Ovarian Activation. As expected, active queens had fully activated ovaries (mean oocyte length, 3.4 ± 0.04 mm) while gynes had inactive ovaries (0.82 ± 0.04 mm). In gynes, oocyte size significantly changed with age (GLMM, F3,16 = 68.3, p < 0.001), being smallest on day 3 (0.54 ± 0.02 mm) and peaking on day 10 (1.03 ± 0.02 mm). Oocyte size in workers was significantly affected by social condition and age, with a significant interaction between the two (GLMM, F13,164 = 253.9, p < 0.001 for age, F2,164 = 48.73, p < 0. 001 for social condition, F26,164 = 27.9, p < 0.0001 for interaction). QLBL workers had larger oocytes than both QL and QR workers (mean oocyte length, 1.97 ± 0.15 mm for QLBL, 1.35 ± 0.13 mm for QL and 1.11 ± 0.12 mm for QR groups, n = 70 per group) (post-hoc LSD pairwise contrast, p < 0.001) and QL workers, on average, had larger oocytes than QR workers (post-hoc LSD pairwise contrast, p < 0.001). Oocyte size in all worker groups started increasing on day 2 and reached a plateau on day 8 (post-hoc LSD pairwise contrast, p < 0.001 for all comparisons between days 1–8, p > 0.05 for later time points).
Identification of Gland Constituents. The chemical analyses of the cephalic labial glands showed a total of 79 compounds in queens and workers, of which 53 were conclusively or tentatively identified, and 26 remain unknown at present (Table 2). All compounds were used in subsequent discriminant analyses (Fig. 1) but only known compounds were used in further analyses (Figs. 2–4). The main ions of the unknown compounds are provided as supplementary material (Table S1). The secretion was composed mainly of hydrocarbons ranging from 21 to 33 carbons, fatty acids, wax esters, and terpenoid esters. The majority of the compounds were ubiquitous in all female castes, however 38 compounds (20 fully identified) were present only in active queens (n = 9 compounds, mostly wax esters), gynes (n = 10, mostly terpenoids), queens (both active queens and gynes, n = 9, mostly terpenoids) and workers (n = 10, mostly hydrocarbons and wax esters) (Table 2).
Table 2
Relative percentages of compounds in labial gland secretion presented as means ± SE. For all compounds styled in bold, identification has been confirmed by external standard. UD stands for undetectable. One sample of active queen and 4 samples of workers were not included in the analysis due to a technical issue with these samples.
Compound name
|
Class
|
Rt (min)
|
Mean percentage (%) ± SE
|
Specificity
|
Active queens (n = 19)
|
Gynes (n = 20)
|
QL workers (n = 67)
|
QLBL workers (n = 70)
|
QR workers (n = 69)
|
E-β-farnesene
|
terpenoid
|
12.10
|
UD
|
0.79 ± 0.07
|
UD
|
UD
|
UD
|
Gynes
|
myristic acid
|
free fatty acid
|
17.27
|
0.130 ± 0.04
|
1.02 ± 0.12
|
UD
|
UD
|
UD
|
Queens
|
2-Heptadecanone
|
ketone
|
21.33
|
0.34 ± 0.05
|
0.08 ± 0.01
|
0.41 ± 0.03
|
0.40 ± 0.02
|
0.55 ± 0.03
|
|
unknown 1
|
unknown
|
21.59
|
0.09 ± 0.02
|
UD
|
UD
|
0.11 ± 0.02
|
0.07 ± 0.01
|
|
unknown 2
|
unknown
|
21.70
|
UD
|
UD
|
0.18 ± 0.02
|
0.18 ± 0.02
|
0.22 ± 0.03
|
Workers
|
β-springene
|
terpenoid
|
21.73
|
0.07 ± 0.02
|
0.07 ± 0.02
|
UD
|
UD
|
UD
|
Queens
|
methyl palmitate
|
methyl/ethyl ester
|
21.85
|
0.64 ± 0.15
|
1.22 ± 0.16
|
1.61 ± 0.37
|
0.88 ± 0.13
|
2.10 ± 0.28
|
|
palmitic acid
|
free fatty acid
|
22.98
|
0.39 ± 0.11
|
0.53 ± 0.07
|
0.10 ± 0.01
|
0.04 ± 0.01
|
0.13 ± 0.01
|
|
heneicosane
|
hydrocarbon
|
25.88
|
0.94 ± 0.09
|
1.12 ± 0.12
|
1.38 ± 0.16
|
1.13 ± 0.12
|
1.25 ± 0.11
|
|
methyl oleate
|
methyl/ethyl ester
|
26.05
|
0.90 ± 0.26
|
0.08 ± 0.02
|
0.08 ± 0.01
|
0.07 ± 0.02
|
0.22 ± 0.03
|
|
oleic acid + stearic acid
|
free fatty acid
|
28.02
|
9.76 ± 1.29
|
2.20 ± 0.28
|
2.05 ± 0.22
|
3.20 ± 0.33
|
2.81 ± 0.34
|
|
(Z)-9-tricosene
|
hydrocarbon
|
29.55
|
15.31 ± 1.52
|
4.18 ± 0.60
|
24.11 ± 0.67
|
21.79 ± 0.68
|
22.14 ± 0.68
|
|
tricosane
|
hydrocarbon
|
30.10
|
8.28 ± 0.50
|
2.68 ± 0.30
|
10.50 ± 0.29
|
10.74 ± 0.35
|
8.31 ± 0.20
|
|
(Z)-9-tetracosene
|
hydrocarbon
|
31.55
|
1.17 ± 0.11
|
0.27 ± 0.03
|
1.51 ± 0.03
|
1.40 ± 0.04
|
1.33 ± 0.03
|
|
tetracosane
|
hydrocarbon
|
32.06
|
0.22 ± 0.02
|
0.05 ± 0.00
|
0.42 ± 0.01
|
0.43 ± 0.01
|
0.34 ± 0.01
|
|
(Z)-9-pentacosene
|
hydrocarbon
|
33.56
|
18.11 ± 1.33
|
9.73 ± 0.86
|
29.91 ± 0.40
|
28.96 ± 0.48
|
25.72 ± 0.51
|
|
pentacosane
|
hydrocarbon
|
34.00
|
3.52 ± 0.29
|
1.15 ± 0.06
|
7.50 ± 0.20
|
7.36 ± 0.20
|
6.06 ± 0.18
|
|
octyl palmitoleate + decyl myristoleate
|
wax ester
|
34.70
|
0.24 ± 0.03
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
octyl palmitate + decyl myristate
|
wax ester
|
35.33
|
1.37 ± 0.20
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
(Z)-9-hexacosene
|
hydrocarbon
|
35.36
|
UD
|
0.24 ± 0.01
|
0.35 ± 0.01
|
0.38 ± 0.02
|
0.33 ± 0.02
|
|
hexacosane
|
hydrocarbon
|
35.91
|
0.07 ± 0.01
|
0.26 ± 0.03
|
0.11 ± 0.00
|
0.10 ± 0.00
|
0.08 ± 0.00
|
|
(Z)-9-heptacosene
|
hydrocarbon
|
37.22
|
2.47 ± 0.43
|
4.55 ± 0.27
|
5.29 ± 0.28
|
5.04 ± 0.31
|
4.62 ± 0.33
|
|
heptacosane
|
hydrocarbon
|
37.63
|
0.70 ± 0.10
|
0.97 ± 0.08
|
1.72 ± 0.09
|
1.56 ± 0.09
|
1.44 ± 0.09
|
|
octyl oleate
|
wax ester
|
38.62
|
9.38 ± 1.19
|
0.07 ± 0.02
|
0.13 ± 0.04
|
0.32 ± 0.10
|
0.16 ± 0.03
|
|
unknown 3
|
unknown
|
38.77
|
UD
|
0.01 ± 0.00
|
UD
|
UD
|
UD
|
Gynes
|
dodecyl myristate
|
wax ester
|
38.79
|
UD
|
UD
|
0.01 ± 0.00
|
0.02 ± 0.00
|
0.02 ± 0.00
|
Workers
|
decyl palmitate + octyl stearate
|
wax ester
|
38.88
|
0.32 ± 0.07
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
(Z)-9-octacosene
|
hydrocarbon
|
38.91
|
UD
|
0.11 ± 0.01
|
0.31 ± 0.04
|
0.26 ± 0.03
|
0.26 ± 0.03
|
|
unknown 4
|
unknown
|
39.06
|
UD
|
0.17 ± 0.03
|
UD
|
UD
|
UD
|
Gynes
|
farnesyl laurate
|
terpenoid
|
39.41
|
0.14 ± 0.04
|
0.70 ± 0.10
|
UD
|
UD
|
UD
|
Queens
|
octacosane
|
hydrocarbon
|
39.42
|
UD
|
UD
|
0.06 ± 0.01
|
0.08 ± 0.01
|
0.04 ± 0.00
|
Workers
|
squalene
|
terpenoid
|
39.66
|
0.09 ± 0.01
|
UD
|
0.11 ± 0.02
|
0.16 ± 0.03
|
0.14 ± 0.02
|
|
(Z)-9-nonacosene
|
hydrocarbon
|
40.65
|
1.56 ± 0.25
|
2.81 ± 0.22
|
2.71 ± 0.13
|
2.68 ± 0.18
|
2.38 ± 0.17
|
|
nonacosane
|
hydrocarbon
|
40.99
|
0.31 ± 0.04
|
0.24 ± 0.02
|
0.83 ± 0.03
|
0.77 ± 0.04
|
0.74 ± 0.04
|
|
geranyl linoleate
|
terpenoid
|
41.73
|
UD
|
0.45 ± 0.04
|
0.03 ± 0.00
|
0.03 ± 0.00
|
0.08 ± 0.00
|
|
farnesyl myristate
|
terpenoid
|
41.75
|
0.20 ± 0.06
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
decyl oleate
|
wax ester
|
41.91
|
3.92 ± 0.52
|
UD
|
0.06 ± 0.02
|
0.17 ± 0.05
|
0.10 ± 0.02
|
|
dodecyl palmitate
|
wax ester
|
42.10
|
0.18 ± 0.15
|
UD
|
0.02 ± 0.00
|
0.03 ± 0.00
|
0.06 ± 0.01
|
|
dihydrofarnesyl myristate
|
terpenoid
|
42.10
|
UD
|
0.05 ± 0.01
|
UD
|
UD
|
UD
|
Gynes
|
(Z)-9-triacontene
|
hydrocarbon
|
42.28
|
0.08 ± 0.02
|
0.13 ± 0.01
|
0.10 ± 0.01
|
0.10 ± 0.01
|
0.08 ± 0.00
|
|
unknown 5
|
unknown
|
42.45
|
0.12 ± 0.09
|
0.15 ± 0.02
|
UD
|
UD
|
UD
|
Queens
|
unknown 6
|
unknown
|
42.56
|
0.06 ± 0.02
|
0.11 ± 0.03
|
UD
|
UD
|
UD
|
Queens
|
triacontane
|
hydrocarbon
|
42.65
|
UD
|
UD
|
0.04 ± 0.01
|
0.04 ± 0.01
|
0.06 ± 0.01
|
Workers
|
unknown 7
|
unknown
|
42.7
|
0.05 ± 0.01
|
1.24 ± 0.18
|
UD
|
UD
|
UD
|
Queens
|
(Z)-9-hentriacontene
|
hydrocarbon
|
44.0
|
1.06 ± 0.17
|
0.80 ± 0.08
|
1.58 ± 0.10
|
1.65 ± 0.10
|
1.26 ± 0.05
|
|
hentriacontane
|
hydrocarbon
|
44.16
|
0.14 ± 0.02
|
0.03 ± 0.00
|
0.32 ± 0.01
|
0.29 ± 0.01
|
0.31 ± 0.01
|
|
unknown 8
|
unknown
|
44.46
|
0.10 ± 0.03
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
unknown 9
|
unknown
|
44.58
|
UD
|
UD
|
0.01 ± 0.00
|
0.03 ± 0.00
|
0.05 ± 0.00
|
Workers
|
unknown 10
|
unknown
|
44.79
|
UD
|
2.77 ± 0.33
|
UD
|
UD
|
UD
|
Gynes
|
dihydrofarnesyl palmitoleate
|
terpenoid
|
44.89
|
UD
|
0.05 ± 0.01
|
UD
|
UD
|
UD
|
Gynes
|
dodecyl oleate
|
wax ester
|
44.89
|
0.52 ± 0.08
|
UD
|
0.19 ± 0.02
|
0.33 ± 0.04
|
0.43 ± 0.07
|
|
dihydrofarnesyl palmitate
|
terpenoid
|
45.25
|
UD
|
0.15 ± 0.02
|
UD
|
UD
|
UD
|
Gynes
|
dodecyl linoleate
|
wax ester
|
45.35
|
UD
|
UD
|
0.03 ± 0.00
|
0.04 ± 0.00
|
0.03 ± 0.00
|
Workers
|
farnesyl palmitoleate
|
terpenoid
|
45.5
|
0.25 ± 0.04
|
3.02 ± 0.26
|
UD
|
UD
|
UD
|
Queens
|
dotriacontane
|
hydrocarbon
|
45.76
|
UD
|
UD
|
0.08 ± 0.01
|
0.12 ± 0.02
|
0.19 ± 0.01
|
Workers
|
farnesyl palmitate
|
terpenoid
|
45.88
|
0.18 ± 0.05
|
3.22 ± 0.35
|
UD
|
UD
|
UD
|
Queens
|
unknown 11
|
unknown
|
46.3
|
0.15 ± 0.09
|
0.11 ± 0.01
|
0.01 ± 0.00
|
0.10 ± 0.02
|
0.07 ± 0.02
|
|
unknown 12
|
unknown
|
46.45
|
0.17 ± 0.11
|
UD
|
0.03 ± 0.00
|
0.07 ± 0.01
|
0.08 ± 0.01
|
|
unknown 13
|
unknown
|
46.63
|
UD
|
0.28 ± 0.03
|
UD
|
UD
|
UD
|
Gynes
|
(Z)-9-tritriacontene
|
hydrocarbon
|
46.86
|
0.11 ± 0.02
|
0.40 ± 0.05
|
0.19 ± 0.02
|
0.20 ± 0.02
|
0.16 ± 0.01
|
|
myristoleyl oleate + oleyl myristoleate
|
wax ester
|
47.68
|
0.58 ± 0.04
|
UD
|
0.06 ± 0.01
|
0.10 ± 0.02
|
0.15 ± 0.01
|
|
dihydrofarnesyl linoleate
|
terpenoid
|
47.92
|
UD
|
1.41 ± 0.20
|
UD
|
UD
|
UD
|
Gynes
|
farnesyl oleate
|
terpenoid
|
48.65
|
1.95 ± 0.33
|
31.43 ± 2.16
|
0.20 ± 0.02
|
0.29 ± 0.03
|
0.46 ± 0.03
|
|
unknown 14
|
unknown
|
48.75
|
UD
|
UD
|
0.25 ± 0.04
|
0.45 ± 0.06
|
0.84 ± 0.07
|
Workers
|
farnesyl linoleate
|
terpenoid
|
49.5
|
0.38 ± 0.05
|
0.74 ± 0.04
|
UD
|
UD
|
UD
|
Queens
|
unknown 15
|
unknown
|
49.5
|
UD
|
UD
|
0.18 ± 0.05
|
0.19 ± 0.03
|
0.43 ± 0.03
|
Workers
|
hexadecyl oleate
|
wax ester
|
51.86
|
5.92 ± 1.72
|
8.53 ± 0.95
|
2.60 ± 0.33
|
4.16 ± 0.49
|
7.23 ± 0.45
|
|
unknown 16
|
unknown
|
52.75
|
2.78 ± 0.75
|
4.13 ± 0.44
|
1.24 ± 0.16
|
1.82 ± 0.19
|
3.44 ± 0.21
|
|
unknown 17
|
unknown
|
52.98
|
0.62 ± 0.20
|
0.67 ± 0.07
|
0.24 ± 0.03
|
0.19 ± 0.02
|
0.31 ± 0.03
|
|
unknown 18
|
unknown
|
53.37
|
0.19 ± 0.04
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
unknown 19
|
unknown
|
53.62
|
0.11 ± 0.03
|
UD
|
0.25 ± 0.03
|
0.30 ± 0.08
|
0.48 ± 0.04
|
|
unknown 20
|
unknown
|
53.85
|
UD
|
0.17 ± 0.02
|
UD
|
UD
|
UD
|
Gynes
|
unknown 21
|
unknown
|
54.11
|
1.08 ± 0.34
|
1.61 ± 0.21
|
0.52 ± 0.06
|
0.66 ± 0.07
|
1.35 ± 0.09
|
|
unknown 22
|
unknown
|
54.29
|
0.24 ± 0.06
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
unknown 23
|
unknown
|
54.53
|
0.33 ± 0.03
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
unknown 24
|
unknown
|
54.67
|
0.21 ± 0.09
|
UD
|
UD
|
UD
|
UD
|
Active Q
|
unknown 25
|
unknown
|
54.83
|
UD
|
UD
|
0.24 ± 0.02
|
0.42 ± 0.04
|
0.44 ± 0.02
|
Workers
|
unknown 26
|
unknown
|
55.05
|
UD
|
0.14 ± 0.03
|
0.05 ± 0.01
|
0.01 ± 0.00
|
0.14 ± 0.01
|
|
oleyl oleate and other long esters
|
wax ester
|
57.26
|
1.04 ± 0.30
|
2.90 ± 0.43
|
0.10 ± 0.02
|
0.12 ± 0.01
|
0.30 ± 0.02
|
|
Discriminant Analyses. The cephalic labial gland profiles of all bees were analyzed using discriminant analysis using relative quantities of substances. Four discriminant functions significantly discriminated between gynes, active queens, and workers using this analysis, with the first two functions explaining 99.8% of the variance. Function 1 (eigenvalue = 2127.76, canonical correlation = 1, percent of explained variance = 94.3%; Wilk’s λ304 < 0.0001, χ2 = 2841.65, p < 0.001) discriminated between gynes and all other bees and had the highest correlation values with farnesyl and dihydrofarnesyl esters, while function 2 (eigenvalue = 123.29, canonical correlation = 0.99, percent of explained variance = 5.5%; Wilk’s λ225 = 0.001, χ2 = 1381.79, p < 0.001), discriminated between active queens and all other bees, and had the highest correlation values with wax esters, particularly octyl esters (Fig. 1A).
Because large differences between castes may have obscured differences between treatments and ages in workers, we analyzed these data separately. Two discriminant functions significantly discriminated between QR, QL, and QLBL treatments in this analysis. Function 1 (eigenvalue = 4.626, canonical correlation = 0.91, percent of explained variance = 69.3%; Wilk’s λ100 = 0.059, χ2 = 469.74, p < 0.001) discriminated between QR and QLBL workers and had the highest correlation values with methyl palmitate, dodecyl linoleate, geranyl linoleate, octacosane, (Z)-9-triacontene and an unknown compound characterized by a base peak at m/z = 95 (unknown 26 in Table 2) while function 2 (eigenvalue = 2.04, canonical correlation = 0.82, percent of explained variance = 30.6%; Wilk’s λ49 = 0.32, χ2 = 183.8, p < 0.001), discriminated between QL workers and the other two groups, and had the highest correlation values with farnesyl linoleate, hexadecyl oleate, oleyl oleate, and other long-chain wax esters as well as two other unknown compounds characterized by a base peak at m/z = 95 (unknowns 14 and 16 in Table 2) (Fig. 1B).
Thirteen discriminant functions significantly discriminated between workers of different age groups, with the first two functions explaining 83.2% of the variance. Function 1 (eigenvalue = 23.65, canonical correlation = 0.98, percent of explained variance = 67.3%; Wilk’s λ650 < 0.0001, χ2 = 1519.26, p < 0.001) discriminated between all ages from 1 to 14 days in workers, while function 2 (eigenvalue = 5.58, canonical correlation = 0.92, percent of explained variance = 15.9%; Wilk’s λ588 = 0.002, χ2 = 1006.8, p < 0.001), discriminated between workers at 3–6 days of age and workers at younger and older ages. Both functions had the highest correlation values with the most abundant hydrocarbon compounds, with short- and long-chain hydrocarbons displaying negative and positive correlation coefficients, respectively (Fig. 1C).
The two discriminant functions significantly discriminated between workers with oocytes at different stages of activation with the first two functions explaining 95% of the variance in reproduction. Function 1 (eigenvalue = 3.72, canonical correlation = 0.89, percent of explained variance = 62.7%; Wilk’s λ150 = 0.056, χ2 = 475.45, p < 0.001) separated workers with undeveloped ovaries (stage 1) from all other workers and had highest correlation values with the major hydrocarbon compounds (heneicosane, tricosane, tetracosane, heptacosane, nonacosane and the corresponding alkenes) with short- and long-chain hydrocarbons displaying negative and positive correlation coefficients, respectively.
Based on the discriminant analysis information, further analyses of the labial gland secretions were done using three major classes of compounds: 1) terpenoid compounds, comprising farnesene, farnesyl esters, dihydrofarnesyl esters, and geranyl esters, 2) wax esters, with the alcohol moiety chain lengths ranging from 8 to 18 carbons and the acid moiety chain lengths ranging from 14 to 22 carbons, 3) hydrocarbons with chain lengths ranging from 21 to 33 carbons. The relative proportion of each compound class in the total secretion was calculated and used in further analyses. The ratio of short- (≤ 24 carbons) to long-chain hydrocarbons (≥ 26 carbons) was also calculated.
Terpenoid Components. GLM analysis revealed that the proportions of terpenoid components differed significantly between all groups and were highest in gynes, where they comprised up to 68% of the total secretion, and lowest in QL workers (0.1% of the total secretion) (GLM, Wald χ24 = 327.67, p < 0.0001 for group, p < 0.0001 for all post hoc comparisons) (Fig. 2A). Among gynes of different ages, terpenoid compound proportions peaked on days 6 and 10 and declined on day 14, without covariance with oocyte size or interaction between oocyte size and age (GLMM, F3,12 = 9.19, p = 0.002 for age, F1,12 = 1.49, p = 0.24 for oocyte size, F3,12 = 0.4, p = 0.75 for interaction) (Fig. 2B).
Wax Ester Components. The proportion of wax esters was highest in active queens (on average 23%) and lowest in QL workers (on average 2.9%) (GLM, Wald χ24 = 211.44, p < 0.0001 for group, p < 0.05 for all post hoc comparisons). However, the composition of wax esters differed between groups, with octyl esters being almost exclusively present in active queens and dodecyl ester proportions being highest in active queens, QR, and QLBL workers and undetectable in gynes, which almost exclusively produced long-chain esters (> 32 carbons in total) (Fig. 3). Wax ester proportion in gynes was not significantly explained by either age or by oocyte size (GLMM, F3,12 = 1.82, p = 0.19 for age, F1,12 = 1.1, p = 0.31 for oocyte size, F3,12 = 0.47, p = 0.70 for interaction).
Following our finding on caste differences in abundance of different wax esters we performed a discriminant analysis based solely on ester compounds. Four discriminant functions significantly discriminated between gynes, active queens, and workers using this analysis, with the first two functions explaining 97% of the variance. Function 1 (eigenvalue = 12.55, canonical correlation = 0.96, percent of explained variance = 83.5%; Wilk’s λ56 = 0.016, χ2 = 910.05, p < 0.001) discriminated between active queens and all other bees and had highest correlation values with octyl esters, while function 2 (eigenvalue = 2.02, canonical correlation = 0.81, percent of explained variance = 13.5%; Wilk’s λ39 = 0.22, χ2 = 310.01, p < 0.001), discriminated between gynes and all other bees and had highest correlation values with very long chain esters (> 32 carbons). Workers of different treatment groups could not be discriminated based on ester composition.
Differences in Compound Classes Between Workers of Different Ages and Treatments. Based on the results of the discriminant analysis, we tested whether treatment group, age, and ovary size predicted the relative proportion of wax esters and the series of unknown compounds with m/z 95 mass spectral base peak. Wax ester proportion was significantly predicted by age and treatment group, being highest in QR workers and at later ages (day 11 and later) but not by ovary size, with significant interaction between age and treatment and age and ovary size (GLMM, F13,148 = 3.72, p < 0.0001 for age, F2,148 = 31.11, p < 0.0001 for treatment, F26,148= 3.75, p < 0.0001 for interaction between age and treatment, F1,148 = 1.13, p = 0.28 for covariance with ovary size, F2,148 =1.76, p = 0.17 for interaction between treatment and ovary size and F13,148 =1.8, p = 0.047 for interaction between age and ovary size). The proportion of unidentified compounds with m/z = 95-base peak compounds was significantly predicted by age, treatment group (highest in QR workers) and ovary size, with significant interaction between age and treatment (GLMM, F13,148 = 3.79, p < 0.0001 for age, F2,148 = 38.99, p < 0.0001 for treatment, F26,148= 3.97, p < 0.0001 for interaction between age and treatment, F1,148 = 10.88, p = 0.001 for covariance with ovary size, F2,148 =1.45, p = 0.23 for interaction between treatment and ovary size and F13,148 =1.73, p = 0.05 for interaction between age and ovary size).
Hydrocarbon Composition and Ovarian Development. In line with a previous study (Orlova et al. 2020), the short- to long-chain hydrocarbon ratio was highest in active queens (5.2 ± 0.16) and lowest in gynes (0.82 ± 0.09) (GLM, Wald χ24 = 295.66, p < 0.0001 for group, post-hoc LSD: p < 0.0001 for active queen vs. other groups, p < 0.0001 for gyne vs. other groups, p > 0.05 for comparisons between worker treatments). In workers, short- to long-chain hydrocarbon ratio was on average 3.2 ± 0.08 and was significantly predicted by age and treatment group and ovary size, peaking on day 8 and being initially higher in QLBL and QL workers, and then in QR workers at later ages with significant interaction between age and treatment and age and ovary size (GLMM, F13,148 = 7.90, p < 0.0001 for age, F2,148 = 11.61, p < 0.0001 for treatment, F26,148= 5.61, p < 0.0001 for interaction between age and treatment, F1,148 = 50.91, p < 0.0001 for covariance with ovary size, F2,148 = 2.08, p = 0.12 for interaction between treatment and ovary size and F13,148 =6.86, p < 0.0001 for interaction between age and ovary size). In gynes, short- to long-chain hydrocarbon ratio peaked on day 14 and displayed no covariance with oocyte size, but there was significant interaction between oocyte size and age (GLMM, F3,12 = 51.04, p < 0.0001 for age, F1,12 = 3.2, p = 0.098 for oocyte size, F3,12 = 12.16, p = 0.001 for interaction). When the relationship between short to long CHC ratio was analyzed separately using regression curve estimation, polynomial regression with cubic fit proved the best fitting curve (R = 0.57, R2 = 0.325, F3,202=32.39, p < 0.0001) (Fig. 4).