The volatiles from fresh feces of Przewalski’s horse at the pre-oviposition, oviposition, and post-oviposition stages of G. pecorum
Throughout the stages of pre-oviposition (PREO), oviposition (OVIP), and post-oviposition (POSO) of G. pecorum, 70 volatiles were identified in fresh feces of Przewalski’s horse. Among them, 46, 48, and 52 volatiles were identified at PREO, OVIP, and POSO, respectively, and 29 volatiles were common at all three stages. In addition, 4, 5, and 9 volatiles were common between PREO and OVIP, OVIP and POSO, as well as PREO and POSO, whereas 4, 10, and 9 volatiles were unique at the single stage of PREO, OVIP, and POSO, respectively (Table 1; Fig. S1). According to relative content, the two main chemical classes of volatiles were aromatic hydrocarbons and alkenes, that is, their respective contents in a sample were both more than 25% of the total content. Except alcohols which exhibited significant difference between PREO and POSO (One-way ANOVA, F = 8.400, df = 2, P = 0.018), there was no significant difference in all other pairwise comparisons among the nine chemical classes at three stages (One-way ANOVA or Kruskal–Wallis test: P > 0.05) (Fig. 1). Non-metric multidimensional scaling (NMDS) analysis revealed certain extent of overlap (Fig. 2), while one-way analysis of similarity (ANOSIM) indicated that there were significant differences among the three stages (R = 0.5391, P = 0.008).
Volatile classes detected from fresh feces of Przewalski’s horse at the stages of PREO, OVIP, and POSO of Gasterophilus pecorum. PREO, OVIP, and POSO represent fresh feces at the stages of pre-oviposition, oviposition, and post-oviposition of Gasterophilus pecorum, respectively. Data are mean (n = 3) ± SE. Different letters indicate significant differences at P < 0.05.
Multivariate analysis of the volatiles from fresh feces of Przewalski’s horse at the stages of PREO, OVIP, and POSO of Gasterophilus pecorum by the non-metric multidimensional scaling (NMDS) method. PREO, OVIP, and POSO represent fresh feces at the stages of pre-oviposition, oviposition, and post-oviposition of Gasterophilus pecorum, respectively.
Among the eight volatiles which were the five most abundant volatiles at least at one of the three stages, toluene, 1,6-Octadiene,3,7-dimethyl-,(S)-, and caprolactam were identified at all three stages. For each of these three volatiles, its relative contents at three stages revealed no significant differences (One-way ANOVA: P > 0.05). Furthermore, acetic acid was common to PREO and POSO, but no difference was observed between them (Independent t test, t = 0.137, df = 4, P = 0.897) (Table 1).
Of particular concern among the eight volatiles mentioned above, ammonium acetate and butanoic acid were unique to OVIP, the critical stage of oviposition. Although not one of the five most abundant volatiles, another nine volatiles were also specific to OVIP, of which hexanoic acid, cyclopentasiloxane,decamethyl- and cyclohexene,3-methyl-6-(1-methylethyl)- were higher than 1% in relative content (Table 1).
Among the 47 volatiles common to two or three stages, only six volatiles were significantly different in relative contents. Of which, D-limonene was higher at PREO than at OVIP (One-way ANOVA: F = 11.936, df = 2, P = 0.012) or POSO (P = 0.012), and 1-butanol was higher at OVIP than at PREO (One-way ANOVA: F = 8.175, df = 2, P = 0.024) or POSO (P = 0.04). Relative contents of the other four volatiles were less than 1% (Table 1).
The volatiles from feces of Przewalski’s horse with different freshness states at the OVIP stage of G. pecorum
Totally, 83 volatiles were detected from fresh feces (Fresh), semi-fresh feces (Semi-fresh), and dry feces (Dry) at the OVIP stage of G. pecorum. Of which, 48, 41 and 28 volatiles were identified in Fresh, Semi-fresh and Dry, and 7 volatiles were common to all three feces with different freshness states. In addition, 14, 3 and 3, were common between Fresh and Semi-fresh, Semi-fresh and Dry, as well as Fresh and Dry, whereas 24, 17, and 15 were unique to Fresh, Semi-fresh, and Dry, respectively (Table 2; Fig. S2). Aromatic hydrocarbons and alkenes, acids and ketones, as well as alcohols and aldehydes were the two main chemical classes of Fresh, Semi-fresh, and Dry in respective. Except esters and ‘others’ which showed no significant difference in the feces, there were significant differences among other seven classes in at least one pairwise comparison of the three freshness states (One-way ANOVA, Independent t-test or Kruskal–Wallis test: P < 0.05) (Fig. 3). NMDS analysis revealed no overlap among the three states, and noticeably, three data points per states were all identical (Fig. 4). Moreover, ANOSIM indicated there were significant differences among the three states (R = 1, P = 0.005).
Volatile classes detected from feces of Przewalski’s horse with different freshness states at the OVIP stage of Gasterophilus pecorum. Fresh, Semi-fresh, and Dry represent fresh, semi-fresh, and dry feces at the oviposition (OVIP) stage of Gasterophilus pecorum, respectively. Data are mean (n = 3) ± SE. Different letters indicate significant differences at P < 0.05.
Multivariate analysis of the volatiles from feces of Przewalski’s horse with the three different freshness states at the OVIP stage of Gasterophilus pecorum by the non-metric multidimensional scaling (NMDS) method. Fresh, Semi-fresh, and Dry represent fresh, semi-fresh, and dry feces at the oviposition (OVIP) stage of Gasterophilus pecorum, respectively.
Of the twelve volatiles which were one of the five most abundant volatiles at least in one state, only caprolactam occurred in all three states, which was not significantly different among Fresh, Semi-fresh and Dry (One-way ANOVA: F = 4.876, df = 2, P > 0.05). Besides, butanoic acid was common to Semi-fresh and Fresh, and its level in the former was higher than that in the latter (Independent t-test, t = –3.018, df = 4, P = 0.039) (Table 2).
It is worthy to note that toluene, 1,6-octadiene,3,7-dimethyl-,(S)-, and ammonium acetate were unique to Fresh (i.e., OVIP), and acetophenone, acetic acid, and 1,5,9-undecatriene,2,6,10-trimethyl-,(Z)- were unique to Semi-fresh among the twelve volatiles mentioned above. Although not one of the five most abundant volatiles, another twenty-two volatiles were also unique to Fresh, of which cyclohexene,3-methyl-6-(1-methylethyl)-, butanoic acid,2-methyl-, 1,4-cyclohexadiene,1-methyl-4-(1-methylethyl)-, hexanoic acid, cyclopentasiloxane,decamethyl- and pentanal were higher than 1% in relative content. Similarly, another sixteen volatiles were also unique to Semi-fresh, of which propanoic acid,2-methyl-, 1-pentanol, 2-pentanone, 2-butanone,3-hydroxy-, 2-hexanone, 2-hexanone,5-methyl-, 2-heptanone,5-methyl-, 2-octanone and ethanol,2-nitro-,propionate (ester) were higher than 1% (Table 2).
Among the 27 volatiles common to two or three states, except for butanoic acid and acetophenone which belonged to the five most abundant volatiles, only twelve volatiles were significantly different in relative content in at least one pairwise comparison of the three states. Of which, 1H-indene,1-methylene- was higher in Dry than in Fresh (Independent t-test, t = − 9.288, df = 4, P = 0.001), limonene was higher in Dry than in Semi-fresh (Independent t-test, t = − 3.747, df = 4, P = 0.020), so was 3,4-Dihydroxybenzyl alcohol,tris(trimethylsilyl)- (Independent t-test, t = − 9.007, df = 4, P = 0.001). Nonanal and heptanal were higher in Dry than in Semi-fresh (One-way ANOVA: P < 0.001 and P = 0.002) or Fresh (P < 0.001 and 0.001) with significant differences between Semi-fresh and Fresh (P = 0.030 and 0.009). Decanal and octanal were higher in Dry than in Semi-fresh (One-way ANOVA: P = 0.042 and 0.002) or Fresh (P = 0.042 and 0.002). 5,9-Undecadien-2-one,6,10-dimethyl-,(E)- was higher in Dry than in Semi-fresh (One-way ANOVA: F = 28.826, df = 2, P = 0.011) or Fresh (P < 0.001), with significant difference between the latter two (P = 0.011). 5-Hepten-2-one,6-methyl- was higher in Dry than in Semi-fresh (Independent t-test, t = − 4.545, df = 4, P = 0.010). Relative content of other three volatiles were less than 1% (Table 2).
Source: Ecology - nature.com
