Submicronic Fungal Bioaerosols: High-Resolution Microscopic Characterization and Quantification

Submicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in mold-contaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min⁻¹ airflow. The overall median numbers of aerosolized submicronic particles were 2 × 10⁵ cm⁻², 2.6 × 10³ cm⁻², and 0.9 × 10³ cm⁻² for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.     

The Volatome of Aspergillus fumigatus

Early detection of invasive aspergillosis is absolutely required for efficient therapy of this fungal infection. The identification of fungal volatiles in patient breath can be an alternative for the detection of Aspergillus fumigatus that still remains problematic. In this work, we investigated the production of volatile organic compounds (VOCs) by A. fumigatus in vitro, and we show that volatile production depends on the nutritional environment. A. fumigatus produces a multiplicity of VOCs, predominantly terpenes and related compounds. The production of sesquiterpenoid compounds was found to be strongly induced by increased iron concentrations and certain drugs, i.e., pravastatin. Terpenes that were always detectable in large amounts were α-pinene, camphene, and limonene, as well as sesquiterpenes, identified as α-bergamotene and β-trans-bergamotene. Other substance classes that were found to be present in the volatome, such as 1-octen-3-ol, 3-octanone, and pyrazines, were found only under specific growth conditions. Drugs that interfere with the terpene biosynthesis pathway influenced the composition of the fungal volatome, and most notably, a block of sesquiterpene biosynthesis by the bisphosphonate alendronate fundamentally changed the VOC composition. Using deletion mutants, we also show that a terpene cyclase and a putative kaurene synthase are essential for the synthesis of volatile terpenes by A. fumigatus. The present analysis of in vitro volatile production by A. fumigatus suggests that VOCs may be used in the diagnosis of infections caused by this fungus.     

Analysis of the Aspergillus fumigatus Biofilm Extracellular Matrix by Solid-State Nuclear Magnetic Resonance Spectroscopy

Aspergillus fumigatus is commonly responsible for lethal fungal infections among immunosuppressed individuals. A. fumigatus forms biofilm communities that are of increasing biomedical interest due to the association of biofilms with chronic infections and their increased resistance to antifungal agents and host immune factors. Understanding the composition of microbial biofilms and the extracellular matrix is important to understanding function and, ultimately, to developing strategies to inhibit biofilm formation. We implemented a solid-state nuclear magnetic resonance (NMR) approach to define compositional parameters of the A. fumigatus extracellular matrix (ECM) when biofilms are formed in RPMI 1640 nutrient medium. Whole biofilm and isolated matrix networks were also characterized by electron microscopy, and matrix proteins were identified through protein gel analysis. The ¹³C NMR results defined and quantified the carbon contributions in the insoluble ECM, including carbonyls, aromatic carbons, polysaccharide carbons (anomeric and nonanomerics), aliphatics, etc. Additional ¹⁵N and ³¹P NMR spectra permitted more specific annotation of the carbon pools according to C-N and C-P couplings. Together these data show that the A. fumigatus ECM produced under these growth conditions contains approximately 40% protein, 43% polysaccharide, 3% aromatic-containing components, and up to 14% lipid. These fundamental chemical parameters are needed to consider the relationships between composition and function in the A. fumigatus ECM and will enable future comparisons with other organisms and with A. fumigatus grown under alternate conditions.     

RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior

Background

Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long.

Results

We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes.

Conclusions

We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth.

Electronic supplementary material

The online version of this article (doi10.1186/s12864-015-1853-1) contains supplementary material, which is available to authorized users.     

Wnt5a contributes to dectin-1 and LOX-1 induced host inflammatory response signature in Aspergillus fumigatus keratitis

Fungal keratitis causes devastating corneal ulcers which can result in significant visual impairment and even blindness. As a ligand that activates the non-canonical Wnt signaling pathways, Wnt5a triggers the production of important inflammatory chemokines and the chemotactic migration of neutrophils. In this study we aimed to characterize the role of Wnt5a production, in situ, in vivo and in vitro in response to fungal keratitis. Wnt5a expression in corneas of Aspergillus fumigatus (A. fumigatus) keratitis patients was determined by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. In vivo and in vitro experiments were then performed in mouse models and THP-1 macrophages cell cultures infected with A. fumigatus, respectively. C57BL/6 mice were pretreated with siRNAs or neutralizing antibodies for dectin-1, LOX-1 and Wnt5a, or inhibitors of erk1/2 and JNK. Changes in Wnt5a expression were assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blot and bioluminescence imaging system image acquisition. We confirmed that corneal Wnt5a expression increased with A. fumigatus keratitis in patients and a murine model. Wnt5a production was dependent on dectin-1 and LOX-1 expression with contributions by Erk1/2 and JNK pathways. Additionally, Wnt5a knockdown revealed decreased levels of MPO, lower neutrophil recruitment, and a higher fungal load in mouse models. Compared with controls, Wnt5a knockdown impaired pro-inflammatory cytokine IL-1β production in response to A. fumigatus exposure. Wnt5a also produces dectin-1 and LOX-1 induced inflammatory signature via effective neutrophil recruitment and inflammatory cytokine production in response to A. fumigatus keratitis. These findings demonstrate that Wnt5a is a critical component of the antifungal immune response.     

The Aspergillus fumigatus Dihydroxyacid Dehydratase Ilv3A/IlvC Is Required for Full Virulence

Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.     

Gene Expression Profiles of Human Dendritic Cells Interacting with Aspergillus fumigatus in a Bilayer Model of the Alveolar Epithelium/Endothelium Interface

The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549) and endothelium (human pulmonary artery epithelial cells, HPAEC) on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC), monocyte-derived DC (moDC) and myeloid DC (mDC), were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA.     

Thermostable cellulase production of Aspergillus fumigatus Z5 under solid-state fermentation and its application in degradation of agricultural wastes

A lignocellulosic decomposing fungus Z5 was isolated and identified as Aspergillus fumigatus, its capacity to produce cellulase was assessed under solid-state fermentation (SSF) using lignocellulosic materials as substrates. Cultivation conditions of A. fumigatus Z5 for cellulase production were optimized, results showed that for carboxymethyl cellulase (CMCase) and filter paper enzyme (FPase), the best condition was 50 °C, 80% initial moisture, initial pH 4.0 and 7% initial inoculum, the average activity of CMCase activity, FPase activity reached 526.3 and 144.6 U g⁻¹ dry weight (dw) respectively, much higher than most of previous reports of this genus. Optimal temperature and pH for the CMCase activity of the crude enzyme were found to be 50 °C and 5.0, respectively. Zymogram analysis showed that eight kinds of CMCase were secreted by A. fumigatus Z5 when cellulose-containing materials were supplied in the culture. The crude enzyme secreted by the strain was further applied to hydrolyze pretreated corn stover and the enzymatic hydrolysate was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of bio-ethanol was 0.112 g g⁻¹ dry substrate (gDS), suggesting that it is a promising fungus in the bio-ethanol production process.     

Pathway of Glycine Betaine Biosynthesis in Aspergillus fumigatus

The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD⁺ to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.     

Structural Requirements for the Activity of the MirB Ferrisiderophore Transporter of Aspergillus fumigatus

Siderophores have been identified as virulence factors in the opportunistic fungal pathogen Aspergillus fumigatus. The 14-pass transmembrane protein MirB is postulated to function as a siderophore transporter, responsible for uptake of the hydroxamate siderophore N,N′,N″-triacetylfusarinine C (TAFC). Our aim was to identify amino acids of A. fumigatus MirB that are crucial for uptake of TAFC. Site-directed mutagenesis was used to create MirB mutants. Expression of wild-type and mutant proteins in the Saccharomyces cerevisiae strain PHY14, which lacks endogenous siderophore transporters, was confirmed by Western blotting. TAFC transport assays using ⁵⁵Fe-labeled TAFC and growth assays with Fe-TAFC as the sole iron source identified alanine 125, tyrosine 577, loop 3, and the second half of loop 7 (Loop7Del2) as crucial for function, since their substitution or deletion abrogated uptake completely. Wild-type MirB transported ferricrocin and coprogen as well as TAFC but not ferrichrysin. MirB was localized by fluorescence microscopy using antisera raised against a MirB extracellular loop peptide. Immunofluorescence microscopy showed that in yeast, wild-type MirB had a punctate distribution under the plasma membrane, as did the A125D and Y577A strains, indicating that the defect in transport of these mutants was unlikely to be due to mislocalization or degradation. MirB immunolocalization in A. fumigatus showed that the transporter was found in vesicles which cycled between the cytoplasm and the plasma membrane and was concentrated at the hyphal tips. The location of MirB was not influenced by the presence of the siderophore TAFC but was sensitive to internal iron stores.     

Dispersal of Aspergillus fumigatus from Sewage Sludge Compost Piles Subjected to Mechanical Agitation in Open Air

Aerosolization of the thermophilous fungal opportunist Aspergillus fumigatus from mechanically agitated compost piles was examined at a pilot-scale sewage sludge composting facility and two other selected test sites. Aerosols of A. fumigatus downwind from stationary compost piles were insignificant in comparison with those downwind from agitated piles. These aerosols were generated by a front-end loader moving and dropping compost. Aerial concentrations of the fungus at distances downwind from the point of emission were used to determine an emission rate for A. fumigatus associated with the moving operations. The maximum emission rate, 4.6 × 10⁶A. fumigatus particles per s, was used to calculate predicted concentrations in an unobstructed plume with restrictive, neutral, and dispersive atmospheric mixing conditions up to 1 km downwind from the emission source.     

Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis

The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.     

Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) from Aspergillus fumigatus and Molecular Basis for the Development of Antifungal Drugs

Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min⁻¹, respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this relatively small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections.     

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H₂O₂ (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P = 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H₂O₂ (P < 0.01), unexpectedly, exogenous gliotoxin relieved H₂O₂-induced growth inhibition in a dose-dependent manner (0 to 10 μg/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ΔgliK²⁶⁹³³ deletion mutant. The A. fumigatus ΔgliK²⁶⁹³³ mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ΔgliK²⁶⁹³³ mutant than in those of the wild type (P = 0.0024 [fold difference, 24] and P = 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ΔgliK²⁶⁹³³ mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P = 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK⁴⁶⁶⁴⁵ mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus.     

Gliotoxinogenic Aspergillus fumigatus in the dairy herd environment

The potential association between hygienic conditions in the environment of lactating cows and the presence of gliotoxinogenic Aspergillus fumigatus strains was studied. Milk samples (individual cow’s milk [ICM], bulk tank milk [BTM]) from 44 dairy farms were sampled. In ICM samples, eight different species of Aspergillus were identified. A. flavus and A. fumigatus were predominant, with 37.8 % and 26.1 % relative densities, respectively. A. fumigatus strains were isolated from 61.4 % of the BTM samples, and 34 % of these strains were able to produce gliotoxin. Principal component analysis was used to associate the presence of A. fumigatus with some hygienic and sanitary practices. A significant and positive correlation was observed between dry cow therapy and forestripping. The presence of A. fumigatus gliotoxin producers in milk was associated with high somatic cells count (SCC) samples. Good hygienic and sanitary practices were associated with absence of A. fumigatus and relatively low SCCs of <250,000 cells/ml. In general, a high percentage of dairy farms were positive for A. fumigatus in BTM samples. This is the first work that indicates the positive effects of adequate hygienic and sanitary practices in dairy herds on the control of A. fumigatus and related species. By reducing the frequency of Aspergillus spp. in the dairy environment, the risk of farm handlers’ exposure and the risk of intramammary fungal infections would also be reduced.     

Characterization of glucuronic acid containing glycolipid in Aspergillus fumigatus mycelium

A glucuronic acid containing glycerolipid was isolated from the filamentous fungi Aspergillus fumigatus. This acidic glycolipid was extracted from the membrane of mycelium and purified by two successive chromatographic steps on DEAE-Sephadex and Silica columns. Chemical structural analysis was performed using methylation, gas-chromatography, gas-chromatography-mass spectrometry, nano-electrospray mass spectrometry and ¹H/¹³C NMR spectra. The corresponding structure is a 3-(O-α-glucuronyl)-1,2-diacyl-sn-glycerol, where acyl chains are mainly C_16:0, C_18:0, C_18:1, and C_18:2. This α-GlcA-diacylglycerol is not present in fungal conidia. This acidic glycerolipid is described here for the first time in a fungal species. Two homologs of UDP-glucose dehydrogenase that convert UDP-glucose into UDP-glucuronic acid, are present in A. fumigatus genome, UGD1 and UGD2. Gene deletion showed that only UGD1 is essential for the biosynthesis of GlcA-DG. However, no particular phenotype has been observed in the Ugd1Δ mutant. Biological function of this acidic glycolipid remains unknown in A. fumigatus.     

The Fungal Exopolysaccharide Galactosaminogalactan Mediates Virulence by Enhancing Resistance to Neutrophil Extracellular Traps

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.     

Production of farnesoic acid and methyl farnesoate by mandibular organs of the crayfish,Procambarus clarkii

The mandibular organs (MO) of crustaceans secrete methyl farnesoate (MF) and farnesoic acid (FA). To better understand the secretory activity of MO, the kinetics of production and release of both compounds were determined in vitro by following incorporation of [2-¹⁴C]acetate and l-[³H-methyl]methionine into MF and [2-¹⁴C]acetate into FA by MO of Procambarus clarkii. MO released more FA than MF but contained more MF. In medium lacking unlabeled acetate, the percentage incorporation of [¹⁴C]acetate into MF, relative to [³H]methionine, was between 21 and 40%, suggesting that there may be an alternative source of C₂ units.MO produce similar amounts of MF at concentrations of acetate from 0.08 to 10 mM. However, the addition of exogenous unlabelled FA to incubation media did not stimulate the biosynthesis of MF, raising the possibility that unlike JH biosynthesis in insects, the last step in MF production may be rate-limiting. Nonetheless, exogenous FA significantly reduced the incorporation of [¹⁴C]acetate into MF, suggesting that the glands use exogenous FA to synthesize MF. The absence of stimulation of FA production by exogenous FA indicates that there is no feedback effect of this product on the early steps in the biosynthetic pathway.