Unraveling the skatole biodegradation process in an enrichment consortium using integrated omics and culture-dependent strategies

3-Methylindole(skatole)is regarded as one of the most offensive compounds in odor emission.Biodegradation is feasible for skatole removal but the functional species and genes responsible for skatole degradation remain enigmatic.In this study,an efficient aerobic skatole-degrading consortium was obtained.Rhodococcus and Pseudomonas were identified as the two major and active populations by integrated metagenomic and metatranscriptomic analyses.Bioinformatic analyses indicated that the skatole downstream degradation wasmainly via the catechol pathway,and upstream degradation was likely catalyzed by the aromatic ring-hydroxylating oxygenase and flavin monooxygenase.Genome binning and gene analyses indicated that Pseudomonas,Pseudoclavibacter,and Raineyella should cooperate with Rhodococcus for the skatole degradation process.Moreover,a pure strain Rhodococcus sp.DMU1 was successfully obtained which could utilize skatole as the sole carbon source.Complete genome sequencing showed that strain DMU1 was the predominant population in the consortium.Further crude enzyme and RT-qPCR assays indicated that strain DMU1 degraded skatole through the catechol ortho-cleavage pathway.Collectively,our results suggested that synergistic degradation of skatole in the consortium should be performed by diverse bacteria with Rhodococcus as the primary degrader,and the degradation mainly proceeded via the catechol pathway.

An unsettling explanation for the failure of skatole-baited ovitraps to capture Culex mosquitoes

Culex mosquitoes are primarily found in temperate and tropical regions worldwide where they play a crucial role as main vectors of filarial worms and arboviruses. In Recife, a northeast city in Brazil, high densities of Culex quinquefasciatus are often found in association with human populated areas. In marked contrast to another part of the city, field tests conducted in the neighborhood of Sitio dos Pintos showed that trapping of mosquitoes in skatole-baited ovitraps did not differ significantly from captures in control (water) traps. Thus, classical and molecular taxonomic approaches were used to analyze the Culex species circulating in Sitio dos Pintos. Results obtained from both approaches agreed on the cocirculation of Culex quinquefasciatus and Culex nigripalpus in three different areas of this neighborhood. What was initially considered as an unexpected failure of this lure turned out to be a more unsettling problem, that is, the first report in Recife of Culex nigripalpus, a vector of Venezuelan equine encephalitis virus and West Nile virus. Unplanned urbanization processes close to remnants of the Atlantic forest, such as observed in Sitio dos Pintos, may have contributed to the introduction of Cx. nigripalpus in urban areas.

Mulberry leaf supplementation inhibits skatole deposition by regulating gut microbiota and upregulating liver cytochrome P4501A1 expression in finishing pigs

Skatole,a strong fecal odor substance,is generated through microbial degradation of tryptophan in the animal hindgut.It easily accumulates in adipose tissue and affects meat quality.In this study,the effect of mulberry leaf supplementation on skatole in finishing pigs was studied.In a 35-day trial,20 finishing pigs(barrows and gilts)were fed with a basal diet or basal diet with 6%mulberry leaves.Growth performance of the pigs(n=10)was automatically recorded by a performance-testing feeder system and 8 pigs in each treatment were slaughtered and sampled for the remaining tests.Skatole and short-chain fatty acids were detected using HPLC and gas chromatography,respectively.Fecal microbiota were analyzed using 16S r RNA gene sequencing.The metabolomics analysis of feces and serum was performed with UHPLC-MS/MS.The major cytochrome P450(CYP)enzymes that catalyze skatole degradation in the liver were tested by using RT-PCR and Western blot.Effects of major bioactive compounds in mulberry leaves on the CYP genes were verified in the hepatic cell line Hep G2 in an in vitro test(n=3).In finishing pigs,mulberry leaf supplementation had no significant effect on the average daily gain,average daily feed intake,and feed conversion ratio(P>0.05),but reduced skatole levels in feces,serum,and backfat(P<0.05),and increased acetic acid levels in feces(P=0.027).Mulberry leaf supplementation decreased the relative abundance of the skatole-producing bacteria Megasphaera and Olsenella(P<0.05).Indole-3acetic acid,the intermediate that is essential for skatole production,was significantly reduced in feces by mulberry leaf supplementation(P<0.05)and was positively correlated with skatole content in feces(P=0.004).In pigs treated with mulberry leaves,liver CYP1A1 expression was increased(P<0.05)and was negatively correlated with skatole content in backfat(P=0.045).The in vitro test demonstrated that mulberry leaf polyphenols and polysaccharides could directly stimulate CYP1A1 expression in hepatic cells.These findings suggest that mulberry leaf supplementation reduces skatole production and deposition in finishing pigs by regulating the gut microbiota and promoting skatole degradation in liver.