Ecology

Volatile contents of S. caucasica shoots during the stages of oviposition by G. pecorum
Overall, 60 volatile compounds were identified in S. caucasica shoots during the preoviposition (I), oviposition (II), and postoviposition (III) stages of G. pecorum. These comprised 16 aldehydes, 14 ketones, 12 esters, 9 alcohols, 3 alkanes, 3 aromatic hydrocarbons, 1 acid, 1 ether, and 1 other. Among them, 35 volatiles were identified in I-L, 36 in II-L, and 37 in III-L. In addition, 18 volatiles were common to I-L, II-L, and III-L; 5 to I-L and II-L; 5 to II-L and III-L; and 2 to I-L and III-L. Ten volatiles were unique to I-L, 8 to II-L, and 12 to III-L (Table 1). The main chemical classes of I-L, II-L, and III-L were alcohols, esters, and others; alcohols and others; and alcohols and esters, respectively (Fig. 1).
Table 1 Volatiles detected from shoots of Stipa caucasica during preoviposition, oviposition, and postoviposition of Gasterophilus pecorum.
Full size table

Figure 1

Volatiles classes from shoots of Stipa caucasica during preoviposition, oviposition, and postoviposition of Gasterophilus pecorum. I-L, II-L, and III-L represent Stipa caucasica shoots during the preoviposition, oviposition, and postoviposition stages of Gasterophilus pecorum. (A) alcohols, (B) esters, (C) aldehydes, (D) ketones, (E) others, (F) acids, (G) alkanes, (H) aromatic hydrocarbons, and (I) ethers. Data are mean (n = 3) ± SE. Different letters indicate significant differences at p  0.05) (Fig. 1A). Of the alcohols, 3-hexen-1-ol,(Z)- had the highest relative contents, 25.68%, 55.65%, and 32.35% in I-L, II-L, and III-L, respectively, with no significant differences among these three (P  > 0.05). The relative content of 1-hexanol was higher in II-L (1.52%) than in III-L (1.01%) (P = 0.002) or I-L (0.89%) (P = 0.001), whereas III-L and I-L showed no significant difference (P  > 0.05). The relative contents of the other volatile alcohols were less than 0.8% (Table 1).
Twelve esters were identified from the three stages of S. caucasica. Among them, three, i.e., acetic acid hexyl ester, ethyl acetate, and acetic acid phenylmethyl ester, were common to all three stages; and four, i.e., 3-cyclohexen-1-ol,acetate, 2(3H)-furanone,5-ethyldihydro-, 3-hexen-1-ol,formate,(Z)-, and acetic acid pentyl ester, were common to two of the three stages. The relative contents of esters were lower in II-L (3.16%) than in III-L (40.61%) or I-L (27.81%) (P = 0.000; P = 0.000), whereas there was no significant difference between III-L and I-L (P  > 0.05) (Fig. 1B). The relative contents of acetic acid hexyl ester in II-L (1.47%) and III-L (1.14%) were not significantly different (P  > 0.05), but were higher in both than in I-L (0.52%) (P = 0.001 and 0.005, respectively). The relative contents of 3-hexen-1-ol,acetate,(Z)- (24.8%), a specific volatile of I-L, and 3-hexen-1-ol,acetate(E)- (38.7%), which was specific to III-L, were highest in esters in stages specifically containing them. The relative content of propanoic acid,2-methyl-,3-hydroxy-2,4,4-trimethylpentyl ester, which was detected only in I-L, was 1.12%, whereas those of the other volatiles in esters were lower than 0.8% (Table 1).
Sixteen aldehydes were identified from the three stages of S. caucasica. Among them, seven, i.e., hexanal, nonanal, decanal, heptanal, undecanal, 2-octenal, (E)-, and 2-heptenal,(Z)-, were common to all three stages; and two, i.e., 3-hexenal and 2,4-hexadienal, (E,E)-, were common to two of the three stages. The relative contents of aldehydes in I-L, II-L, and III-L were 10.83%, 6.84%, and 9.9%, and those of hexanal were 0.62%, 2.38%, and 1.16%, respectively; none of these differences was significant (P  > 0.05) (Fig. 1C). The relative contents of nonanal in I-L (1.45%) and II-L (1.9%) did not differ significantly (P  > 0.05), and both were higher than that in III-L (0.96%) (P  > 0.05 and P = 0.018, respectively). The relative content of decanal was higher in II-L (1.20%) than in I-L (0.78%) (P = 0.043) or III-L (0.65%) (P = 0.016), but those in I-L and III-L did not differ significantly (P  > 0.05). The following two volatiles were present in two of the three stages: 3-hexenal, with higher content in I-L (7.10%) than in III-L (5.03%) (P  > 0.05); and 2,4-hexadienal,(E,E)-, with content higher in II-L (0.3%) than in III-L (0.22%) (P = 0.00). Benzaldehyde was specific to III-L (0.99%), with the relative contents of other volatile aldehydes  0.05), with no significant difference between II-L and III-L (P  > 0.05) (Fig. 1D). The content of 2(5H)-Furanone,5-ethyl- was specific to II-L (2.38%), and the relative contents of the other ketones were  0.05), and both were higher than those for III-L (12.9%) (P = 0.017 and P  > 0.05, respectively) (Fig. 1E). The relative content of acetic acid, the only volatile in the class of acids, was lower in III-L (0.61%) than in II-L (3.36%) or I-L (2.14%) (P = 0.022 and P  > 0.05, respectively); there was no significant difference between the latter two (P  > 0.05). The relative contents of alkanes, aromatic hydrocarbons, and ethers were less than 0.22% (Fig. 1G–I). These included three alkanes, one in I-L and two each in II-L and III-L; three aromatic hydrocarbons, one of them specific to each stage; and one ether, which was not found in III-L (Table 1).
The five volatile compounds with the highest relative contents, in order, during the three stages of S. caucasica were as follows: I-L, caprolactam (30.66%)  > 3-hexen-1-ol,(Z)- (25.68%)  > 3-hexen-1-ol,acetate,(Z)- (24.8%)  > 3-hexenal (7.1%)  > acetic acid (2.14%); II-L, 3-hexen-1-ol,(Z)- (55.65%)  > caprolactam (22.68%)  > acetic acid (3.36%)  > hexanal (2.38%) = 2(5H)-furanone,5-ethyl- (2.38%); III-L, 3-hexen-1-ol,acetate,(E)- (38.7%)  > 3-hexen-1-ol,(Z)- (32.35%)  > caprolactam (12.9%)  > 3-hexenal (5.03%)  > hexanal (1.16%) (Table 1). A total of eight volatiles were detected: two (i.e., 3-hexen-1-ol,(Z)- and caprolactam) were common to the three stages, and three (i.e., acetic acid, 3-hexenal, and hexanal) to two of the three stages. Finally, 2(5H)-furanone,5-ethyl- was in the top 5 volatile compounds of only II-L.
Relative contents of volatiles in three plant species during the oviposition stage of G. pecorum
During the oviposition stage of G. pecorum, a total of 60 volatiles were identified in S. orientalis (II-D), S. caucasica (II-L), and C. latens (II-T). These comprised 18 esters, 13 aldehydes, 11 alcohols, 10 ketones, 2 alkanes, 2 aromatic hydrocarbons, 1 acid, 1 alkene, 1 ether, and 1 other. Of these, 35 were identified in II-D, 36 in II-L, and 27 in II-T. In addition, 11 were common to II-D, II-L, and II-T, 14 to II-D and II-L, and 2 to II-L and II-T; 10 were unique to II-D, 9 to II-L, and 14 to II-T (Table 2). The main chemical classes of II-D and II-L were alcohols and others, and those of II-T were alcohols, esters, and others (Fig. 2).
Table 2 Volatiles detected from shoots of three plant species during oviposition of Gasterophilus pecorum.
Full size table

Figure 2

Volatiles classes from shoots of three plant species during oviposition of Gasterophilus pecorum. II-D, II-L, and II-T represent shoots of Stipa orientalis, Stipa caucasica, and Ceratoides latens during the oviposition stage of Gasterophilus pecorum. (A) alcohols, (B) esters, (C) aldehydes, (D) ketones, (E) others, (F) acids, (G) alkanes, (H) aromatic hydrocarbons, (I) ethers, and (J) alkenes. Data are mean (n = 3) ± SE. Different letters indicate significant differences at p  0.05) (Fig. 2A). The relative content of 3-hexen-1-ol,(Z)- was lower in II-T (14.28%) than in II-L (55.65%) or II-D (44.64%) (P = 0.002 and 0.008), but there was not significant difference between II-L and II-D (P  > 0.05). The relative contents of 1-hexanol and 2-hexen-1-ol,(E)- in II-D, II-L, and II-T were 1.67%, 1.52%, 2.79%, and 0.72%, and 0.59% and 2.66%, respectively; these differences were not significant (P  > 0.05). Finally, 3-hexen-1-ol was specific to II-D (1.57%), and the relative contents of other alcohols were  0.05) (Fig. 2B). The relative content of acetic acid hexyl ester in II-D, II-L, and II-T was 0.4%, 1.47%, and 4.25%, respectively; these differences were not significant (P  > 0.05). The relative content of 2(3H)-furanone, 5-ethyldihydro- was higher in II-T (0.71%) than in II-D (0.27%) or II-L (0.26%) (P = 0.000; P = 0.000), but II-D and II-L were not significantly different (P  > 0.05). Both 3-hexen-1-ol,acetate,(Z)- (13.13%) and propanoic acid,2-methyl-,3-hydroxy-2,4,4-trimethylpentyl ester (1.07%) were unique to II-D, and benzoic acid methyl ester (1.88%), methyl salicylate (2.52%), and cis-3-hexenyl isovalerate (8.45%) were all unique to II-T. The relative contents of other esters were  0.05) (Fig. 2C). The relative contents of hexanal, nonanal, decanal, and heptanal were 0.25–2.38% and were higher in II-L than in II-D or II-T, although the differences were not significant (P  > 0.05). Finally, 3-hexenal (6.57%) was unique to II-D, and benzaldehyde (0.94%) to II-T. The relative contents of other aldehydes were  0.05) (Fig. 2D). Five ketones, i.e., 5-hepten-2-one,6-methyl-, 2(3H)-furanone,dihydro-5-methyl-, 2-hexanone,4-methyl-, 2-undecanone,6,10-dimethyl-, and acetophenone, were common to II-D and II-L, and 2(5H)-furanone,5-ethyl- (2.38%) was unique to II-L. The relative contents of other ketones were  0.05) (Fig. 2E). Acetic acid was the only substance in the class ‘acids,’ and its relative content was lower in II-D (1.44%) than in II-T (3.62%) (P = 0.046) or II-L (3.36%) (P  > 0.05); contents in II-T and II-L did not differ significantly (P  > 0.05). The only alkene, 1,3,6-Octatriene,3,7-dimethyl-, was unique to II-T (12.67%). The relative contents of other alkanes and ethers were  caprolactam (21.76%)  > 3-hexen-1-ol,acetate,(Z)- (13.13%)  > 3-hexenal (6.57%)  > 1-hexanol (1.67%); II-L, 3-hexen-1-ol,(Z)- (55.65%)  > caprolactam (22.68%)  > acetic acid (3.36%)  > hexanal (2.38%) = 2(5H)-furanone,5-ethyl- (2.38%); II-T, caprolactam (34.2%)  > 3-hexen-1-ol,(Z)- (14.28%)  > 1,3,6-octatriene,3,7-dimethyl- (12.67%)  > cis-3-hexenyl isovalerate (8.45%)  > acetic acid hexyl ester (4.25%) (Table 2). Eleven volatiles were included: two (3-hexen-1-ol,(Z)- and caprolactam) were common to all three plant species; the other nine were in the top five of only one species.
Relative contents of volatiles from S. caucasica in different growth periods
From S. caucasica at the early, flourishing, and late growth periods (GP1, GP2, and GP3, respectively), a total of 69 volatile compounds were identified. These comprised 17 ketones, 13 aldehydes, 11 esters, 10 alcohols, 4 alkanes, 4 aromatic hydrocarbons, 2 acids, 2 alkenes, 1 ether, and 5 others. Of these, 35 were found in GP1, 36 in GP2, and 40 in GP3. In addition, 11 were common to all three stages, 10 to both GP2 and GP3, 6 to both GP1 and GP2, and 4 to both GP1 and GP3; 14 were unique to GP1, 9 to GP2, and 15 to GP3 (Table 3). The main chemical classes of GP1 and GP2 were alcohols and others, and those of GP3 were esters and others (Fig. 3).
Table 3 Volatiles detected from shoots of Stipa caucasica during its different growth periods.
Full size table

Figure 3

Volatiles classes from shoots of Stipa caucasica during its different growth periods. GP1, GP2, and GP3 represent Stipa caucasica shoots during the early, flourishing, and late growth periods, respectively. Note that GP2 was actually the same sample as II-L in Figs. 1 and 2. Thus, the three groups had a total of seven rather than nine samples. (A) alcohols, (B) esters, (C) aldehydes, (D) ketones, (E) others, (F) acids, (G) alkanes, (H) aromatic hydrocarbons, (I) ethers, and (J) alkenes. Data are mean (n = 3) ± SE. Different letters indicate significant differences at p  0.05). The 3-hexen-1-ol,(Z)- content, which was the highest among all alcohols, was lower in GP3 (15.42%) than in GP1 (49.5%) or GP2 (55.65%) (P = 0.005 and 0.002, respectively); the latter two did not differ significantly (P  > 0.05). The relative content of 1-hexanol was lower in GP3 (0.59%) than in GP1 (1.98%) or GP2 (1.52%) (P = 0.001 and 0.007, respectively); the latter two were not significantly different (P  > 0.05). The relative contents of other alcohols were  0.05) (Fig. 3B). The relative content of acetic acid hexyl ester was higher in GP2 (1.47%) than in GP1 (0.52%) (P = 0.022) or GP3 (0.98%) (P  > 0.05), with no significant difference between GP1 and GP3 (P  > 0.05). Propanoic acid,2-methyl-,3-hydroxy-2,4,4-trimethylpentyl ester (1.48%) was unique to GP1, and 3-hexen-1-ol,acetate,(Z)- (28.42%) to GP3. The relative contents of other esters were  0.05) (Fig. 3E). The relative contents of the remaining four ‘others’ were  0.05) (Fig. 3C). The relative contents of hexanal and decanal decreased with growth period from 4.77% and 1.38% to 1.51% and 1.06%, respectively; but there were no significant differences between periods (P  > 0.05). Six volatiles were common to two of the three periods. There were no significant differences between the relative contents of 3-hexenal in GP3 (6.13%) and in GP1 (5.50%) (P  > 0.05) or between those of nonanal in GP2 (1.90%) and GP3 (1.43%) (P  > 0.05). Finally, 2-hexenal (4.51%) was unique to GP3, and the relative contents of other aldehydes were  0.05) (Fig. 3D). The relative content of 5-hepten-2-one, 6-methyl- was higher in GP1 (0.7%) than in GP3 (0.33%) (P = 0.020), with no significant difference between that in GP2 (0.45%) and that in GP1 or GP3 (both P  > 0.05). The relative content of 2-undecanone,6,10-dimethyl- was higher in GP1 (3.12%) than in GP2 (0.14%) (P = 0.05). Finally, 2(5H)-furanone,5-ethyl- (2.38%) was specific to GP2, and the relative contents of other ketones were  0.05) (Fig. 3F). The relative content of acetic acid, which was common to all three periods, was higher in GP2 (3.36%) than in GP3 (0.97%) (P = 0.035), but there was no significant difference between GP1 (1.87%) and GP2 or GP3 (both P  > 0.05). The other acid, propanoic acid,2-methyl-,2,2-dimethyl-1- (1%), was specific to GP3 (Table 3).
Four alkanes were identified, and the relative contents of individual alkanes ranged from 0.06% to 0.89%. The relative contents of all alkanes were higher in GP1 (1.56%) than in GP3 (0.15%) (P = 0.022), with no significant difference between GP2 (0.22%) and GP1 or GP3 (both P  > 0.05) (Fig. 3G). Two alkenes were found only in GP3; they had a total relative content of 4.76% (Fig. 3J); one, 1,3,6-octatrine,3,7-dimethyl-, accounted for 4.70% of this total. The relative aromatic hydrocarbon and ether contents were  caprolactam (19.78%)  > 3-hexenal (5.5%)  > hexanal (4.77%)  > 2-undecanone,6,10-dimethyl- (3.12%); GP2, 3-hexen-1-ol,(Z)- (55.65%)  > caprolactam (22.68%)  > acetic acid (3.36%)  > hexanal (2.38%) = 2(5H)-furanone,5-ethyl-(2.38%); GP3, caprolactam (28.8%)  > 3-hexen-1-ol,acetate,(Z)- (28.42%)  > 3-hexen-1-ol,(Z)- (15.42%)  > 3-hexenal (6.13%)  > 1,3,6-octatriene,3,7-dimethyl- (4.70%) (Table 3). Overall, nine volatiles were detected: two (3-hexen-1-ol,(Z)- and caprolactam) were in the top five in all three growth periods, two (3-hexenal and hexanal) in two growth periods, and the other five were in the top five of in only one of the three growth periods. More

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The GM maize material used was the GM insect-resistant maize variety (line) GIF, and the maize was a yellow grain strain provided by the Lai Jinsheng Teacher Laboratory of China Agricultural University. The conventional maize variety Meiyu 11 with white kernels was selected as the pollen receptor of GM maize. The inheritance of the seed (kernel) color can be considered to be a single gene, with one pair of alleles (yellow vs. white). The yellow allele is dominant, and the white allele is recessive. The experimental site was sown at the base of the agricultural GM environmental safety assessment of the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Wujitangxia Village, Maihao Town, Wenchang City, Hainan Province (110° 45′ 44″ E, 19° 32′ 14″ N). Transgenic insect-resistant maize was sown three times, once every other week, so that the pollination period of GM maize overlapped with the silking period of the non-GM maize. Artificial on-demand sowing with three seeds per hole and a 4–5 cm sowing depth was adopted.
Field experiments were carried out during two seasons in 2016–2017 and 2017–2018. In the first planting season of 2016–2017, the farthest investigated distance of flow frequency was 60 m (Fig. 1A, Table 1). According to the results from the first investigation, the frequency of gene flow in the eight directions beyond 30 m was very low, almost zero (Table 1). Thus, in 2017–2018, the farthest investigated distance of flow frequency was adjusted to 30 m. In the second planting season, the total area was approximately 14,000 m2 (Fig. 1B, Table 2). As in Hainan off-season reproduction regions the work of breeding research institutes is particularly intensive, it is generally difficult to meet conventional isolation conditions. At the same time, this area also provided a reference for regions around the world that need close isolation. Therefore, we added bagging measures in the treatment areas during the maize tassel pollination period in the second planting season in order to further reduce the flow frequency.
Figure 1

Design of the experimental area. (A) In the period of 2016–2017, the design of the experimental area included one control area (A) and one isolation area (B). The dimensions of control area A and isolation area B in the figure are the same. (B) In the period from 2017 to 2018, the design of the experimental area included one control area (D) and three isolation areas (A, B and C). The solid line represents the isolation area, and the dashed line represents the control area without isolation devices. A1–A8 and B1–B8 in (A) and A1–A8, B1–B8, C1–C8 and D1–D8 in (B) represent eight directions of NE, N, NW, W, SW, S, SE and E, respectively. The dimensions of control area D and isolation areas A, B and C in the figure are the same. The blue numbers represent the size of the experimental areas. The green arrows represent the main wind direction during flowering.

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In the first year of the experiment, control and treatment areas were set up. The area of the control region was 10,000 m2 (100 m × 100 m). A 100 m2 (10 m × 10 m) plot was designated in the center for GM insect-resistant maize, and non-GM maize was planted around this central area. The treatment area with isolation measures was 10,000 m2 (100 m × 100 m). A 100 m2 (10 m × 10 m) plot was designated in the center for GM insect-resistant maize, and non-GM maize was planted around this area. Colored steel plates were used as an isolation measure. The isolation height was 4 m.
A colored steel plate was the isolation material used in these experiments (Fig. 2). Colored steel plates and steel plates are two different materials. At present, there are many colors of colored steel plates. As for which color was used in our isolation experiments, there was no strict requirement, only a desire to match with the surrounding environment. Colored steel plates have the advantages of having both an organic polymer and a steel plate, and many organic polymers have good colorability, formability, corrosion resistance, decoration and high-strength. This combines with the workability of a steel plate, which can be easily finished by stamping, cutting, bending, deep drawing, and other processing to form virtually any shape. This makes the products made of colored steel plates have excellent practicability, decoration, processing and durability.
Figure 2

Isolation device for natural ecological risk control of GM maize. (A) Schematic of the isolation device; (B) partial diagram of the isolation device; (C) sectional view of figure (B); (D) structural detail diagram of the square card; 1: rectangular steel frame, 1.1: steel frame wall, 1.1a: horizontal steel rod, 1.1b: vertical steel rod, 2: inclined support rod, 3: colored steel plate, 4: door for entry and exit, 5: hot-dip galvanized steel frame. 6: structure of the square card, 6.1: screw.

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When maize was harvested after ripening, the investigated directions of control plots were NE, N, NW, W, SW, S, SE and E, labeled with A1–A8, respectively, and those of the isolation plots were labeled with B1–B8, respectively. The location of GM insect-resistant maize from 1 m, 3 m, 5 m, 10 m, 15 m, 20 m, 30 m, 40 m, 50 m and 60 m was investigated along these eight directions. The farthest investigation distances for NE, NW, SW and SE were 60 m, and other directions were 40 m. Ten maize plants were harvested randomly at each point (the first ear). Plants were marked in the order of P1, P2, P3, … P10, dried and stored for further testing. The total number of kernels harvested per corn ear was recorded.
In the second year of the experiment, one control and three treatments were set up. The control plot and the three treatment areas with isolation measures covered an area of 3500 m2 (50 m × 70 m). A 100 m2 (10 m × 10 m) plot was designated in the center of the plot to plant GM maize, and non-GM maize was planted around this central area. Colored steel plates were used as an isolation measure. Bagging of tassels of transgenic maize plants was performed during the pollination period. No bagging was conducted in the control area.
When the maize was harvested after ripening, the investigated directions of control plots were NE, N, NW, W, SW, S, SE and E, labeled D1, D2, D3, D4, D5, D6, D7 and D8, respectively. Isolation area A was marked A1, A2, A3, A4, A5, A6, A7 and A8 along the same eight directions. Isolation areas B and C were marked with B1, B2, B3, B4, B5, B6, B7 and B8, and C1, C2, C3, C4, C5, C6, C7 and C8, respectively. The location of GM insect-resistant maize from 1 m, 3 m, 5 m, 10 m, 15 m, 20 m and 30 m was investigated along these eight directions. The farthest investigation distances for NE, NW, SW and SE were 30 m, and the farthest investigation distances for N, W, S and E were 20 m. Ten maize plants were harvested randomly at each point (the first ear). Plants were marked in the order of P1, P2, P3, … P10, dried and stored for further testing. The total number of kernels harvested per corn ear was recorded.
The endosperm was identified by dominant and recessive traits. According to the number of endosperm traits of GM insect-resistant maize harvested at different directions and distances from GM insect-resistant maize, the pollen transmission distance and outcrossing rate of GM insect-resistant maize were then determined. This method can only be applied to dominant endosperm traits such as yellow or non-waxy grains.
The outcrossing rate was calculated according to formula (1):

$$ P = frac{N}{T} times 100, $$
(1)

where P is the outcrossing rate percentage (%), N is the number of corn kernels containing exogenous genes (the number of the yellow seeds) per ear of corn in units of granules, and T is the total grains (the number of the yellow seeds and white seeds) per ear in units of granules. The outcrossing rates of exogenous genes in different directions and distances were determined, and then the pollen flow distance was determined.
As descriptive statistics, the arithmetic mean as well the standard deviation of outcrossing rates were calculated. The outcrossing rate at each point (1 m, 3 m, 5 m, … 60 m) in the experiment was the mean of the outcrossing rate (P1, P2, P3, … P10) of 10 corn plants at that point.
Details of the isolation device for gene flow risk control of GM maize
The isolation device for gene flow risk control of GM maize, as shown in Fig. 2, comprises a rectangular steel frame (1). The rectangular steel frame 1 was composed of four steel frame walls (1.1), each of which was composed of multiple horizontal steel poles (1.1a) and vertical steel poles (1.1b). Each vertical steel pole was fixed 20–30 cm deep in the soil, and the angle between the inclined support pole (2) and the vertical steel pole was 30°–45°. The vertical steel pole of the four steel frame walls intersected the horizontal steel pole of the top. There were eight inclined supporting poles at the intersection of the vertical steel pole at the four corners of the rectangular steel frame and the horizontal steel pole at the top of the rectangular steel frame, and one inclined supporting pole was fixed through the square card structure (6). The four-sided steel frame wall of the rectangular steel frame was equipped with a colored steel plate (3), and one side of the isolation device was provided with an entry and exit (4). Horizontal steel bars at the top of the rectangular steel frame were provided with a hot-dip galvanized steel frame (5). The hot-dip supporting steel frame was a quadrilateral, and the four corners of the hot-dip supporting steel frame were fixed in the middle of the horizontal steel pole through the hoop. The clamp structure (6) included a side opening and a hollow rectangular frame. The top of the inclined support rod was obliquely inserted into the square clamp structure and fixed on the vertical steel rod through a screw (6.1). The dimensions of the steel rod and the inclined supporting rod were 6000 mm in length, 40 mm in diameter and 2 mm in thickness, and the colored steel plate was 0.425 mm in thickness. The size of the device and the number of inclined supporting rods were determined according to the actual situation in the field. More

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We found that in breast-fed group, α diversity remained unchanged before 3 months of age, but increased significantly in 6 months of age. Previously studies have reported that faecal bacterial diversity increases with age, indicating a more complex microbial community over time8,9. Studies have shown that infants who are exclusively breast-fed have lower microbial diversity, compared with formula-fed babies whose gut microbiota is more diverse and similar to older children10,11,12. The difference of gut microbial diversity between breast-fed and formula-fed babies is also reported in animal research in tiger cubs13. We also found that among different groups, α diversity was lower in breast-fed group than formula-fed groups in 40 days of age. In adults, low gut microbial diversity has been linked to diseases in recent studies. In infants, breast milk may be the major determinant of a lower gut microbial diversity, because specific bacteria are selected for degrading particular oligosaccharides in breast milk. The predomination of infant-type Bifidobacteria during breastfeeding results in a low bacterial diversity, but it is beneficial for babies’ health. For example, the infant-type Bifidobacteria has a large impact on the maturation of the immune system, which may help reduce the incidence of infections in children. However, some diseases have been associated with a reduced microbial diversity in early life, such as eczema and asthma, which have been linked to low microbial diversity in 1 week–4 months of age. But the low microbial diversity is not coupled to Bifidobacterium abundance in these studies, and no reports have shown negative impacts of breastfeeding on development of asthma or allergies. The causality of lower diversity to diseases remains to be identified. What’s more, research has suggested that an immature gut microbial community can be “repaired” by introduction of adult-like microbes increasing greatly during introduction of solid foods in 6 months of age, which is within the development window of opportunity. Findings in adults cannot be inferred to infants regarding the association of gut microbial diversity with diseases, since the microbial ecosystem and the immune system of infants are quite different from adults4.
Bifidobacterium represented the most predominant genus and Enterobacteriaceae the second in all groups at all time-points in our study. Previous study also indicates that all infants have significant levels of Enterobacteriaceae and Bifidobacteriaceae at family level in 2 months of age. The abundance of a single genus usually constitutes the most in family level evaluation. Roger et al. have indicated that Bifidobacterium accounts for 40–60% on average of the total faecal microbiota of a 2-week old new born10. In our study, in 40 days of age, Bifidobacterium accounted for 46.2% in breast-fed group, and 32.2–33.0% in formula-fed groups, which was precisely classified according to feeding types. Bifidobacterium is present in the first few months and decreases as age goes on to almost zero by 18 months old14. Enterobacteriaceae also decreases with time7,8. This is consistent with the European study of 531 infants, which indicates the decrease trend in Bifidobacteriaceae and Enterobacteriaceae species from 6 weeks of age until 4 weeks after solid foods introduction, regardless of differences in feeding patterns15. We found that in breast-fed group, Bifidobacterium decreased from 46.2% in 40 days to 41.4% in 3 months and 29.9% in 6 months of age. In formula-fed groups, after solid foods introduction, Bifidobacterium decreased from 32.2% in 3 months to 31.7% in 6 months of age in formula A group, but increased from 33.0 to 39.0% in formula B group, indicating that different formulas may have different effects on microbiota. In our study, solid foods were introduced from 4 to 6 months of age, so they affected only the last time point in 6 m. We found that in 40 days of age, Bifidobacterium and Bacteroides were significantly higher, while Streptococcus and Enterococcus copy numbers were significantly lower in breast-fed group than they were in formula A-fed group. Lachnospiraceae was lower in breast-fed group than that in formula B-fed group. Veillonella and Clostridioides were lower in breast-fed group than that in formula A and B-fed groups. In 3 months of age there were less Lachnospiraceae and Clostridioides in breast-fed group than formula-fed groups. Other differences of microbiota were shown in Figs. 5 and 6.
After birth, the most important determinant of infant gut microbial colonization is breastfeeding. Studies have shown that breastfeeding is associated with higher levels of Bifidobacterium1,2,16, which is consistent with our study. The genus Bifidobacterium possesses multiple benefits, such as modulation of the immune system, production of vitamins, remission of atopic dermatitis symptoms in infants and decrease in rotavirus infections and lactose intolerance in children and adults10,17. Bifidobacteria is reported to be associated with diminished risk of allergic diseases18 and excessive weight gain19. Higher level of Bifidobacteria also indicates better immune responses to vaccines20.
Bacteroides is among several beneficial bacteria in the earlier neonatal phase, which has important and specific functions in the development of mucosal immune system6. The early activation of mucosal immune system may provide human body lifelong protection from health disorders6. Bacteroides is also linked with increased diversity and faster maturation of gut2. Koenig has studied 1 baby for 2.5 years after its birth and found that Bacteroides genus is absent before the introduction of solid foods21. However, Yassour M. et al. have reported that many infants present a significant Bacteroides species in the first 6 months, before the introduction of solid foods, in a longitudinal study of 39 children in their first 3 years of life14. We also found that there was Bacteroides in the first 6 months of life in all groups. Bacteroides was significantly higher in breast-fed infants, ranking third in 40 days (0.095) in breast-fed group, but decreased as time went on to 0.059 in 3 m and 0.039 in 6 m.
Besides Bacteroides, other health promoting bacteria like Clostridia has been reported to be vital to provide mucosal barrier homeostasis during the neonatal period, which is necessary in the immature intestine6. Formula-fed infants tend to have a more diverse microbial community with increased Clostridia species9,12, which is in accordance with our finding. We also found Veillonella was lower in breast-fed infants than formula-fed ones. Although there is an analysis indicating that Veillonella has been associated with a lower incidence of asthma, it has not taken feeding patterns into consideration22. So more data are needed to clarify the specific roles of certain bacteria with regard to feeding types.
Studies have shown that breast milk keeps the gut in a condition with a lower abundance of Veillonellaceae, Enterococcaceae, Streptococcaceae9,11,23 and Lachnospiraceae7, which is consistent with our results. Some researchers have indicated that higher level of Streptococcus sp. is seen in patients suffered from type 1 diabetes2. There may be other negative effects of these bacteria, but we still know little about them.
The subsequent big change in diet is the introduction of solid foods in 4–6 months of age, which is largely associated with changes in infant gut microbiota. A case study has found an increase in Bacteroidetes at phylum level after solid foods are introduced21. They have indicated that Bacteroidetes is specialized in the decomposition of complex plant polysaccharides21, and it is also associated with faster maturation of the intestinal microbial community2. In our study, after solid foods introduction, percentage of Bacteroides at genus level increased in formula A-fed group, from 0.023 to 0.028, but kept almost the same from 0.009 to 0.008 in formula B-fed group. While in breast-fed group, a decreased percentage of Bacteroides was found from 0.059 in 3 m to 0.039 in 6 m. The trends are different according to different feeding patterns. Pannaraj et al. believe that daily breastfeeding as a part of milk intake continues to affect the infant gut microbial composition, even after solid foods introduction8. But in our study, differences in gut microbiota between breast-fed group and formula-fed groups were not seen any more after solid foods were introduced. As for studies of gut microbiota, the taxonomic level of bacteria adopted in research may affect the results. We focused on microbiota mainly at genus level, resulting in certain discrepancies with some other articles at phylum or species level.
There were significant differences of microbiota between formula A-fed and formula B-fed groups in our study. We found that Pediococcus was less in formula A-fed group than that in formula B-fed group in 40 days. Many research articles have not taken the differences of formulas into consideration, especially retrospective studies. Even breast-fed group is mixed with formulas in some reports. So there must be some inaccuracies of their findings.
Except for feeding patterns, several factors are associated with the microbiota over the first year of life, which is a key period for the gut colonization, such as the mode of delivery, antibiotic exposure, geographical location, household siblings, and furry pets2,9. During the first days of life, the gut microbiota in infants born by vaginal delivery (VD) is similar to that in maternal vagina and intestinal tract, whereas in infants born by caesarean section delivery (CS) the gut microbiota shares characteristics with that of maternal skin. We noticed that the genera of Bacteroides and Parabacteroides were negatively correlated with CS. This was consistent with findings in many other studies, in which the difference of Bacteroides remains in 4 and 12 months of age7,9, and we also found the negative correlation of Bacteroides with CS existed not only in 40 days but also in 6 months of age. The increased morbidity reported extensively in infants born by CS is likely led by altered early gut colonization partially24. Accumulating data have indicated that antibiotic-mediated gut microbiota turbulence during the vital developmental window in early life period may lead to increased risk for chronic non-infectious diseases in later life24. There is a high detection rate of gut Enterococcus in antibiotic-treated infants in their early postnatal period among 26 infants born in a mean gestational age of 39 weeks25. We also found that the relative abundance of Enterococcus was positively correlated with antibiotics usage. The overgrowth of Enterococcus may be caused by antibiotic selection25.
In conclusion, by a larger cohort study than before, differences in gut microbiota among infants who were fed exclusively by breast milk or a single kind of formulas were obtained from this study, contributing further to our understanding of early gut microbial colonization, with more solid data than previous studies with mixed feeding patterns. Faecal diversity was lower in breast-fed infants than formula-fed ones in early life period, but increased significantly after solid foods introduction. A low diversity of the gut microbiota in early life appeared to characterize a healthy gut, if caused by breastfeeding, which was different from theories in adults. There were differences in bacterial composition in infants according to different feeding types, and even different formulas had different effects on microbiota, which we could not ignore in future research. This study presented initial data facilitating further research that will help us understand the importance of breastfeeding to gut microbiota in early life period. More

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