Marinade injection of pork as a possible technological strategy to reduce boar taint: Response and attitude of the consumer.

Sensory evaluation by consumers allows validation of a solution for the food industry to overcome boar taint and market entire male pork with high levels of boar taint considered "unfit for human consumption". One possible technological strategy for improvement is the injection marination of entire male pork to help minimise the impact of boar taint and improve its marketability. The responses of 120 regular pork consumers, to entire males with high levels of boar taint and castrated pork, both injection-marinated, were evaluated. The results showed a similar response between entire and castrated male pork regardless of sex, and the detection of unpleasant odours and flavours (as farm/animal), thus offering a new alternative to the pork sector to mask the boar taint and revalue this pork. However, it was observed that consumer information on castration and animal welfare of entire male pork is scarce, and it is important to increase their knowledge so that they can place a value on this technological strategy with entire male pork and thus increase their purchase intention.

Impact of dietary digestible aromatic amino acid levels and stachyose on growth, nutrient utilization, and cecal odorous compounds in broiler chickens.

This study evaluated the impact of dietary digestible aromatic amino acid (DAAA) levels and stachyose on growth, nutrient utilization and cecal odorous compounds in broiler chickens. A 3×2 two-factor factorial design: Three dietary DAAA levels (1.40, 1.54, 1.68%) supplemented with either 5 g/kg of stachyose or without any stachyose were used to create 6 experimental diets. Each diet was fed to 6 replicates of 10 birds from d 22 to 42. Findings revealed that broilers receiving a diet with 1.54% DAAA levels supplemented with 5 g/kg stachyose exhibited a significant boost in average daily gain and improved utilization of crude protein, ether extract, tryptophan, and methionine compared to other diet treatments (P < 0.05). As the dietary DAAA levels increased, there was a significant rise in the concentrations of indole, skatole, p-methylphenol, and butyric acid in the cecum of broilers (P < 0.05). The addition of stachyose to diets reduced concentrations of indole, skatole, phenol, p-methylphenol, acetic acid and propionic acid in the cecum (P < 0.05). The lowest concentrations of indole, phenol, p-methylphenol, volatile fatty acids and pH in cecum of broilers were observed in the treatment which diet DAAA level was 1.40% with stachyose (P < 0.05). In conclusion, dietary DAAA levels and stachyose had significant interactions on the growth, main nutrient utilization and cecal odorous compounds in broilers. The dietary DAAA level was 1.54% with 5 g/kg of stachyose can improve the growth performance, nutrient utilization. However, the dietary DAAA level was 1.40% with stachyose was more beneficial to decrease the cecal odor compound composition in broilers.

Magnolol-driven microbiota modulation elicits changes in tryptophan metabolism resulting in reduced skatole formation in pigs.

Skatole of gut origin has garnered significant attention as a malodorous pollutant due to its escalating emissions, recalcitrance to biodegradation and harm to animal and human health. Magnolol is a health-promoting polyphenol with potential to considerably mitigate the skatole production in the intestines. To investigate the impact of magnolol and its underlying mechanism on the skatole formation, in vivo and in vitro experiments were conducted in pigs. Our results revealed that skatole concentrations in the cecum, colon, and faeces decreased by 58.24% (P = 0.088), 44.98% (P < 0.05) and 43.52% (P < 0.05), respectively, following magnolol supplementation. Magnolol supplementation significantly decreased the abundance of Lachnospira, Faecalibacterium, Paramuribaculum, Faecalimonas, Desulfovibrio, Bariatricus, and Mogibacterium within the colon (P < 0.05). Moreover, a strong positive correlation (P < 0.05) between skatole concentration and Desulfovibrio abundance was observed. Subsequent in silico studies showed that magnolol could dock well with indolepyruvate decarboxylase (IPDC) within Desulfovibrio. Further in vitro investigation unveiled that magnolol addition led to less indole-3-pyruvate diverted towards the oxidative skatole pathway by the potential docking of magnolol towards IPDC, thereby diminishing the conversion of substrate into skatole. Our findings offer novel targets and strategies for mitigating skatole emission from the source.

A study on the association between gut microbiota, inflammation, and type 2 diabetes.

Type 2 diabetes mellitus (T2DM) was reported to be associated with impaired immune response and alterations in microbial composition and function. However, the underlying mechanism remains elusive. To investigate the association among retinoic acid-inducible gene-I-like receptors (RLRs) signaling pathway, intestinal bacterial microbiome, microbial tryptophan metabolites, inflammation, and a longer course of T2DM, 14 patients with T2DM and 7 healthy controls were enrolled. 16S rRNA amplicon sequencing and untargeted metabolomics were utilized to analyze the stool samples. RNA sequencing (RNA-seq) was carried out on the peripheral blood samples. Additionally, C57BL/6J specific pathogen-free (SPF) mice were used. It was found that the longer course of T2DM could lead to a decrease in the abundance of probiotics in the intestinal microbiome. In addition, the production of microbial tryptophan derivative skatole declined as a consequence of the reduced abundance of related intestinal microbes. Furthermore, low abundances of probiotics, such as Bacteroides and Faecalibacterium, could trigger the inflammatory response by activating the RLRs signaling pathway. The increased level of the member of TNF receptor-associated factors (TRAF) family, nuclear factor kappa-B (NF-κB) activator (TANK), in the animal colon activated nuclear factor kappa B subunit 2 (NFκB2), resulting in inflammatory damage. In summary, it was revealed that the low abundances of probiotics could activate the RLR signaling pathway, which could in turn activate its downstream signaling pathway, NF-κB, highlighting a relationship among gut microbes, inflammation, and a longer course of T2DM. KEY POINTS: Hyperglycemia may suppress tryptophanase activity. The low abundance of Bacteroides combined with the decrease of Dopa decarboxylase (DDC) activity may lead to the decrease of the production of tryptophan microbial derivative skatole, and the low abundance of Bacteroides or reduced skatole may further lead to the increase of blood glucose by downregulating the expression of glucagon-like peptide-1 (GLP1). A low abundance of anti-inflammatory bacteria may induce an inflammatory response by triggering the RLR signaling pathway and then activating its downstream NF-κB signaling pathway in prolonged T2DM.

Development and validation of a classification model for boar taint detection in pork fat samples.

This study aims to characterize a complete volatile organic compound profile of pork neck fat for boar taint prediction. The objectives are to identify specific compounds related to boar taint and to develop a classification model. In addition to the well-known androstenone, skatole and indole, 10 other features were found to be discriminant according to untargeted volatolomic analyses were conducted on 129 samples using HS-SPME-GC×GC-TOFMS. To select the odor-positive samples among the 129 analyzed, the selection was made by combining human nose evaluations with the skatole and androstenone concentrations determined using UHPLC-MS/MS. A comparison of the data of the two populations was performed and a statistical model analysis was built on 70 samples out of the total of 129 samples fully positive or fully negative through these two orthogonal methods for tainted prediction. Then, the model was applied to the 59 remaining samples. Finally, 7 samples were classified as tainted.

Microbiome-metabolomics analysis reveals abatement effects of itaconic acid on odorous compound production in Arbor Acre broilers.

BACKGROUND: Public complaints concerning odor emissions from intensive livestock and poultry farms continue to grow, as nauseous odorous compounds have adverse impacts on the environment and human health. Itaconic acid is a metabolite from the citric acid cycle of the host and shows volatile odor-reducing effects during animal production operations. However, the specific role of itaconic acid in decreasing intestinal odorous compound production remains unclear. A total of 360 one-day-old chicks were randomly divided into 6 treatment groups: control group (basal diet) and itaconic acid groups (basal diet + 2, 4, 6, 8 and 10 g/kg itaconic acid). The feeding experiment lasted for 42 d. RESULTS: Dietary itaconic acid supplementation linearly and quadratically decreased (P < 0.05) the cecal concentrations of indole and skatole but did not affect (P > 0.05) those of lactic, acetic, propionic and butyric acids. The cecal microbial shift was significant in response to 6 g/kg itaconic acid supplementation, in that the abundances of Firmicutes, Ruminococcus and Clostridium were increased (P < 0.05), while those of Bacteroidetes, Escherichia-Shigella and Bacteroides were decreased (P < 0.05), indicative of increased microbial richness and diversity. Furthermore, a total of 35 significantly (P < 0.05) modified metabolites were obtained by metabolomic analysis. Itaconic acid decreased (P < 0.05) the levels of nicotinic acid, nicotinamide, glucose-6-phosphate, fumatic acid and malic acid and increased (P < 0.05) 5-methoxytroptomine, dodecanoic acid and stearic acid, which are connected with the glycolytic pathway, citrate acid cycle and tryptophan metabolism. Correlation analysis indicated significant correlations between the altered cecal microbiota and metabolites; Firmicutes, Ruminococcus and Clostridium were shown to be negatively correlated with indole and skatole production, while Bacteroidetes, Escherichia-Shigella and Bacteroides were positively correlated with indole and skatole production. CONCLUSIONS: Itaconic acid decreased cecal indole and skatole levels and altered the microbiome and metabolome in favor of odorous compound reduction. These findings provide new insight into the role of itaconic acid and expand its application potential in broilers.

Change in fat skatole and indole content in lambs switched from a concentrate-based diet indoors to alfalfa grazing for various durations before slaughter.

Meat from lambs fattened on alfalfa is at risk of excessive pastoral flavours due to high levels of in-fat volatile indolic compounds (especially, skatole). Skatole has also been identified as a potential marker of interest for authenticating pasture-fed lamb meat. Here, we investigated the change in skatole and indole concentrations in kidney fat from lambs switched from an indoor-fed concentrate based diet to outdoor alfalfa grazing for various durations (0, 21, 42, 63 days) before slaughter. The study used a total of 219 lambs over 3 consecutive years. Kidney-fat skatole and indole concentrations increased from as early as 21 days on alfalfa, and then reached a plateau. Similarly, the proportion of lambs that had a kidney fat-skatole concentration above 0.15 µg/g liquid fat, a value that has been established as a sensory rejection threshold for pork, increased significantly from as early as 21 days on alfalfa and then reached a plateau. This value was reached or exceeded in a significant proportion of lambs fattened on alfalfa pastures (45.1%). However, skatole was not detected in kidney fat from 20 out of 164 alfalfa-fattened lambs (i.e., 12.2%) but was detected in 15 out of 55 concentrate-fed lambs (i.e., 27.3%). We thus conclude that while skatole content in kidney fat can inform on dietary changes made shortly before slaughter, it does not have the discrimination power needed to reliably authenticate pasture-fed lamb meat, let alone duration of finishing on pasture.

Consumer perception of entire male pork coated with spiced edible films as a new product to mask boar taint.

Pork production has undergone a shift towards the rearing of entire male pigs, however, its meat might carry with the presence of boar taint and it would be considered "unfit for human consumption". To offer a new alternative to the pork sector tailored to the needs of consumers, a viable option would be the use of edible spiced gelatin films to help minimize boar taint and improve its marketability. The responses of 120 regular meat consumers to entire pork with high levels of boar taint and castrated pork free of boar taint, both coated with spiced gelatin films were evaluated. They showed a similar response between entire and castrated male pork coated with spiced films, regardless of whether consumers usually detected unpleasant odours (as farm/animal) when consuming pork or not. Therefore, the new spiced films offer a new range of products to consumers as they contribute to the improvement of the sensory quality of entire male pork, especially among consumers who tend to buy new products.

Natural Product Skatole Ameliorates Lipotoxicity-Induced Multiple Hepatic Damage under Hyperlipidemic Conditions in Hepatocytes.

Skatole (3-methylindole, 3MI) is a natural-origin compound derived from plants, insects, and microbial metabolites in human intestines. Skatole has an anti-lipid peroxidation effect and is a biomarker for several diseases. However, its effect on hepatocyte lipid metabolism and lipotoxicity has not been elucidated. Hepatic lipotoxicity is induced by excess saturated free fatty acids in hyperlipidemia, which directly damages the hepatocytes. Lipotoxicity is involved in several metabolic diseases and hepatocytes, particularly affecting nonalcoholic fatty liver disease (NAFLD) progression. NAFLD is caused by the accumulation of fat by excessive free fatty acids (FFAs) in the blood and is accompanied by hepatic damage, such as endoplasmic reticulum (ER) stress, abnormal glucose and insulin metabolism, oxidative stress, and lipoapoptosis with lipid accumulation. Hepatic lipotoxicity causes multiple hepatic damages in NAFLD and has a directly effect on the progression from NAFLD to nonalcoholic steatohepatitis (NASH). This study confirmed that the natural compound skatole improves various damages to hepatocytes caused by lipotoxicity in hyperlipidemic conditions. To induce lipotoxicity, we exposed HepG2, SNU-449, and Huh7 cells to palmitic acid, a saturated fatty acid, and confirmed the protective effect of skatole. Skatole inhibited fat accumulation in the hepatocytes, reduced ER and oxidative stress, and recovered insulin resistance and glucose uptake. Importantly, skatole reduced lipoapoptosis by regulating caspase activity. In conclusion, skatole ameliorated multiple types of hepatocyte damage induced by lipotoxicity in the presence of excess free fatty acids.

Skatole-induced p38 and JNK activation coordinately upregulates, whereas AhR activation partially attenuates TNFα expression in intestinal epithelial cells.

Increased tumor necrosis factor α (TNFα) expression in intestinal epithelial cells (IECs) plays a major role in the development and progression of inflammatory bowel disease (IBD) and colorectal cancer (CRC). The present study aimed to clarify the relationship between TNFα and skatole, a tryptophan-derived gut microbiota metabolite. The aryl hydrocarbon receptor (AhR) antagonist CH223191 promoted, whereas the p38 inhibitor SB203580 suppressed the increase in TNFα mRNA and protein expression induced by skatole in intestinal epithelial Caco-2 cells. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 repressed only the increased TNFα protein expression, whereas the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 did not affect increased TNFα expression at any level. A neutralizing antibody against TNFα partially inhibited skatole-induced cell death. Overall, these results suggested that TNFα expression is increased by the concerted actions of skatole-activated p38 and JNK, and that TNFα exerts autocrine/paracrine actions on IECs despite partial suppression by activated AhR. Therefore, skatole might play an important role in the development and progression of IBD and CRC via increased TNFα expression.

Lactobacillus rhamnosus MN-431 Metabolic Tryptophan Alleviates Complementary Food-Induced Diarrhea through PXR-NF-κB Pathway and AHR-Th17 Cell Response Pathways.

SCOPE: Lactobacillus rhamnosus MN-431 tryptophan broth culture (MN-431 TBC) can prevent complementary food-induced diarrhea (CFID). However, it is not clear whether this effect is related to indole derivatives. METHODS AND RESULTS: In this study, the anti-CFID effects of different components in MN-431 TBC including MN-431 cells, unfermented tryptophan broth, and supernatant of MN-431 TBC (MN-431 TBS) are investigated. Only MN-431 TBS can significantly prevent CFID, indicating that indole derivatives produced by MN-431 can exert antidiarrheal effects. Intestinal morphological analysis reveals that MN-431 TBS can increase the number of goblet cells, height of ileal villi, and length of rectal glands while also increasing the expression of ZO-1 in colon. Furthermore, HPLC analysis reveals the indole derivatives in MN-431 TBS are IAld and skatole. Cell experiments demonstrate that MN-431 TBS promotes the transcription of aryl hydrocarbon receptor (AHR) and pregnane X receptor (PXR), comparable to the synergistic effect of IAld and skatole. MN-431 TBS can activate AHR and reduces the concentrations of Th17 cell-inflammatory factors IL-17A and IL-21 in intestine and IL-17F, IL-21, and IL-22 in serum. MN-431 TBS can also activate PXR and reduces the concentrations of TNF-α and IL-6 in intestine and serum. CONCLUSION: MN-431 TBS, containing IAld and skatole, can exert anti-CFID effects through the AHR-Th17 and PXR-NF-κB pathways.

2-Oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole, a contributor to the odour of boar-tainted pork meat, mediated by porcine liver microsomal cytochromes P450 1A2, 2A19, 2E1, and 3A22.

The 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole (a contributor to boar taint) mediated by minipig liver microsomes and recombinant P450 enzymes expressed in bacterial membranes were investigated.At low substrate concentrations of 10 µM, the formation rates of indole-3-carbinol, 6-hydroxyskatole, and the sum of 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole in male minipig liver microsomes were significantly lower than those in female minipig liver microsomes.Compensatory 3-methyloxindole and indole-3-carbinol formation in minipig liver microsomes, which lack 6-hydroxyskatole formation in males, was mediated partly by liver microsomal P450 1A2 and P450 1A2/2E1, respectively. These enzymes were suppressed by typical P450 inhibitors in female minipig liver microsomes.Among the 14 pig P450 forms evaluated, P450 2A19 was the dominant form mediating 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole formation from skatole at substrate concentrations of 100 µM. Positive cooperativity was observed in 3-methyloxindole formation from skatole mediated by male minipig liver microsomes and by pig P450 3A22 with Hill coefficients of 1.2-1.5.These results suggest high skatole 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation activities of pig P450 2A19 and compensatory skatole oxidations mediated by pig P450 1A2, 2E1, or 3A22 in male minipig liver microsomes.

Novel gels and films to mask boar taint in entire male pork.

Boar taint masking strategies were developed using hydrocolloids and spices to produce edible gels and films. Carrageenan (G1) and agar-agar (G2) were used for the gels and gelatin (F1) and alginate+maltodextrin (F2) for the films. The strategies were applied to both castrated (control) and entire male pork with high levels of androstenone and skatole. The samples were evaluated sensorially by a trained tasting panel through quantitative descriptive analysis (QDA). Lower hardness and chewiness of entire male pork, with high levels of boar taint compounds object of study, were found with the carrageenan gel, due to the better carrageenan gel adherence to the loin. The films showed that the gelatin strategy tended to have a certain "sweet" taste, and a higher overall masking than the alginate+maltodextrin film. In conclusion, a trained tasting panel found that gelatin film masked boar taint the most, followed by the alginate+maltodextrin film and the carrageenan-based gel.

Effect of whole foods on the microbial production of tryptophan-derived aryl hydrocarbon receptor agonists in growing pigs.

Effects of whole foods on the microbial production of tryptophan-derived aryl hydrocarbon receptor (AhR) ligands in the intestine were investigated in a pig model. Ileal digesta and faeces of pigs after feeding of eighteen different foods were analyzed. Indole, indole-3-propionic acid, indole-3-acetic acid, indole-3-lactic acid, kynurenine, tryptamine, and indole-3-aldehyde were identified in ileal digesta, which were also identified in faeces but at higher concentrations except indole-3-lactic acid, together with skatole, oxindole, serotonin, and indoleacrylic acid. The panel of tryptophan catabolites in ileal digesta and faeces varied across different foods. Eggs induced the highest overall concentration of catabolites in ileal digesta dominated by indole. Amaranth induced the highest overall concentration of catabolites in faeces dominated by skatole. Using a reporter cell line, we observed many faecal samples but not ileal samples retained AhR activity. Collectively, these findings contribute to food selection targeting AhR ligands production from dietary tryptophan in the intestine.

Can sensory boar taint levels be explained by fatty acid composition and emitted volatile organic compounds in addition to androstenone and skatole content?

This study aimed at understanding which molecules were responsible for the differences existing in boar taint sensory evaluation. The latter was therefore linked to the results of skatole and androstenone chemical analyses, fatty acid composition and VOC profiles of heated backfat. This study confirmed that some discrepancy exists between chemical analysis and sensory evaluation of tainted backfats. Significant correlations between human nose scores and fatty acid composition were not revealed. Strong correlations between emissions and contents in skatole and androstenone were found. Oxidation products of polyunsaturated fatty acids, with fatty odor descriptors, were found to be more present in the VOC profiles of boar fat considered untainted through the human nose methodology. Weak coefficient of determination for partial least square regression indicates that other factors, yet unknown, are responsible for sensory evaluation outcomes. These findings hence support the idea that high human nose score is mainly due to boar taint compounds rather than general differences in VOC profiles.

Skatole: A thin red line between its benefits and toxicity.

Skatole (3-methylindole) is a heterocyclic compound naturally found in the feces of vertebrates and is produced by certain flowers. Skatole has been used in specific products of the perfume industry or as a flavor additive in ice cream. Additionally, skatole is formed by tryptophan pyrolysis of tobacco and has been demonstrated to be a mutagen. Skatole-induced pulmonotoxicity was reliably described in ruminants and rodents, but no studies have been conducted in humans. Initially, we provide basic knowledge and a historical overview of skatole. Then, skatole bacterial formation in the intestine is described, and the importance of the microbiome during this process is evaluated. Increased skatole concentrations could serve as a marker for intestinal disease development. Therefore, the human molecular targets of skatole that may have significant effects on various processes in the human body are described. Ultimately, we suggest a link between skatole intestinal formation in humans and skatole-induced pulmonotoxicity, which should be explored further in the future.

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 was mainly 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.

Transcriptomic profiling reveals the molecular responses of Rhodococcus aetherivorans DMU1 to skatole stress.

Skatole is a typical malodor compound in animal wastes. Several skatole-degrading bacterial strains have been obtained, whereas the molecular response of strains to skatole stress has not been well elucidated. Herein, the skatole degradation by a Gram-positive strain Rhodococcus aetherivorans DMU1 was investigated. Strain DMU1 showed high efficiency in skatole degradation under the conditions of 25-40 °C and pH 7.0-10.0. It could utilize various aromatics, including cresols, phenol, and methylindoles, as the sole carbon source for growth, implying its potential in the bioremediation application of animal wastes. Transcriptomic sequencing revealed that 328 genes were up-regulated and 640 genes were down-regulated in strain DMU1 when grown in the skatole-containing medium. Skatole increased the gene expression levels of antioxidant defense systems and heat shock proteins. The expression of ribosome-related genes was significantly inhibited which implied the growth inhibition of skatole. A rich set of oxidoreductases were changed, and a novel gene cluster containing the flavoprotein monooxygenase and ring-hydroxylating oxygenase genes was highly up-regulated, which was probably involved in skatole upstream degradation. The upregulation pattern of this gene cluster was further verified by qRT-PCR assay. Furthermore, skatole should be mainly degraded via the catechol ortho-cleavage pathway with cat25170 as the functional gene. The gene cat25170 was cloned and expressed in E. coli BL21(DE3). Pure enzyme assays showed that Cat25170 could catalyze catechol with Km 9.96 µmol/L and kcat 12.36 s-1.

Single amino acid residue mediates reciprocal specificity in two mosquito odorant receptors.

The southern house mosquito, Culex quinquefasciatus, utilizes two odorant receptors, CquiOR10 and CquiOR2, narrowly tuned to oviposition attractants and well conserved among mosquito species. They detect skatole and indole, respectively, with reciprocal specificity. We swapped the transmembrane (TM) domains of CquiOR10 and CquiOR2 and identified TM2 as a specificity determinant. With additional mutations, we showed that CquiOR10A73L behaved like CquiOR2. Conversely, CquiOR2L74A recapitulated CquiOR10 specificity. Next, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold and AlphaFold and docked skatole and indole using RosettaLigand. These modeling studies suggested space-filling constraints around A73. Consistent with this hypothesis, CquiOR10 mutants with a bulkier residue (Ile, Val) were insensitive to skatole and indole, whereas CquiOR10A73G retained the specificity to skatole and showed a more robust response than the wildtype receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10A73L, CquiOR2, and CquiOR2L74I were insensitive to 3-ethylindole, whereas CquiOR2L74A and CquiOR2L74G gained activity. Additionally, CquiOR10A73G gave more robust responses to 3-ethylindole than CquiOR10. Thus, we suggest the specificity of these receptors is mediated by a single amino acid substitution, leading to finely tuned volumetric space to accommodate specific oviposition attractants.