Chitin Content Research Articles

Breaking down barriers: comprehensive functional analysis of the Aspergillus niger chitin synthase repertoire

BackgroundMembers of the fungal kingdom are heterotrophic eukaryotes encased in a chitin containing cell wall. This polymer is vital for cell wall stiffness and, ultimately, cell shape. Most fungal genomes contain numerous putative chitin synthase encoding genes. However, systematic functional analysis of the full chitin synthase catalogue in a given species is rare. This greatly limits fundamental understanding and potential applications of manipulating chitin synthesis across the fungal kingdom.ResultsIn this study, we conducted in silico profiling and subsequently deleted all predicted chitin synthase encoding genes in the multipurpose cell factory Aspergillus niger. Phylogenetic analysis suggested nine chitin synthases evolved as three distinct groups. Transcript profiling and co-expression network construction revealed remarkably independent expression, strongly supporting specific role(s) for the respective chitin synthases. Deletion mutants confirmed all genes were dispensable for germination, yet impacted colony spore titres, chitin content at hyphal septa, and internal architecture of submerged fungal pellets. We were also able to assign specific roles to individual chitin synthases, including those impacting colony radial growth rates (ChsE, ChsF), lateral cell wall chitin content (CsmA), chemical genetic interactions with a secreted antifungal protein (CsmA, CsmB, ChsE, ChsF), resistance to therapeutics (ChsE), and those that modulated pellet diameter in liquid culture (ChsA, ChsB). From an applied perspective, we show chsF deletion increases total protein in culture supernatant over threefold compared to the control strain, indicating engineering filamentous fungal chitin content is a high priority yet underexplored strategy for strain optimization.ConclusionThis study has conducted extensive analysis for the full chitin synthase encoding gene repertoire of A. niger. For the first time we reveal both redundant and non-redundant functional roles of chitin synthases in this fungus. Our data shed light on the complex, multifaceted, and dynamic role of chitin in fungal growth, morphology, survival, and secretion, thus improving fundamental understanding and opening new avenues for biotechnological applications in fungi.

Gas foamed scaffolds as smart 3D structures in skin tissue engineering

Globally, wound care is a major problem that poses big issues for national healthcare systems. Consequently, the development of wound dressings, systems applied over a wound to prevent complications and speed up recovery, is receiving increasing attention. Electrospun nanofibrous scaffolds gained great interest in the medical field due to their many advantages and beneficial characteristics. However, they are formed by of densely packed fiber layers, which render the available space between adjacent fibers minimal. In this study, nanometric fibers from pullulan polymeric blends were developed using cricket powder as novel component, due to its high chitin and chitosan content. Hydroxyapatite was also added due to its biocompatibility and capability to interact with the host tissues. The 2D mats based on electropsun fibers in membrane were converted to a 3D structure by gas foaming, involving NaBH4. The obtained 3D systems were characterized for their morphology and composition, and mechanical properties. The scaffolds biocompatibility was tested on normal human dermal fibroblasts and mesenchymal stem cells, and cell adhesion was assessed. TNF-α secretion was thereafter characterized to evaluate the anti-inflammatory effects of the scaffolds on macrophages, and their antioxidant activity was also investigated. Finally, an evaluation of the scaffolds’ safety and efficacy in vivo on a murine incisional and burn model was performed. The results obtained not only revealed greater structural and mechanical integrity of the gas-foamed scaffolds containing cricket powder, but also higher cytocompatibility towards different cell lines together with anti-inflammatory and antioxidant properties, thereby making them innovative tools to improve the process of wound healing.

Photoactivated 9-methylacridine destroys midgut tissues of Aedes aegypti larvae by targeting ROS-mediated apoptosis in the mitochondrial pathway of midgut cells

An aromatic ring-containing compound with a wide range of biological activities, 9-methylacridine (AD-9-Me) is a precursor for the synthesis of various drugs. However, its photoactivation properties and mechanism of damage as a photo activator against Aedes aegypti are unknown. The toxic effects of AD-9-Me on Aedes aegypti mosquitoes were determined under light and non-light conditions. The results showed that the toxicity of AD-9-Me to mosquito larvae was significantly higher than that of the dark treatment after 24 h of light exposure; AD-9-Me was mainly distributed in the midgut of larvae, after 24 h of treatment, it can cause an increase in calcium ion concentration, reactive oxygen species (ROS) eruption and ROS accumulation by blocking the ROS elimination pathway in midgut cells. This in turn caused an increase in protein carbonyl and malondialdehyde (MDA) content, a decrease in mitochondrial membrane potential (MMP), a disruption of the barrier function of midgut tissues, a significant decrease in midgut weight and chitin content, which induced the up-regulation of AeDronc, AeCaspase8 and AeCaspase7 genes, leading to apoptotic cell death. In this study, we confirmed that AD-9-Me has photoactivation activity and mainly acts on the midgut of mosquito larvae, which can generate a large amount of ROS in the cells of the midgut and induce apoptosis to occur, resulting in the disruption of the function of the tissues of mosquito larvae, accelerating the death and delaying the development of the mosquito larvae.

Optimizing Chitin Extraction and Chitosan Production from House Cricket Flour

Chitin and its derivative, chitosan, have diverse applications in fields such as agriculture, medicine, and biosensors, amongst others. Extraction is primarily conducted from marine sources, such as crustaceans, which have been the focus of process optimization studies. However, there are other sources that are more readily available, such as insects, where insufficient research has been conducted. The house cricket (Acheta domesticus) is a promising source for chitin extraction because of its high chitin content, availability, and short lifespan. Modern chemical chitin extraction methods have not been standardized due to the use of different reagents, molar concentrations, temperatures, and reaction times across publications. Therefore, in this study, the composition of Acheta domesticus cricket flour was determined: 2.62% humidity, 4.3% ash content, 56.29% protein, 13.35% fat, 23.44% carbohydrates, and 15.71% crude fiber content. After a drying, defatting, demineralization, deproteinization, and bleaching process, chitin extraction was performed, and chitosan was obtained via a deacetylation reaction. The demineralization process was standardized at 30 °C for 3 h using HCl 2 M, resulting in 95.85 ± 0.012%. The deproteinization process was optimized at 80 °C for 45 min using NaOH 2.56 M, yielding 43.23 ± 1.25%. Finally, the identity and physicochemical characteristics of the compounds were confirmed and determined through characterization with Fourier-Transform Infrared Spectroscopy, X-ray Diffraction, Scanning Electron Microscopy, and Differential Scanning Calorimetry.

Candida glabrata hospital isolate from Lebanon reveals micafungin resistance associated with increased chitin and resistance to a cell-surface-disrupting agent

This study aimed to identify the resistance mechanisms to micafungin and fluconazole in a clinical isolate of Candida glabrata. The isolate was whole-genome sequenced to identify amino acid changes in key proteins involved in antifungal resistance, and the isolate was further characterised by pathogenicity-related phenotypic assays that supported the sequencing results. Amino acid substitutions were detected in 8 of 17 protein candidates. Many of these substitutions were novel, including in CHS3, CHS3B, and KRE5, which are involved in the development of micafungin resistance. Regarding fluconazole resistance, overexpression of efflux pumps was observed. Our isolate did not exhibit an increased virulence potential compared with the control strain; however, a significant increase in chitin content and potential to resist the cell surface disruptant sodium dodecyl sulphate was observed. This clinical Candida glabrata isolate experienced a change in cell wall architecture, which correlates with the development of micafungin resistance.

Physical impediment to sodium houttuyfonate conversely reinforces β-glucan exposure stimulated innate immune response to Candida albicans.

Candida albicans is a dimorphic opportunistic pathogen in immunocompromised individuals. We have previously demonstrated that sodium houttuyfonate (SH), a derivative of medicinal herb Houttuynia cordata Thunb, was effective for antifungal purposes. However, the physical impediment of SH by C. albicans β-glucan may weaken the antifungal activity of SH. In this study, the interactions of SH with cell wall (CW), extracellular matrix (EM), CW β-glucan, and a commercial β-glucan zymosan A (ZY) were inspected by XTT assay and total plate count in a standard reference C. albicans SC5314 as well as two clinical fluconazole-resistant strains Z4935 and Z5172. After treatment with SH, the content and exposure of CW β-glucan, chitin, and mannan were detected, the fungal clearance by phagocytosis of RAW264.7 and THP-1 was examined, and the gene expressions and levels of cytokines TNF-ɑ and IL-10 were also monitored. The results showed that SH could be physically impeded by β-glucan in CW, EM, and ZY. This impediment subsequently triggered the exposure of CW β-glucan and chitin with mannan masked in a time-dependent manner. SH-induced β-glucan exposure could significantly enhance the phagocytosis and inhibit the growth of C. albicans. Meanwhile, the SH-pretreated fungal cells could greatly stimulate the cytokine gene expressions and levels of TNF-ɑ and IL-10 in the macrophages. In sum, the strategy that the instant physical impediment of C. albicans CW to SH, which can induce the exposure of CW β-glucan may be universal for C. albicans in response to physical deterrent by antifungal drugs.

Knockdown of the Expression of Two Trehalase Genes with RNAi Disrupts the Trehalose and Chitin Metabolism Pathways in the Oriental Armyworm, Mythimna separata.

Trehalose is an important carbohydrate substance in insect hemolymph. Chitin is the main component of cuticle and peritrophic matrix in insects. Trehalase (Tre) catalyzes the decomposition of trehalose. Few studies of trehalase in lepidopteran insects have been conducted. Here, the functions of soluble Tre (Tre1) and membrane-bound Tre (Tre2) in the growth and development of Mythimna separata were investigated. We cloned and identified Tre1 and Tre2 cDNA sequences in M. separata. Analysis expression revealed that MsTre1 and MsTre2 were highly expressed in midgut and integument, respectively. The expression of MsTre1 and MsTre2 was highest in the pupal stage. We used RNA interference (RNAi) to inhibit Tre expression in M. separata larvae. Injection of dsMsTre1 or dsMsTre2 resulted in abnormal phenotypes and impeded normal molting. Silencing of MsTre1 and MsTre2 resulted in significant changes in the expression of genes in the trehalose and chitin metabolism pathways, significantly increased the trehalose and glycogen content, and significantly decreased MsTre1 and MsTre2 activity, the glucose content, and the chitin content in midgut and integument. Silencing of MsTre1 slowed larval molting, and the new cuticle was significantly thinner. These results indicate that RNAi of Tre may be useful for control strategies against M. separata.

The Extraction of Chitin from Indonesian Shells and its Potential as Zinc Ion Batteries Supporting Material

<p>Indonesia is an archipelagic country that has a very wide water area. One of the results of aquaculture in Indonesia is shrimp. Shrimp production results based on data from the Ministry of Maritime Affairs and Fisheries in 2022 amounted to 8,3 tons. This makes Indonesia one of the largest shrimp-exporting countries in Southeast Asia. The part of the shrimp that is often consumed by the public is the meat. So that the heads, tails, and shells that have been peeled are often discarded. Shrimp waste besides being able to pollute the environment can also disturb the comfort of local residents, so efforts are needed to overcome it. Shrimp shell contains 27.6% minerals, 34.9% protein, and 18.1% chitin. The chitin content in shrimp shells can be processed into materials for zinc-ion batteries, namely into chitosan which has non-toxic properties and can be used as polymer electrolytes that have good conductivity values through the deacetylation process. The main ingredient used is shrimp shell powder from shrimp waste that has been cleaned and dried. In this study, there are three steps of procedures, namely the deproteination method using NaOH base solution, demineralization using HCl acid solution, and testing for the presence of chitin with the Van Wesslink color reaction. In this experiment, the yields obtained were 0.38, 0.1, 0.12, dan 0.47-gram chitin/gram shrimp shell. The big difference in the yield produced is due to the different types of shrimp waste used.</p>

Rates of diagenesis of tropical insectivorous bat guano accumulations: implications for potential paleoenvironmental reconstruction

Cave guano deposits are increasingly being recognized as valuable repositories of paleo-climatic and paleo-environmental information. However, that value is constrained by rates of guano decomposition, and these rates have not been previously well-studied. Here we show that field and laboratory studies of deep insectivorous bat guano sequences in the caves of Borneo demonstrate the extreme dependence of decomposition rate on guano water content. Under tropical conditions, moist guanos exhibit decomposition coefficients (k) values of ~ 0.01, implying the loss of most of the organic content on decadal timescales. Under similar temperatures but drier conditions, k values drop to ~3 x 10-6, permitting significant organic (chitin) content to persist for tens of thousands of years. One of the implications of these findings for paleo-environmental records older than the limit of carbon dating is that a value for k can be calculated based on a single carbon date, which then allows an estimate of likely age at base of deposit.

Fluorescence and Biochemical Assessment of the Chitin and Chitosan Content of Cryptococcus.

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.

ИЗУЧЕНИЕ ПОТЕНЦИАЛА ЛИЧИНКИ HERMETIA ILLUCENS И ЕЕ ГИДРОЛИЗАТОВ ДЛЯ ИСПОЛЬЗОВАНИЯ В КАЧЕСТВЕ ИСТОЧНИКА БЕЛКА И ЖИРА В АКВАБИОТЕХНОЛОГИИ

The problem of a high-quality source of protein and fat is relevant for the development of aquaculture. An alternative substitute for animal components is the larva of the black soldier fly Hermetia illucens. Due to the presence of chitin, the components of the larvae are not completely absorbed by fish. The purpose of the study was to assess the nutritional value of proteins and lipids of the Hermetia illucens larva after high-temperature hydrolysis. As a result, water-soluble and water-insoluble protein supplements and a fat supplement were obtained from the larva. The chemical composition of raw materials and additives, the amino acid composition of protein additives and their balance for salmon as well as fatty acid composition of the lipid supplement and the ratio of the main groups of fatty acids have been established. An increased content of calcium, phosphorus, iron, magnesium, zinc, and potassium in the water-insoluble additive is shown. A decrease in the chitin content in protein-containing supplements has been established. It is recommended to use additives obtained from the larvae in the composition of fish feed in aquaculture.

Estimation of applicability of protein hydrolysate of black soldier fly (Hermetia illucens) larvae prepared using enzymatic hydrolysis as a fish feed component

The special advantage of black soldier fly (BSF) (Hermetia illucens) larvae for use as feed is their high protein content and low chitin content, which makes the larvae easier to digest, making them nutritious for fish, farm animals and birds. It has been reported that BSF larvae contain all the amino acids necessary for animal protein, including the essential amino acids, also macro- and micronutrients. The technological parameters of BSF larvae protein hydrolysis procedure were optimized. Some physicochemical parameters of the hydrolysate and the yield of hydrolysis were estimated for the hydrolysate prepared according to the optimized parameters. The sterlet (Acipenser ruthenus) starter feed having 10% of its fishmeal portion substituted with BSF larvae protein hydrolysate has been tested. The BSF larvae protein hydrolysate may be included in fish feed formulations, especially in starter feeds.

Function analysis and characterisation of a novel chitinase, MdCht9, in Musca domestica.

Insect chitinases have been proposed as potential targets for pest control. In this work, a novel group IV chitinase gene, MdCht9, from Musca domestica was found to have multiple functions in the physiological activity, including chitin regulation, development and antifungal immunity. The MdCht9 gene was cloned and sequenced, its phylogeny was analysed and its expression was determined in normal and 20E treated larvae. Subsequently, RNA interference (RNAi)-mediated MdCht9 knockdown was performed, followed by biochemical assays, morphological observations and transcriptome analysis. Finally, the recombinant protein MdCht9 (rMdCht9) was purified and tested for anti-microbial activity and enzyme characteristics. The results showed that MdCht9 consists of three domains, highly expressed in a larval salivary gland. RNAi silencing of MdCht9 resulted in significant down-regulation of chitin content and expression of 15 chitin-binding protein (CBP) genes, implying a new insight that MdCht9 might regulate chitin content by influencing the expression of CBPs. In addition, more than half of the lethality and partial wing deformity appeared due to the dsMdCht9 treatment. In addition, the rMdCht9 exhibited anti-microbial activity towards Candida albicans (fungus) but not towards Escherichia coli (G-) or Staphylococcus aureus (G+). Our work expands on previous studies of chitinase while providing a potential target for pest management.

Regulatory effect of trehalose metabolism on chitin synthesis in Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) as determined using RNAi

Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is an important pest of crops that causes huge losses to grain production. Trehalose plays an important regulatory role in chitin synthesis. In this study, the mRNA expression of the trehalose synthase (TPS), soluble trehalase (TRE1), and membrane-bound trehalase (TRE2) genes in S. frugiperda were silenced by RNAi to reveal the mechanism by which trehalose metabolism regulates chitin synthesis. The results showed that the survival rate of S. frugiperda at different developmental stages decreased in varying degrees following RNAi, and abnormal phenotypes were detected. TRE1 activity was extremely significantly inhibited after dsTRE1 and dsTRE2 injection, and TRE2 activity was extremely significantly inhibited after dsTPS and dsTRE2 injection. After silencing different trehalose metabolism-related regulatory genes, the trehalose content increased, whereas the glucose content decreased. Chitin synthase A (CHSA) gene was upregulated at 24 h after dsTPS and dsTRE1 injection. CHSA expression was significantly inhibited at 48 h after dsTRE1 injection. Chitin synthase B (CHSB) gene expression was significantly higher at 24 h after dsTRE1 injection. Furthermore, CHSB expression was significantly inhibited at 48 h after dsTPS, dsTRE1, or dsTRE2 injection. RNAi of TRE1 or TRE2 led to a significant decrease in chitin content. The results suggested that inhibiting the mRNA expression of genes related to trehalose metabolism in S. frugiperda could disrupt normal trehalose metabolism, thereby regulating the mRNA expression of downstream chitin synthase genes and further affecting chitin synthesis.

A combinatorial approach of Monarda citriodora essential oil (MEO) and linalool vapors to control fruit rot of Citrus limon caused by a new pathogen, Aspergillus foetidus, and its underlying mode of action.

Citrus limon (lemon) is a widely cultivated citrus fruit. Significant postharvest losses due to fungi plague its production. Environmental and human health hazards have made the application of synthetic fungicides unsuitable. Despite the previous reports of antifungal activities of essential oil (EO) vapors, their synergistic combinations are understudied. Synergistic vapor combinations are advantageous due to less concentration of active components. This study aimed to isolate and identify postharvest fungal pathogens lemon and to evaluate the antifungal effects of synergistic Monarda citriodora EO (MEO)-constituent vapor combinations in vivo and in vitro. Postharvest fungal pathogens of lemon (C. limon) were isolated from various infected samples. The most pathogenic isolate was identified through morphology and its ITS-based rRNA gene sequencing as Aspergillus foetidus (O4). This is the first report of A. foetidus as a postharvest pathogen of lemon. The minimum fungicidal concentrations (MFCs) of MEO vapors treatment against O4 were 1346.15µL/L air. For carvacrol, hexanal, and linalool, MFC was same (96.16µL/L air). Checkerboard assays demonstrated that 1/4 MFC of MEO (336.54µL/L air) and 1/4 MFC of linalool (24.04µL/L air) (M+L) were synergistic against O4. M+L vapors reduced the O4 growth on lemons during storage by 64%±1.50% and preserved their quality (low weight loss %, unchanged pH, increased ascorbic acid content). Propidium iodide staining, ergosterol content analysis, calcofluor white staining and chitin content analysis revealed the integrity loss of the O4 plasma membrane and cell wall. 2',7'-Dichlorofluorescin diacetate staining revealed accumulation of intracellular reactive oxygen species (ROS), and scanning electron microscopy (SEM) analysis exposed the M+L treated mycelia with malformations. M+L vapors offer protection for lemons from A. foetidus and preserve their quality during storage.

Reduction of histone proteins dosages increases CFW sensitivity and attenuates virulence of Candida albicans

Histone proteins are important components of nucleosomes, which play an important role in regulating the accessibility of DNA and the function of genomes. However, the effect of histone proteins dosages on physiological processes is not clear in the human fungal pathogen Candida albicans. In this study, we found that the deletion of the histone protein H3 coding gene HHT21 and the histone protein H4 coding gene HHF1 resulted in a significant decrease in the expression dosage of the histone proteins H3 and H4, which had a significant impact on the localization of the histone protein H2A and plasmid maintenance. Stress sensitivity experiments showed that the mutants hht21Δ/Δ, hhf1Δ/Δ and hht21Δ/Δhhf1Δ/Δ were more sensitive to cell wall stress induced by Calcofluor White (CFW) than the wild-type strain. Further studies showed that the decrease in the dosage of the histone proteins H3 and H4 led to the change of cell wall components, increased chitin contents, and down-regulated expression of the SAP9, KAR2, and CRH11 genes involved in the cell wall integrity (CWI) pathway. Overexpression of SAP9 could rescue the sensitivity of the mutants to CFW. Moreover, the decrease in the histone protein s dosages affected the FAD-catalyzed oxidation of Ero1 protein, resulting in the obstruction of protein folding in the ER, and thus reduced resistance to CFW. It was also found that CFW induced a large amount of ROS accumulation in the mutants, and the addition of ROS scavengers could restore the growth of the mutants under CFW treatment. In addition, the reduction of the histone proteins dosages greatly weakened systemic infection and kidney fungal burden in mice, and hyphal development was significantly impaired in the mutants under macrophage treatment, indicating that the histone proteins dosages is very important for the virulence of C. albicans. This study revealed that histone proteins dosages play a key role in the cell wall stress response and pathogenicity in C. albicans.

Assessment of hydrolysis of partially defatted black soldier fly larvae meal in diets for Nile tilapia Oreochromis niloticus

The chitin content of black soldier fly (Hermetia illucens) larvae meal (BSFM) is known to negatively affect its digestibility by fish. Nile tilapia (Oreochromis niloticus) tolerates well BSFM in the diet. However, chemical hydrolysis of dietary BSFM has not previously been attempted for this species. In this study, based upon a low-fishmeal control diet (40% crude protein and 9% crude fat), three levels of dietary protein, 25%, 50%, and 75%, were replaced by partially defatted BSFM, subjected or not to a prior hydrolysis treatment with formic acid and fed to Nile tilapia juveniles (initial body weight: 0.26 ± 0.00 g). The hydrolysis treatment did not modify greatly the proximate and amino acid composition of the diets, nor did it improve their in vivo apparent digestibility coefficients of dry matter or crude protein. However, it significantly increased the contents of arginine, taurine, and tyrosine, and significantly reduced the contents of methionine, valine, aspartic acid, and glutamic acid in muscle tissue of Nile tilapia. The hydrolysis treatment also decreased fish growth significantly. In contrast, for diets not containing hydrolyzed BSFM, not only did fish fed all three levels of protein replacement grow satisfactorily, but the diets with the levels of replacement of 50% and 75%, with final weights of 23.04 and 23.11 g, respectively, elicited superior fish performance, as compared to the diet with 25% replacement (final weight of 20.09 g). In addition, all three protein replacement diets produced significantly greater growth than the control diet (final weight of 17.72 g). The actual levels of inclusion of BSFM were, respectively, 14.11%, 28.21% and 42.33% of diet, the latter being the highest inclusion level of BSFM reported to date for successful growth of Nile Tilapia.

Structural and Functional Alterations Caused by Aureobasidin A in Clinical Resistant Strains of Candida spp.

Candida species are one of the most concerning causative agents of fungal infections in humans. The treatment of invasive Candida infections is based on the use of fluconazole, but the emergence of resistant isolates has been an increasing concern which has led to the study of alternative drugs with antifungal activity. Sphingolipids have been considered a promising target due to their roles in fungal growth and virulence. Inhibitors of the sphingolipid biosynthetic pathway have been described to display antifungal properties, such as myriocin and aureobasidin A, which are active against resistant Candida isolates. In the present study, aureobasidin A did not display antibiofilm activity nor synergism with amphotericin B, but its combination with fluconazole was effective against Candida biofilms and protected the host in an in vivo infection model. Alterations in treated cells revealed increased oxidative stress, reduced mitochondrial membrane potential and chitin content, as well as altered morphology, enhanced DNA leakage and a greater susceptibility to sodium dodecyl sulphate (SDS). In addition, it seems to inhibit the efflux pump CaCdr2p. All these data contribute to elucidating the role of aureobasidin A on fungal cells, especially evidencing its promising use in clinical resistant isolates of Candida species.

Evaluation of mealworm meal as an alternative to fish meal in the diet for giant grouper (Epinephelus lanceolatus) based on the protein level adjusted by the nitrogen content in chitin

The crude protein content of insects may be overestimated due to the nitrogen (N) content in the chitin therein. The present study evaluated the replacement of fishmeal with mealworm meal (MWM) in the diet on the growth performance and body composition of giant grouper, Epinephelus lanceolatus. Before the feeding trial, the chitin and crude protein contents of the MWM were analyzed as 13.21% and 74.63% (N × 6.25), and the corrected protein content of MWM meal was found to be 69.29%. Based on the adjusted protein level, MWM was substituted at the levels of 0%, 10%, 20%, and 30% for the fishmeal protein in the diets. Four experimental diets were each fed to three groups of juvenile giant grouper (initial weight: 20.65 ± 0.08 g) via a recirculation system for 8 weeks. The weight gain, protein retention, protein efficiency ratio, feed efficiency, survival, and whole-body amino acid and fatty acid profiles were not affected (p > 0.05) by the dietary treatments. The fish in–fish out ratios decreased linearly with increasing MWM inclusion levels. Plasma triglyceride concentration decreased after increasing the replacement levels. The results indicate that the actual protein content in MWM can be adjusted via the nitrogen content in the ingredient, as well as by adjusting the chitin content. MWM can be used to replace fishmeal protein up to 30% in giant grouper diets without any negative effect on growth performance, and this can also reduce the dependence on the marine resources required to provide aquafeed to achieve the goal of sustainable aquaculture.

Role of mitochondrial farnesyltransferase gene in the prevention of the food spoilage fungi Aspergillus flavus by the antimicrobial natural preservative perillaldehyde

Aspergillus flavus, one of the most widely distributed and abundant genus of Aspergillus worldwide, poses an evident threat as a source of food contamination in grains and cereals. Perillaldehyde (PAE), a volatile essential oil extracted from the stem and leaves of Perilla frutescens L., exhibits potent antifungal activity. In our study, we investigated the role of Cox10, a key enzyme in the heme A synthesis pathway crucial for maintaining mitochondrial function. We found that cox10 is a sensitive gene of A. flavus in response to PAE by gene expression assay and GFP fluorescent localization tagging, and then we found that the deletion of this gene affects the growth and development of A. flavus, but the drug resistance is elevated. Through transcriptome sequencing and its experimental validation, the molecular mechanisms of stress triggered by the deletion of cox10 were further clarified, such as the decrease in intracellular drug content due to the increase in the expression of drug efflux proteins, and the increase in the thickness of cell wall due to the increase in the content of cell wall chitin. Clearly, cox10 plays a critical role in regulating various cellular processes of A. flavus, including growth, reproduction, development, as well as pathogenicity and drug resistance. These significant findings establish a solid theoretical foundation for the development of environmentally friendly, safe, and effective antifungal agents to combat A. flavus contamination.