Chitin Degradation Research Articles

Screening of chitin degrading bacteria from the gut of Asian common toad Duttaphrynus melanostictus: Implication for valorization of chitin rich seafood waste

Chitin is a structural component of many invertebrates and the second most abundant biopolymer after cellulose. Owing to its high insolubility, waste management of chitin rich seafood has severe environmental consequences. Despite the value added bioconversion of waste chitin into commodity products like reducing sugars, its biodegradation by bacteria is still lacking. To this end, we have isolated and characterized chitin degrading bacteria from the gut of common Asian toad, Duttaphrynus melanostictus . The isolates, FG4 and ND3 demonstrated highest chitin degradation activities of 1.44 ± 0.129 U/mL and 1.269 ± 0.138 U/mL respectively. Molecular phylogeny using 16S rRNA gene sequence affiliated FG4 to Stenotrophomonas maltophila and ND3 to Bacillus paramycoides , respectively. Among the tested substrates, highest chitinase activities of 4.210 ± 0.070 U/mL and 1.724 ± 0.494 U/mL were exhibited by S. maltophila FG4 on prawn shells (PS) and chitin flakes (CF), respectively. Similarly, the highest substrate degradation ratio of prawn shells and chitin flakes were 49.52% and 38.15% by S. maltophila FG4. Furthermore, scanning electron microscopy revealed adhesion of bacteria and creation of pores on the substrates. The Fourier transform infrared spectroscopy (FTIR) and X - ray diffraction analysis revealed hydrolysis of prawn shells and chitin flakes by bacteria. Further, the high-resolution mass spectrometric (HRMS) analysis confirmed production of N-acetyl D glucosamine by S. maltophila FG4 on prawn shells. Thus, the present study identifies D. melanostictus gut as a unique reservoir to isolate chitinolytic bacteria that may have potential industrial and therapeutic applications. • First report of bioprospection of amphibian guts for bacteria capable of chitin hydrolysis. • Gut chitinolytic isolates could utilize sea food wastes as sole carbon source. • Applications in production of valuable byproducts such as N-acetyl-D-glucosamine.

Comparative Proteomic Analysis of Bacillus subtilis and Aspergillus niger in Black Soldier Fly Co-Fermentation

Black soldier fly larvae have gained popularity as an organic waste bio-conversional tool and fodder protein replacement in recent decades. It can consume all kinds of animal feces, kitchen waste and agricultural waste with great efficiency and transform them into high-value insect protein, fatty acids, and amino acids, which makes the larva a good substitute for costly fish meal and bean pulp in animal diets. However, excess chitin in the larva skin limits its application as an animal feed additive, consequently, employing fermentation with zymocytes to remove the chitin is necessary. In this study, we raised black soldier fly larvae (BSFL) with different carbon sources, such as chicken feces, straws and glucose, and examined the growth condition; we applied Bacillus subtilis and Aspergillus niger to co-ferment BSFL paste to analyze its nutrition changes. Data revealed that among the four kinds of cultures, the body weight of the corn powder group increased most rapidly; the wood chip group was the most underweight; however, it increased faster than others before day 4, and contained the least fat. Label-free quantitative proteomic analysis revealed that the expression of multiple enzymes from B. subtilis and A. niger involved in polysaccharide hydrolysis, amino acid biosynthesis and fatty acid metabolism, such as peptidase of S8 family, maltogenic α-amylase, oligo-1,6-glucosidase and lysophospholipase like protein changed significantly compared to the control group. Production detection showed that free amino acids, acid-soluble proteins, and short-chain fatty acids increased after fermentation; 13 out of 17 amino acids were increased and total free amino acids were increased from 0.08 g/100 g to 0.3 g/100 g; organic acids increased by 4.81 to 17 fold through fermentation, respectively; the actual protein content declined from 3.03 g/100 g to 1.81 g/100 g, the peptide content increased from 1.3 g/100 g to 2.46 g/100 g, the chitin degradation rate was 40.3%, and fat decreased 30% (p < 0.05). These findings might provide important information for future applications of black soldier fly larvae in different carbon waste recycling measures and material for animal feed/organic fertilizer after fermentation.

Higher pH is associated with enhanced co-occurrence network complexity, stability and nutrient cycling functions in the rice rhizosphere microbiome.

The rice rhizosphere microbiota is crucial for crop yields and nutrient use efficiency. However, little is known about how co-occurrence patterns, keystone taxa and functional gene assemblages relate to soil pH in the rice rhizosphere soils. Using shotgun metagenome analysis, the rice rhizosphere microbiome was investigated across 28 rice fields in east-central China. At higher pH sites, the taxonomic co-occurrence network of rhizosphere soils was more complex and compact, as defined by higher average degree, graph density and complexity. Network stability was greatest at medium pH (6.5 < pH < 7.5), followed by high pH (7.5 < pH). Keystone taxa were more abundant at higher pH and correlated significantly with key ecosystem functions. Overall functional genes involved in C, N, P and S cycling were at a higher relative abundance in higher pH rhizosphere soils, excepting C degradation genes (e.g. key genes involved in starch, cellulose, chitin and lignin degradation). Our results suggest that the rice rhizosphere soil microbial network is more complex and stable at higher pH, possibly indicating increased efficiency of nutrient cycling. These observations may indicate routes towards more efficient soil management and understanding of the potential effects of soil acidification on the rice rhizosphere system.

A pathway for chitin oxidation in marine bacteria

Oxidative degradation of chitin, initiated by lytic polysaccharide monooxygenases (LPMOs), contributes to microbial bioconversion of crystalline chitin, the second most abundant biopolymer in nature. However, our knowledge of oxidative chitin utilization pathways, beyond LPMOs, is very limited. Here, we describe a complete pathway for oxidative chitin degradation and its regulation in a marine bacterium, Pseudoalteromonas prydzensis. The pathway starts with LPMO-mediated extracellular breakdown of chitin into C1-oxidized chitooligosaccharides, which carry a terminal 2-(acetylamino)−2-deoxy-D-gluconic acid (GlcNAc1A). Transmembrane transport of oxidized chitooligosaccharides is followed by their hydrolysis in the periplasm, releasing GlcNAc1A, which is catabolized in the cytoplasm. This pathway differs from the known hydrolytic chitin utilization pathway in enzymes, transporters and regulators. In particular, GlcNAc1A is converted to 2-keto-3-deoxygluconate 6-phosphate, acetate and NH3 via a series of reactions resembling the degradation of D-amino acids rather than other monosaccharides. Furthermore, genomic and metagenomic analyses suggest that the chitin oxidative utilization pathway may be prevalent in marine Gammaproteobacteria.

Chitinase (CHI) of Spodoptera frugiperda affects molting development by regulating the metabolism of chitin and trehalose

Chitin is the main component of insect exoskeleton and midgut peritrophic membrane. Insect molting is the result of the balance and coordination of chitin synthesis and degradation in chitin metabolism under the action of hormones. In this study, a 678 bp dsRNA fragment was designed and synthesized according to the known CHI (Chitinase) sequence of Spodoptera frugiperda. It was injected into the larvae to observe the molting and development of S. frugiperda. At the same time, the activities of trehalase and chitinase, the contents of trehalose, chitin and other substances were detected, and the expression of related genes in the chitin synthesis pathway was determined. The results showed that CHI gene was highly expressed at the end of each instar, prepupa and pupal stage before molting; At 12 and 24 h after dsRNA injection of CHI gene of S. frugiperda, the expression of CHI gene decreased significantly, and the chitinase activity decreased significantly from 12 to 48 h. The expression of chitin synthase (CHSB) gene decreased significantly, and the chitin content increased significantly. Some larvae could not molt normally and complete development, leading to certain mortality. Secondly, after RNAi of CHI gene, the content of glucose and glycogen increased first and then decreased, while the content of trehalose decreased significantly or showed a downward trend. The activities of the two types of trehalase and the expression levels of trehalase genes decreased first and then increased, especially the trehalase activities increased significantly at 48 h after dsCHI injection. And trehalose-6-phosphate synthase (TPS), glutamine: fructose-6-phosphate amidotransferase (GFAT), UDP-N-acetylglucosamine pyrophosphorylases (UAP), hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and phosphoacetylglucosamine mutase (PAGM) all decreased significantly at 24 h, and then increased or significantly increased at 48 h. These results indicated that when the expression of chitinase gene of S. frugiperda was inhibited, it affected the degradation of chitin in the old epidermis and the formation of new epidermis, and the content of chitin increased, which led to the failure of larvae to molt normally. Moreover, the chitin synthesis pathway and trehalose metabolism were also regulated. The relevant results provide a theoretical basis for screening target genes and developing green insecticides to control pests by using the chitin metabolism pathway.

Identification and functional analysis of two potential RNAi targets for chitin degradation in Holotrichia parallela Motschulsky (Insecta Coleoptera)

Chitin metabolism enzymes are safe and desirable targets for pest management. β-N-acetylglucosaminidase (NAG) and N-acetylglucosamine kinase (NAGK) are involved in chitin degradation. NAG is the main glycosidase that works synergistically with chitinases. NAGK is a key enzyme for the generation of UDP-Nacetylglucosamine (UDP-GlcNAc) and for the conversion of GlcNAc into GlcNAc 6-phosphate (GlcNAc-6-P). In this study, NAG and NAGK genes were identified from Holotrichia parallela, a polyphagous soil pest that causes serious damage to crops. The spatiotemporal expression investigated by RT-qPCR indicated that the two genes are expressed in all larval developmental stages. HpNAG is highly expressed in the integument and HpNAGK overexpressed in the midgut. After injection of dsHpNAG and dsHpNAGK, a significant RNAi effect was found after 72 h and larvae stopped growing. The survival rates of larvae were 13.3% and 16.7%, respectively. RNAi of HpNAG and HpNAGK regulated the expression levels of chitin metabolism-related genes, indicating that these two genes could be critical in the chitin metabolism. Furthermore, silencing HpNAG and HpNAGK reduced the thickness of the cuticle, and decreased its content of chitin. The study will lay a foundation for further clarifying the mechanism of chitin metabolism and provide potential targets for the biological control of H. parallela larvae.

Effect of zinc and iron oxide nanoparticles on plant physiology, seed quality and microbial community structure in a rice-soil-microbial ecosystem

In this study, we assessed the impact of zinc oxide (ZnO) and iron oxide (FeO) (<36 nm) nanoparticles (NPs) as well as their sulphate salt (bulk) counterpart (0, 25, 100 mg/kg) on rice growth and seed quality as well as the microbial community in the rhizosphere environment of rice. During the rice growing season 2021–22, all experiments were conducted in a greenhouse (temperature: day 30 °C; night 20 °C; relative humidity: 70%; light period: 16 h/8 h, day/night) in rice field soil. Results showed that low concentrations of FeO and ZnO NPs (25 mg/kg) promoted rice growth (height (29%, 16%), pigment content (2%, 3%)) and grain quality parameters such as grains per spike (8%, 9%), dry weight of grains (12%, 14%) respectively. As compared to the control group, the Zn (2%) and Fe (5%) accumulations at their respective low concentrations of NP treatments showed stimulation. Interestingly, our results showed that at low concentration of both the NPs the soil microbes had more diversity and richness than those in the bulk treated and control soil group. Although a number of phyla were affected by the presence of NPs, the strongest effects were observed for change in the abundance of the three phyla for Proteobacteria, Actinobacteria, and Planctomycetes. The rhizosphere environment was notably enriched with potential streptomycin producers, carbon and nitrogen fixers, and lignin degraders with regard to functional groups of microorganisms. However, microbial communities mainly responsible for chitin degradation, ammonia oxidation, and nitrite reduction were found to be decreased. The results from this study highlight significant changes in several plant-based endpoints, as well as the rhizosphere soil microorganisms. It further adds information to our understanding of the nanoscale-specific impacts of important micronutrient oxides on both rice and its associated soil microbiome.

Isolation, characterization, and genome sequencing of a novel chitin deacetylase producing Bacillus aryabhattai TCI-16.

Chitin deacetylase (CDA) is a chitin degradation enzyme that catalyzes the conversion of chitin to chitosan by the deacetylation of N-acetyl-D-glucosamine residues, playing an important role in the high-value utilization of waste chitin. The shells of shrimp and crab are rich in chitin, and mangroves are usually recognized as an active habitat to shrimp and crab. In the present study, a CDA-producing bacterium, strain TCI-16, was isolated and screened from the mangrove soil. Strain TCI-16 was identified and named as Bacillus aryabhattai TCI-16, and the maximum CDA activity in fermentation broth reached 120.35 ± 2.40 U/mL at 36 h of cultivation. Furthermore, the complete genome analysis of B. aryabhattai TCI-16 revealed the chitin-degrading enzyme system at genetic level, in which a total of 13 putative genes were associated with carbohydrate esterase 4 (CE4) family enzymes, including one gene coding CDA, seven genes encoding polysaccharide deacetylases, and five genes encoding peptidoglycan-N-acetyl glucosamine deacetylases. Amino acid sequence analysis showed that the predicted CDA of B. aryabhattai TCI-16 was composed of 236 amino acid residues with a molecular weight of 27.3 kDa, which possessed a conserved CDA active like the known CDAs. However, the CDA of B. aryabhattai TCI-16 showed low homology (approximately 30%) with other microbial CDAs, and its phylogenetic tree belonged to a separate clade in bacteria, suggesting a high probability in structural novelty. In conclusion, the present study indicated that the novel CDA produced by B. aryabhattai TCI-16 might be a promising option for bioconversion of chitin to the value-added chitosan.

Probing the Structural Details of Chitin Nanocrystal–Water Interfaces by Three‐Dimensional Atomic Force Microscopy (Small Methods 9/2022)

Chitin Nanocrystal Structure In article number 2200320, Yurtsever, Fukuma, and co-workers provided the molecular-scale structural details of chitin nanocrystals in water by combining 3D atomic force microscopy experiments and molecular dynamics simulations. The characterization of chitin nanocrystal surfaces at the molecular scale could pave the way for assessing the chemical and enzymatic activities on chitin nanocrystals, offering an understanding of the mechanisms of chitin degradation.

Soil metatranscriptome demonstrates a shift in C, N, and S metabolisms of a grassland ecosystem in response to elevated atmospheric CO2.

Soil organisms play an important role in the equilibrium and cycling of nutrients. Because elevated CO2 (eCO2) affects plant metabolism, including rhizodeposition, it directly impacts the soil microbiome and microbial processes. Therefore, eCO2 directly influences the cycling of different elements in terrestrial ecosystems. Hence, possible changes in the cycles of carbon (C), nitrogen (N), and sulfur (S) were analyzed, alongside the assessment of changes in the composition and structure of the soil microbiome through a functional metatranscriptomics approach (cDNA from mRNA) from soil samples taken at the Giessen free-air CO2 enrichment (Gi-FACE) experiment. Results showed changes in the expression of C cycle genes under eCO2 with an increase in the transcript abundance for carbohydrate and amino acid uptake, and degradation, alongside an increase in the transcript abundance for cellulose, chitin, and lignin degradation and prokaryotic carbon fixation. In addition, N cycle changes included a decrease in the transcript abundance of N2O reductase, involved in the last step of the denitrification process, which explains the increase of N2O emissions in the Gi-FACE. Also, a shift in nitrate () metabolism occurred, with an increase in transcript abundance for the dissimilatory reduction to ammonium () (DNRA) pathway. S metabolism showed increased transcripts for sulfate () assimilation under eCO2 conditions. Furthermore, soil bacteriome, mycobiome, and virome significantly differed between ambient and elevated CO2 conditions. The results exhibited the effects of eCO2 on the transcript abundance of C, N, and S cycles, and the soil microbiome. This finding showed a direct connection between eCO2 and the increased greenhouse gas emission, as well as the importance of soil nutrient availability to maintain the balance of soil ecosystems.

RNAi Suppression of Hormone Receptor HR3 Blocks Larval Molting and Metamorphosis in the Cigarette Beetle, Lasioderma serricorne

Hormone receptor 3 (HR3), an early-late gene of the 20-hydroxyecdysone (20E) signaling pathway, plays a critical role in insect metamorphosis and development. In this study, we identified and characterized an HR3 gene (LsHR3) from the cigarette beetle, Lasioderma serricorne. The open reading frame of LsHR3 is 1581 bp encoding a 527 amino acid protein that contains a conserved DNA binding domain and a ligand binding domain. LsHR3 was mainly expressed in the fourth-instar larvae, prepupae, and pupae and showed high expression in the fat body. The expression of LsHR3 was induced by 20E, while it was significantly suppressed by silencing of six 20E synthesis and signaling pathway genes. RNA interference (RNAi)-aided knockdown of LsHR3 in the fourth-instar larvae disrupted the larval–pupal molting and caused 100% mortality. The 20E titer of LsHR3-depletion larvae was decreased, and expressions of five 20E synthesis genes were dramatically decreased. Silencing LsHR3 reduced chitin content and downregulated the expression of genes involved in chitin synthesis and degradation. Hematoxylin and eosin staining of abdominal cuticle showed that no apolysis occurred after silencing LsHR3. These results suggest that LsHR3-mediated 20E signaling is involved in the regulation of chitin metabolism during the molting process of L. serricorne, and targeting this gene by RNAi has potential in controlling this pest.

Fungal dynamic changes in naturally fermented 'Kyoho' grape juice.

The 'Kyoho' grape (Vitaceae, Plantae) has large ears, plenty of flesh, and rich nutrition and is planted across a large area in China. There are few reports on this variety in winemaking, especially on the dynamic changes of fungi in the wine fermentation broth. In this study, we used the 'Kyoho' grapes as raw materials and adopted a high throughput to analyze dynamic changes in fungal species composition of the natural fermentation broth at four time points: day 1 (D1P), day 3 (D3P), day 5 (D5P), and day 15 (D15P). Changes in fungal metabolic pathways and dominant yeasts were also analyzed. A total of 78 families, 110 genera, and 137 species were detected, in the natural fermentation broth samples. Forty-nine families, 60 genera, and 72 species were found in the control check (CK). A total of 66 differential metabolic pathways were enriched; of those, 41 were up-regulated compared to CK, such as CDP-diacylglycerol biosynthesis I (PWY 5667), chitin degradation to ethanol (PWY 7118), and the super pathway of phosphatidate biosynthesis (PWY 7411). Changes in fungal metabolic pathways were in line with the dynamic changes of dominant yeast species in the whole process of fermentation. Pichia kluyveri, P. membranifaciens, and Citeromyces matritensis are the dominant species in the later stages of natural fermentation. These yeast species may play vital roles in the 'Kyoho' wine industry in the future.

Degradation‐induced microbiome alterations may aggravate soil nutrient loss in subalpine meadows

AbstractGrassland degradation has become a serious environmental problem worldwide. However, the link between grassland degradation and soil functional microorganisms remains unclear. Here, we investigated the effects of subalpine meadow degradation on the taxonomic and functional communities of the soil microbiome on Mount Wutai on the eastern margin of the Loess Plateau, China, using shotgun metagenomic sequencing. The results showed that vegetation degradation decreased most soil nutrients (e.g., soil organic carbon [SOC, −48.9%], total nitrogen [TN, −15.2%] and carbon [TC, −38.3%]), leading to significant changes in the taxonomic (r = 0.892, p &lt; 0.001) and functional compositions (r = 0.972, p &lt; 0.001) of the microbial communities (explaining 66.6% and 64.5% of the variance, respectively). The structural equation model (SEM) showed that variations in the soil nutrient status and plant parameters caused by degradation resulted in changes in the microbial community structure and function. The variations in the plant and microbial communities during degradation altered carbon (C) and nitrogen (N) cycling separately or interactively. Among the C degradation‐related genes, the relative abundances of cellulose degradation, sugar utilization and chitin degradation genes in moderately degraded (MD) and heavily degraded (HD) meadows were significantly higher than those in nondegraded (ND) meadows (p &lt; 0.05). The abundance of denitrification genes (nirK, nirS, norB and norC) and nitrification genes (pmoA/amoA, pmoB/amoB, pmoC/amoC and hao) was higher in all degraded meadows, which increased the risk of soil N loss. Our findings revealed the negative feedback of microbes due to subalpine meadow degradation, during which metabolically active microbes tended to accelerate soil nutrient loss.

Chitinolytic enzymes contribute to the pathogenicity of Aliivibrio salmonicida LFI1238 in the invasive phase of cold-water vibriosis

BackgroundAliivibrio salmonicida is the causative agent of cold-water vibriosis in salmonids (Oncorhynchus mykiss and Salmo salar L.) and gadidae (Gadus morhua L.). Virulence-associated factors that are essential for the full spectrum of A. salmonicida pathogenicity are largely unknown. Chitin-active lytic polysaccharide monooxygenases (LPMOs) have been indicated to play roles in both chitin degradation and virulence in a variety of pathogenic bacteria but are largely unexplored in this context.ResultsIn the present study we investigated the role of LPMOs in the pathogenicity of A. salmonicida LFI238 in Atlantic salmon (Salmo salar L.). In vivo challenge experiments using isogenic deletion mutants of the two LPMOs encoding genes AsLPMO10A and AsLPMO10B, showed that both LPMOs, and in particular AsLPMO10B, were important in the invasive phase of cold-water vibriosis. Crystallographic analysis of the AsLPMO10B AA10 LPMO domain (to 1.4 Å resolution) revealed high structural similarity to viral fusolin, an LPMO known to enhance the virulence of insecticidal agents. Finally, exposure to Atlantic salmon serum resulted in substantial proteome re-organization of the A. salmonicida LPMO deletion variants compared to the wild type strain, indicating the struggle of the bacterium to adapt to the host immune components in the absence of the LPMOs.ConclusionThe present study consolidates the role of LPMOs in virulence and demonstrates that such enzymes may have more than one function.

Metagenomic analysis reveals the microbial degradation mechanism during kitchen waste biodrying

Biodrying is a treatment to remove moisture using bio-heat generated during organic degradation. Organic matter degradation and microbial metabolism were studied during the whole kitchen waste biodrying, using metagenomic analysis. After the 25-day biodrying process, carbohydrate, protein and lipid contents decreased by 83.7%, 27.8% and 79.3%, respectively, and their degradation efficiencies increased after the thermophilic phase. Lipase activity exceeded 10 mmol d−1 g−1 throughout biodrying. Cellulase and lipase activities recovered by 2.21% and 5.77%, respectively, after the thermophilic phase, while the protease activity had a maximum increment of 347%. Metabolic analysis revealed that carbohydrate, amino acid and lipid metabolism was possibly inhibited by the high temperature, but the relative abundances of related predicted functions recovered by more than 0.9%, 7% and 11%, respectively, by the end of biodrying. Protein function prediction suggests that β-oxidation, fatty acid biosynthesis, and the degradation of cellulose and chitin were possibly enhanced during the thermophilic phase. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that leucine, isoleucine and lysine could ultimately degraded to acetyl-CoA. Weissella, Aeribacillus and Bacillus were the genera with the most enriched functional genes during the whole biodrying process. These findings help elucidate the microbial degradation processes during biodrying, which provides further scientific support for improving the application of biodrying products.

Transcription factors Vrf1 and Hox7 coordinately regulate appressorium maturation in the rice blast fungus Magnaporthe oryzae

Magnaporthe oryzae infects rice, wheat and other grass crops through appressoria. The formation of the appressorium is regulated by the external environment, signal transduction pathways, and transcription factors. Transcription factors Vrf1 and Hox7 are involved in the regulation of appressorium formation. In this study, we demonstrate that Vrf1 and Hox7 play vital roles in coordinately regulating appressorium maturation. In strain 70–15, deletion of VRF1 resulted in the inability to continue melanization and maturation of the incipient appressorium, and deletion of HOX7 also resulted in defects in appressorium melanization and maturation. The defects in appressorium formation in Δhox7Δvrf1 were similar to those in Δhox7 and Δvrf1. The gene expression profiles of the incipient appressoria at 5 h post-inoculation (hpi) showed that the expression levels of 704 genes (25.94 % of all differentially expressed genes in the three mutants) were significantly downregulated (606 genes) or upregulated (98 genes). In the appressoria of Δhox7, Δvrf1, and Δhox7Δvrf1 at 5 hpi, the expression level of genes related to cell wall remodeling was changed. Genes for melanin synthesis, chitin and glucan degradation, and extracellular cell wall degrading enzyme were significantly downregulated, while genes for chitin and glucan synthesis were upregulated. After 8 hpi, the incipient appressoria of Δhox7, Δvrf1, and Δhox7Δvrf1 regerminated and formed swollen hyphal-like structures with multiple nuclei. The ratio of the nuclear number of the hyphal-like structures of Δhox7, Δhox7Δvrf1, and Δvrf1 was close to 6:4:2 at 24 hpi. Therefore, although Vrf1 and Hox7 are somewhat functionally different, they synergistically regulate appressorium maturation in M. oryzae.

SfDicer2 RNA Interference Inhibits Molting and Wing Expansion in Sogatella furcifera.

Simple SummaryEndoribonuclease 2 (Dicer2) plays various physiological roles in the RNA interference (RNAi) pathway by fragmenting double-stranded RNA to generate small interfering RNA, which then mediates gene silencing. In this study, the role of Dicer2 in the regulation of molting and wing expansion in Sogatella furcifera (white-backed planthopper) was investigated. In particular, SfDicer2-mediated RNAi resulted in wing deformities and lethal modifications in S. furcifera, which are attributable to the significant inhibition of chitin synthesis and degradation and wing expansion genes. This study provides insights into the biological functions of Dicer2 in insects, which can aid in RNAi-mediated pest control.Endoribonuclease 2 (Dicer2) is a key nicking endonuclease involved in the small interfering RNA biosynthesis, and it plays important roles in gene regulation and antiviral immunity. The Dicer2 sequence was obtained using the transcriptomic and genomic information of Sogatella furcifera (Horváth), and the spatiotemporal characteristics and functions of molting and wing expansion regulation were studied using real-time quantitative polymerase chain reaction and RNA interference (RNAi) technology. The expression of SfDicer2 fluctuated during the nymphal stage of S. furcifera. Its expression decreased significantly over the course of molting. SfDicer2 exhibited the highest transcript level in the nymphal stage and adult fat body. After SfDicer2 was silenced, the total mortality rate was 42.69%; 18.32% of the insects died because of their inability to molt. Compared with the effects of dsGFP or water, 44.38% of the insects subjected to the silencing of SfDicer2 exhibited wing deformities after successful eclosion. After SfDicer2 RNAi, the expression of chitinase, chitin deacetylase, trehalase, chitin synthase 1, and wing expansion-related genes was significantly inhibited. These findings indicate that SfDicer2 controls molting by affecting genes associated with chitin synthesis and degradation and regulates wing expansion by altering the expression of wing expansion-related genes in S. furcifera.

Comparative Genomic Analysis of Seven Vibrio alginolyticus Strains Isolated From Shrimp Larviculture Water With Emphasis on Chitin Utilization.

The opportunistic pathogen Vibrio alginolyticus is gaining attention because of its disease-causing risks to aquatic animals and humans. In this study, seven Vibrio strains isolated from different shrimp hatcheries in Southeast China were subjected to genome sequencing and subsequent comparative analysis to explore their intricate relationships with shrimp aquaculture. The seven isolates had an average nucleotide identity of ≥ 98.3% with other known V. alginolyticus strains. The species V. alginolyticus had an open pan-genome, with the addition of ≥ 161 novel genes following each new genome for seven isolates and 14 publicly available V. alginolyticus strains. The percentages of core genes of the seven strains were up to 83.1–87.5%, indicating highly conserved functions, such as chitin utilization. Further, a total of 14 core genes involved in the chitin degradation pathway were detected on the seven genomes with a single copy, 12 of which had undergone significant purifying selection (dN/dS < 1). Moreover, the seven strains could utilize chitin as the sole carbon-nitrogen source. In contrast, mobile genetic elements (MGEs) were identified in seven strains, including plasmids, prophages, and genomic islands, which mainly encoded accessory genes annotated as hypothetical proteins. The infection experiment showed that four of the seven strains might be pathogenic because the survival rates of Litopenaeus vannamei postlarvae were significantly reduced (P < 0.05) when compared to the control. However, no obvious correlation was noted between the number of putative virulence factors and toxic effects of the seven strains. Collectively, the persistence of V. alginolyticus in various aquatic environments may be attributed to its high genomic plasticity via the acquisition of novel genes by various MGEs. In view of the strong capability of chitin utilization by diverse vibrios, the timely removal of massive chitin-rich materials thoroughly in shrimp culture systems may be a key strategy to inhibit proliferation of vibrios and subsequent infection of shrimp. In addition, transcontinental transfer of potentially pathogenic V. alginolyticus strains should receive great attention to avoid vibriosis.

Knockdown of UDP-N-acetylglucosamine pyrophosphorylase and chitin synthase A increases the insecticidal efficiency of Lufenuron to Spodoptera exigua

Spodoptera exigua (Lepidoptera, Noctuidae) has been responsible for causing considerable and widespread agricultural losses worldwide. Owing to strong selective pressure, S. exigua showed increased resistance to Lufenuron (LUF). Consequently, RNA interference (RNAi)-based insecticides had more benefits than chemical insecticides. Therefore, to enhance the insecticidal activity of LUF to S. exigua, in the present study, we aimed to elucidate the impact of double-stranded RNAs (dsRNAs) on S. exigua larval susceptibility to LUF. First, the transcriptome of S. exigua was sequenced following the treatment with LUF. By comparing the upregulated and downregulated GO enrichment, chitin binding and chitin metabolic processes were the significantly enriched pathways. According to transcriptome sequencing, 8 genes associated with chitin biosynthesis, 8 chitin degradation genes, and 17 cuticle protein genes were obtained. UDP-N-acetylglucosamine pyrophosphorylase (UAP) and Chitin synthase A (CHSA) showed significantly downregulated expression after treatment with different sublethal doses of LUF. Downregulation of UAP increased mortality from 31.97% to 47.91% when the larvae were exposed to LUF. A significant increase in the mortality of S. exigua from 30.63% to 50.19% was observed following LUF administration after dsCHSA. In addition, the expression analysis of genes associated with chitin biosynthesis was significantly changed after LUF treatment, dsRNAs-RNAi, and their combination (LUF-dsRNAs). Significant differences were observed in the chitin content between the control group at 72 h after treatments. Results of the present study can help further elucidate the understanding of the combined effects of RNAi and LUF on S. exigua. Additionally, this research provides a suitable foundation for future studies with the aim to develop an efficient method of delivery for large-scale pest control in the fields.

Nuclear receptor HR3 mediates transcriptional regulation of chitin metabolic genes during molting in Tribolium castaneum.

Chitin, a major component of insect cuticles, plays a critical role in insect molting and morphogenesis. Thus, coordination of chitin remodeling during insect development requires tight transcriptional control of the chitin metabolism genes involved in chitin synthesis, assembly and degradation. However, the molecular mechanism underlying transcriptional coordination of chitin metabolism genes during beetle development is not yet completely understood. We cloned the full-length cDNA encoding hormone receptor 3 (TcHR3) from Tribolium castaneum and showed a critical role of TcHR3 in modulating chitin metabolism gene expression during molting. Genome-wide transcriptome analysis of HR3-deficient old larvae using RNA sequencing analysis revealed a positive correlation between TcHR3 and transcription of chitin metabolism genes involved in chitin synthesis and degradation. In addition, HR3 overexpression significantly induced the gene promoter activity of N-acetylglucosaminidase 1 (NAG1) involved in chitin degradation and UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) involved in chitin synthesis. Chromatin immunoprecipitation analysis revealed that HR3 could directly bind to HR3-response element of NAG1 and UAP1 promoters. Finally, HR3-deficient late instar larvae and prepupae exhibited defects in larval-larval and larval-pupal molting, respectively, leading to eventual larval death because developing larvae were trapped inside the old cuticle as a result of abnormal chitin metabolism. TcHR3 is a transcriptional regulator of chitin metabolic genes for molting of T.castaneum. Controlling the molting system by TcHR3 might be a new management strategy for selective control of red flour beetle infestation. © 2022 Society of Chemical Industry.