Fabp) has been suggested as a candidate that interacts with the chitin synthase Krotzkopf verkehrt (Kkv) in Drosophila melanogaster. Here, using split-ubiquitin membrane yeast two-hybrid and pull-down assays, we have demonstrated that the Fabp-B splice variant physically interacts with Kkv in vitro. The global knockdown of Fabp in D. melanogaster using RNA interference (RNAi) induced lethality at the larval stage. Moreover, in tissue-specific RNAi experiments, silenced Fabp expression in the epidermis and tracheal system caused a lethal larval phenotype. Fabp knockdown in the wings resulted in an abnormal wing development and uneven cuticular surface. In addition to reducing the chitin content in the first longitudinal vein of wings, Fabp silencing also caused the loss of procuticle laminate structures. This study revealed that Fabp plays an important role in chitin synthesis and contributes to a comprehensive understanding of the complex insect chitin biosynthesis.

RNA Interference of Chitin Synthase 2 Gene in Liriomyza trifolii through Immersion in Double-Stranded RNA.

Simple SummaryLiriomyza trifolii is an important insect pest that infects many horticultural crops and vegetables, displaying strong interspecific competitiveness and inflicting serious harm. Here, chitin synthase 2 transcript was studied, and a prepupal immersion dsRNA delivery method was established for L. trifolii. The dsRNA can enter in the prepupal stage and play a role in the pupal stage, resulting in a decrease in the CHS2 expression level and eclosion rate. This study is an important supplement to the research on the RNAi of L. trifolii, enhances knowledge of the function of chitin synthase 2 in Liriomyza species, and may also provide a new idea for its control strategies.Liriomyza trifolii is an important invasive pest that infects horticultural vegetables, displaying a strong competitive advantage and showing great potential for inflicting harm. Chitin synthase is one of the key enzymes in insect chitin metabolism and plays an important role in insect growth and development. In this study, a chitin synthase (CHS) transcript of L. trifolii was cloned, and the results showed that LtCHS belongs to the CHS2 family. The expression analysis indicated the presence of the highest abundance of LtCHS2 in the pupae at different developmental stages but showed no significant difference among different tissues in the adult. Furthermore, a dsRNA immersion method was developed for RNA interference (RNAi) in L. trifolii using LtCHS2 transcript. RNAi can significantly reduce the expression of LtCHS2 in pupae, and the emergence rate of the pupae was significantly lower than that of the control. The results provide a theoretical basis for exploring the role of chitin synthase gene in L. trifolii and proposing new pest control strategies.

E74 knockdown represses larval development and chitin synthesis in Hyphantria cunea

E74 is a key transcription factor induced by 20E, which plays a broad role in many physiological events during insect growth and development, including vitellogenesis, organ remodeling and new tissue formation, programmed cell death and metamorphosis. However, whether it is involved in regulating insect chitin biosynthesis remains largely unclear. Here, the E74 gene was identified for the first time from Hyphantria cunea, a notorious defoliator of forestry. Thereafter, the role of HcE74 in regulating growth, development and chitin synthesis in H. cunea larvae was evaluated. Bioinformatics analysis showed that HcE74 shared the highest identity (95.53%) with E74A of Spodoptera litura, which belonged to Ets superfamily. The results of RNAi bioassay showed that the larval mortality on 6 d after HcE74 knockdown was up to 51.11 ± 6.94%. Meanwhile, a distinct developmental deformity phenotype was found when HcE74 was silenced. These results indicated that HcE74 plays an important role in the development and molting of H. cunea larvae. Moreover, HcE74 knockdown also significantly decreased the expression of four key genes related to chitin synthesis, including glucose-6-phosphate isomerase (HcG6PI), UDP-N-acetylglucosamine pyrophosphorylase (HcUAP), chitin synthetase A (HcCHSA), and chitin synthetase B (HcCHSB). As a result, the content of chitin in midgut and epidermis decreased by 0.54- and 0.08-fold, respectively. Taken together, these results demonstrated that HcE74 not only plays a critical role in the growth and molting of H. cunea larvae, but also probably participates in the transcriptional regulation of genes involved in chitin biosynthesis.