Chemoecology
https://doi.org/10.1007/s00049-023-00386-y
CHEMOECOLOGY
ORIGINAL ARTICLE
After chemo‑metamorphosis: p‑menthane monoterpenoids
characterize the oil gland secretion of adults of the oribatid mite,
Nothrus palustris
Günther Raspotnig1 · Michaela Bodner1 · David Fröhlich1 · Julia Blesl1 · Edith Stabentheiner1 · Olaf Kunert2
Received: 5 April 2023 / Accepted: 5 July 2023
© The Author(s) 2023
Abstract
The oil gland secretion of the oribatid mite Nothrus palustris is known to show the phenomenon of juvenile–adult polymorphism, i.e., juvenile instars produce secretions predominated by geranial, whereas adults secrete dehydrocineole along
with a number of chemically unidentified compounds. We here re-analyzed the secretions of adult N. palustris by GC–MS
and NMR spectroscopy, eventually identifying the unknown compounds as p-menthane monoterpenoids. The major components were two isomeric 6-isopropenyl-3-methyl-cyclohex-3-en-1-yl formates (= p-1,8-menthadien-5-yl formates), which
accounted for about 75% of the secretion. These were accompanied by five additional, only partly identified p-menthanes
(or p-methane-derivatives), all of which represented minor or trace components. In addition, adult secretions contained two
C21-hydrocarbons, 1,12-heneicosadiene (major) and a heneicosatriene (minor). Menthane monoterpenoids represent a novel
sub-class of terpene compounds in the oil gland secretions of Oribatida. In case of N. palustris, we assume that both geranial
and p-menthane monoterpenoids arise via the mevalonate pathway which obviously shows a split at the level of geranyl
pyrophosphate, leading to geranial in juveniles and to p-menthanes in adults. The significance of methane occurrence in oil
glands as well as the taxonomic distribution of juvenile–adult polymorphism in oribatid oil gland secretions is discussed. The
latter phenomenon—i.e., “chemo-metamorphosis” of secretions—is not known from early- and middle-derivative Oribatida
nor from Astigmata, but appears to be more common in some derivative desmonomatan and brachypyline oribatid groups.
Keywords Opisthonotal glands · Astigmatid compounds · Dehydrocineole · Geranial · Chemosystematics · p-1,8Menthadien-5-ol
Introduction
Oil glands constitute paired opisthosomal exocrine glands,
that characterize the majority of Oribatida (“glandulate
Oribatida”) and the Astigmata (Norton 1998). If present in
a species, oil glands occur in all ontogenetic stages, from
larvae to adult individuals (Van der Hammen 1980), representing sources of various pheromones, repellents, and
Communicated by Marko Rohlfs.
* Günther Raspotnig
guenther.raspotnig@uni-graz.at
1
Institute of Biology, University of Graz, Graz, Austria
2
Institute of Pharmaceutical Sciences, University of Graz,
Graz, Austria
antimicrobial compounds (Shimano et al. 2002; Raspotnig
2006; Kuwahara 1991, 2004; Heethoff et al. 2011a).
The secretions of oil glands have mainly been studied in
early- and middle-derivative Oribatida (e.g., Sakata and Norton 2001, 2003; Raspotnig et al. 2001, 2004, 2005a, b, 2008,
2009; Heethoff et al. 2018) and, even more extensively, in
Astigmata (e.g., Kuwahara et al. 1975; Kuwahara 2004).
A major chemical trait in these groups is a combination of
specific hydrocarbons, terpenes, and aromatics which are
arranged in species-specific chemical profiles (Sakata and
Norton 2001; Raspotnig et al. 2001). Such profiles do not
appear to change dramatically during ontogenetic development, so that all stages of a particular species—from larvae
up to adult individuals—show the same oil gland chemistry.
In Collohmannia gigantea Sellnick 1922, for instance, adults
produce a blend of 2-hydroxy-6-methyl benzaldehyde, neral,
geranial, neryl formate, γ-acaridial, tri- and pentadecane,
which is very similar in juveniles (Raspotnig et al. 2001;
13
Vol.:(0123456789)
G. Raspotnig et al.
Raspotnig 2006). A lack of juvenile–adult polymorphism is
also found in the desmonomatans Archegozetes longisetosus
Aoki, 1965 (Sakata and Norton 2003), Platynothus peltifer
(C.L. Koch 1839) (Raspotnig et al. 2005b) as well as in different species of Trhypochthonius Berlese 1904 (Raspotnig
et al. 2004; Heethoff et al. 2011b). To the best of our knowledge, there is no evidence of altered compositions of secretions during ontogeny in the Astigmata either (Kuwahara
1991, 2004).
About 20 years ago, a report on a first incidence of juvenile–adult polymorphism of oil gland secretions in Oribatida
was published: When investigating the oil glands of the desmonomatan Nothrus palustris (C.L. Koch 1839), Shimano
et al. (2002) found that juvenile stages—but not adults—produced an alarm pheromone, geranial. Adult secretions, by
contrast, contained a rich set of novel compounds, of which
only two, namely dehydrocineole and a C21-hydrocarbon
(heneicosadiene), were identified.
We here re-analyzed the secretion of N. palustris with the
aim to identify the remaining compounds. We eventually
show that it is a blend of p-menthane monoterpenoids that
replace juvenile geranial and predominate in the secretions
of N. palustris after the final moult.
Materials and methods
About 215 adult individuals and 14 juveniles of N. palustris
were extracted (by Berlese–Tullgren extraction) from sieved
soil samples collected during the years 2022/2023 at different locations in Styria, Austria, namely (1) near the “Fischteich” in Passail, Styria, Austria (N 47.2755; E 15.5326);
(2) in Pirching am Traubenberg (N 46.9330; E 15.6111), and
(3) in Heiligenkreuz am Waasen (N 46.9644; E 15.5829).
Forty adult individuals from different populations were used
to prepare individual whole-body extracts (single individuals were extracted in 20 µl methylene chloride for 15 min).
Crude extracts, containing extruded oil gland secretion, were
used for gas chromatography–mass spectrometry (GC–MS).
To determine the double-bond position of unsaturated
hydrocarbons, 16 individuals were extracted in 100 µl hexane for 15 min. The remaining adult individuals (about 160)
were used to prepare a pooled extract in 720 µl deuterated
chloroform (CDCl3) for nuclear magnetic resonance spectroscopy (NMR). Additionally, ten extracts from juvenile
stages were prepared: one pooled larval extract (five larvae
in 20 µl methylene chloride) and three individual extracts
from each nymphal stage (proto-, deuto-, and tritonymphs;
each individual in 20 µl methylene chloride).
Instrumentation/conditions for GC–MS: We measured
on a Trace GC-DSQI instrument from Thermo (Vienna,
Austria). The GC was equipped with a ZB-5 capillary column (30 m × 0.25 mm × 0.25 µm) which was heated as
13
follows: 50 °C for 1 min, then increase by 10 °C/min to
300 °C, and a 5 min isothermal hold. Helium (at a constant
flow rate of 1.2 ml/min) was the carrier gas. The injector
was kept at 240 °C; the transfer line at 310 °C. The MS
worked in electron impact (EI) mode at 70 eV. The ion
source was at 200 °C; we scanned ions from mass/charge
ratio 40–500.
Some measurements (particularly those for the localization of double bonds, see text) were done on a 5977B GC/
MSD (coupled to an 8890 GC) from Agilent (Vienna, Austria). We used two series connected HP-5MS ultra inert capillary columns, each 15 m × 0.25 mm × 0.25 µm, at helium
flow rates of 1.0 and 1.2 ml/min, respectively, and the same
MS parameters as listed above. For the detection of the
DMDS derivatives, we relied on a slightly longer temperature program, starting at 40 °C, ramping by 10 °C/min to
300 °C, and keeping 300 °C for 15 min.
Instrumentation/conditions for NMR: NMR experiments
(1D 1H, and 2D COSY, HSQC, HMBC, and HSQC-TOCS)
were performed with a Bruker 700 MHZ Avance II NMR
spectrometer (Rheinstetten, Germany) equipped with a cryoprobe. For the HSQC, a version with multiplicity editing
was used.
Data evaluation/reference compounds: GC–MS data were
evaluated with XCalibur 2.07 (Thermo) and MassHunter
Workstation 10.0 (Agilent). For a first approach to identify
compounds, we used the NIST05 mass spectrometric library.
To fully identify compound A (dehydrocineole),
2,3-dehydro-1,8-cineole was synthesized according to
already published procedures (Carman et al. 2005; Brenna
et al. 2013). For compounds B and C, GC–MS data in combination with NMR data were used for identification. The
hydrocarbons D and VI were identified by their mass spectra, and the positions of double bonds in compound D were
determined by DMDS derivatization (Carlson et al. 1989;
Fröhlich et al. 2022). The remaining minor compounds
(I–V) were tentatively identified by GC–MS data only.
Normal alkane retention indices (Van den Dool and Kratz
1963) were calculated using an alkane-standard (C8-C40).
Relevant compounds for the synthesis of dehydrocineole
[eucalyptol, N-bromo succinimide, dibenzoyl peroxide,
azobis(isobutyronitrile)] as well as the alkane standard were
purchased from Sigma (Vienna, Austria); tetrahydrofuran,
dimethylformamide, and dimethylsulfoxide were from Roth
(Graz, Austria); α-terpineol was from TCI Europe (Eschborn, Germany).
To visualize position and morphology of oil gland pores,
scanning electron micrographs (SEM) were taken as follows:
a clean, adult individual of N. palustris was fixed in ethanol, dehydrated in 100% ethanol and acetone, air dried overnight, then mounted onto Aluminium pin stubs (Agar Scientific, Biedermannsdorf, Austria), sputtercoated with gold
(Agar Sputter Coater; Christine Gröpl, Tulln, Austria), and
After chemo‑metamorphosis: p‑menthane monoterpenoids characterize the oil gland secretion…
investigated with a Hitachi FlexSEM1000 (Tokyo, Japan)
using 20 kV acceleration voltage in the high-vacuum mode.
a
500 µm
b
30 µm
Fig. 1 Scanning electron micrographs of an adult individual of
Nothrus palustris showing the location of oil gland pores (a: arrows)
and details of right pore (b)
Results
Oil glands of N. palustris are large paired glands located
beneath slight bulges at the distal edges of the notogaster
(Fig. 1a). They open dorso-laterally at each edge of the notogaster via a small, mouth-shaped pore surrounded by smooth
cuticular lips (pore diameter: about 15 µm) (Fig. 1b).
Whole-body extracts of adult individuals consistently
showed four major peaks (A-D), together amounting for
about 90% of the extract profile (Fig. 2, Table 1). The proton
NMR spectrum (Fig. 3) of the extract (160 adults) was dominated by the three compounds A, B, and C. Their molecular constitutions were determined in mixture by complete
assignments of their resonances in the 2D NMR spectra
(Table 2).
The EI mass spectrum and the NMR data of peak A (M+
at m/z 152, base ion at m/z 109) indicated 2,3-dehydro1,8-cineole, as already identified from adult extracts in a
previous study (Shimano et al. 2002). The identity of the
compound as 2,3-dehydro-1,8-cineole was confirmed by
synthesis.
Peaks B and C appeared to be isomeric compounds,
exhibiting indistinguishable mass spectra (Table 1).
Molecular ions in both components were weak, but could
be detected at m/z 180 (intensity < 1%). Major fragment
ions were observed at m/z 134, 119, 105, 91, and 77, corresponding to a p-menthane monoterpenoid structure. Hits
B
100
90
80
Relative Abundance
C
70
60
A
50
40
30
IV
V
20
D
VI
II
I
10
III
0
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
Time (min)
Fig. 2 Characteristic chemical profile of the oil gland secretion of
adult Nothrus palustris. Major compounds (black): peak A (2,3-dehydro-1,8-cineole), B ((1R,6R)-or (1R,6R)-p-1,8-methadien-5-yl formate), C ((1R,6S)-or (1S,6R)-p-1,8- methadien-5-yl formate), and D
(1,12-heneicosadiene). Minor compounds (red): peak I (menthanehydrocarbon C10H14, possibly menthatriene isomer 1), peak II (men-
thane-hydrocarbon C10H14, possibly menthatriene isomer 2), peak III
(mono-oxygenated p-menthane C10H16O, possibly p-1,8-menthadien5-ol), peak IV (mono- or doubly oxygenated p-menthane C12H20O or
C11H16O2, isomer 1), peak V ((mono- or doubly oxygenated p-menthane C12H20O or C11H16O2, isomer 2), and peak VI (heneicosatriene)
13
G. Raspotnig et al.
Table 1 Gas chromatographic—mass spectrometric data to extract components of adult Nothrus palustris
Peak no RI*
EI-fragmentation pattern [m/z (rel. Int.)]
Major peaks
A
995
152(6), 124(21), 109(100), 94(21), 91(9), 79(38), 77(13),
43(13)
B
1267 180(< 1), 134(67), 119(100), 117(8), 107(6), 106(13),
105(30), 93(24), 92(31), 91(45), 84(16), 83(29),
79(14), 77(16), 41(11)
C
1288 180(< 1), 134(67), 119(100), 117(8), 107(6), 106(11),
105(25), 93(22), 92(24), 91(41), 84(9), 83(13), 79(14),
77(14), 67(6), 65(5), 55(6), 41(9)
D
2069 292(13), 180(4), 166(7), 152(8), 138(13), 137(11),
124(23), 123(24), 110(35), 109(41), 97(46), 96(85),
95(67), 83(73), 82(100), 81(84), 70(19), 69(75),
68(48), 67(65), 57(28), 56(29), 55(86), 54(33), 43(28),
41(44)
Minor peaks (tentatively identified)
I
1009 134(61), 119(100), 117(17), 115(14), 105(31), 93(21),
92(35), 91(97), 79(14), 77(32), 65(9), 41(8)
II
1029 134(70), 119(72), 117(12), 115(7), 106(14), 105(26),
103(8), 93(14), 92(27), 91(100), 79(23), 77(34), 65(9),
51(6), 41(9)
III
1213 152(5), 137(100), 134(20), 119(87), 117(9), 109(63),
108(61), 107(35), 106(19), 105(28), 97(25), 95(19),
94(23), 92(25), 91(72), 84(76), 83(70), 82(20), 81(27),
79(44), 77(35), 69(25), 68(22), 67(37), 65(13), 56(15),
55(19), 53(17), 43(11), 41(32)
IV
1280 180(< 1), 165(< 1), 152(2), 137(8), 134(100), 119(77),
117(13), 115(9), 112(31), 106(14), 105(26), 94(10),
93(23), 92(39), 91(75), 84(71), 83(48), 79(22), 77(30),
67(19), 65(12), 41(17)
V
1302 180(< 1), 165(< 1), 152(1), 137(4), 134(100), 121(6),
119(64), 117(10), 115(7), 112(19), 107(11), 106(12),
105(22), 94(20), 93(23), 92(22), 91(63), 84(47),
83(32), 79(30), 77(27), 67(8), 65(10), 55(7), 41(12)
VI
2069 290(5), 191(2), 163(2), 150(7), 149(6), 138(16), 135(16),
124(12), 123(17), 122(14), 121(22), 110(32), 109(27),
107(13), 96(52), 93(27), 83(21), 82(69), 81(100),
80(57), 79(29), 77(14), 69(25), 68(44), 67(98), 57(13),
55(48), 54(18), 43(10), 41(30)
Rel.
abundance**
Identified as
24
2,3-Dehydro-1,8-cineole
34
8
(4R,5R)- or (4S,5S)-p-1,8-menthadien-5-yl formate
= (1R,6R)- or (1S,6S)-6-isopropenyl-3-methyl-cyclohex3-en-1-yl) formate
(4R,5S)- or (4S,5R)-p-1,8-menthadien-5-yl formate
= (1R,6S)- or (1S,6R)-6-isopropenyl-3-methyl-cyclohex3-en-1-yl) formate
1,12-Heneicosadiene (1,12-C21:2)
<1
p-Menthane monoterpene C10H14 (isomer1)
<1
p-Menthane monoterpene C10H14 (isomer 2)
1
Oxygenated p-menthane monoterpenoid C10H16O
(probably p-1,8-menthadien-5-ol)
2
Oxygenated p-menthane monoterpenoid C10H18O2
(isomer 1)
4
Oxygenated p-menthane monoterpenoid C10H18O2
(isomer 2)
<1
Heneicosatriene (C21:3)
25
*normal alkane retention index (Van den Dool and Kratz 1963). **%peak area of total extract components
from NIST05 could not be confirmed. However, the fragment at m/z 134 (M+—46: C10H14+), probably arising from
the neutral loss of formic acid from the molecular ion,
tentatively indicated an ester structure. Assignment of the
NMR resonances led to two isomeric p-1,8-menthadien5-yl formates—comprising a limonene backbone with a
formyl group in position 5—with different carbon chemical shifts at the two asymmetric carbons C-4 and C-5 and
the methylene group C-3 indicating the presence of isomers or two pairs of enantiomers with different relative
configurations in the extract. The NMR resonances with
the higher intensities were correlated with compound B
(higher peak area in GC) and the NMR resonances with
13
lower intensity, consequently, with compound C. While
both compounds have not been described in literature yet,
the chemical synthesis of a homologue (4S,5R)-acetate has
already been reported (Brenna et al. 2006). As the carbon
resonance values of the synthetic compound (Table 2) are
in very good agreement with compound C in the extract,
the relative configuration of C has to be (4S,5R) and the
relative configuration of B has to be (4R,5R). On the basis
of the NMR spectra, the compounds B and C determined
as (1R,6R)- or (1S,6S)-6-isopropenyl-3-methyl-cyclohex3-en-1-yl formate for compound B [= (4R,5R)- or
(4S,5S)-p-1,8-menthadien-5-yl-formate] and (1R,6S)or (1S,6R)- 6-isopropenyl-3-methyl-cyclohex-3-en-1-yl
After chemo‑metamorphosis: p‑menthane monoterpenoids characterize the oil gland secretion…
Fig. 3 Proton NMR spectrum
(700 MHz, CDCl3) of the N.
palustris chloroform extract.
Resonances of the three
assigned compounds A–C are
labeled accordingly
H 2O
CHCl3
AA
A
B
B
C
C
B
B
A
A
B
B C
C
8.5
8.0
7.5
7.0
6.5
6.0
5.5
BC
B
C
C
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
H [ppm]
Table 2 NMR spectrometric
data (700 MHz, 175 MHz,
CDCl3) of extract components
A–C of adult Nothrus palustris
and literature data of the acetate
homologue of C
Atom
A
B
C
(4S, 5R) homologue
of C (synthetic
acetate)
δC
δH, (J in Hz)
δC
δH, (J in Hz)
δC
δH, (J in Hz)
δC
δH, (J in Hz)
1
2
70.9
135.2
129.8
120.5
–
5.52
130.8
120.2
–
5.37 brs
120.6
5.35 t (4.6)
3
135.3
–
6.10 dd
(8.2, 1.0)
6.46 t (7.3)
25.1
30.2
2.10 – 2.15
39.4
20.3
46.8
72.0
2.32 – 2.45
5.05
6
31.1
2.32 – 2.45
23.5
146.2
112.7
10
formate
28.1
1.28 s
2.40
2.08
1.66 s
–
4.83 brs
4.81 brs
1.70 s
8.05 s
36.6
24.7
74.2
29.1
2.39
2.15
1.67 s
–
4.87 brs
4.76
1.79 s
8.04 s
35.7
7
8
9
2.28
2.02
1.20 tdd
(12.3, 5.1, 2.3)
1.73
1.26
1.33 s
–
0.98 s
2.16
2.16
2.50
5.17
30.8
4
5
2.34
2.08
2.28
5.50
1.65—1.72
–
4.75 m
4.75 m
1.65—1.72
formate [= (4S,5R)- or (4R,5S)-p-1,8-menthadien-5-ylformate] for compound C, respectively.
Compound D was identified as a C21-alkadiene, mainly by
interpretation of its mass spectrum (M+ at m/z 292; Table 1).
The positions of the double bonds were determined by
DMDS derivatization (Table 3, Fig. 4). Initially, a 6,9-heneicosadiene—as proposed by Shimano et al. (2002)—was
expected. Diadducts of alkadienes with double bonds
separated by only one CH2 unit (as in 6,9-heneicosadiene)
42.6
69.2
36.3
23.1
144.9
111.3
22.4
160.8
46.1
71.5
23.0
145.3
112.6
19.3
160.8
20.1
show the formation of cyclic thioethers, along with a loss
of 62 amu (CH3SCH3) by adduct formation, as described
in detail in Raspotnig et al. (2005b). In the present case,
such a loss (theoretically leading to a fragment at m/z 418:
292 + 4 × SCH3—62) was not observed, indicating that the
two double bonds were not adjacent.
We indeed observed a molecular ion an m/z 480
(= 292 plus 4 × SCH 3) along with characteristic losses
of SCH3-groups from M+ at m/z 432 (M+—48), m/z 386
13
G. Raspotnig et al.
Table 3 Localization of double-bond position: diagnostic ions in the DMDS derivative of heneicosadiene (peak D)
m/z
[A]+
[D]+
[B—94]+
[B—48]+
[B]+
[C—94]+
[C—48]+
[C]+
[M—141]+
[M—94]+
[M—48]+
[M]+
61
173
325
371
–
213
259
307
339
386
432
480
Diagnostic fragments were interpreted following Carlson et al. (1988), as outlined in detail in the text and shown in Fig. 4. Fragment ion [B]+,
probably instable by containing three SCH3 groups and theoretically at m/z 419, was not detected
[D]+
[A]+
H3CS
DMDS
SCH3
H2C
CH
[M]+
CH
HC
heneicosa-1,12-diene
H3CS
SCH3
[B]+
[D]+
x105
[C-48]+
173
3
[C]+
259
2,8
2,6
2,4
[B-94]+
[C-94]+
[A]+
2,2
325
2
1,8
[M-48]+
480
371
432
339
307
290
247
197
179
163
137
151
75
0,4
0,2
[B-48]+
123
87
41
0,8
0,6
[M]+
109
1
[M-94]+
[C]+
1,2
386
213
69
83
55
61
1,4
95
[M-141]+
1,6
0
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
360
380
400
420
440
460
480
500
Counts vs. Mass-to-Charge (m/z)
Fig. 4 Localization of double bonds in the Nothrus-heneicosadiene.
Diagnostic ions of the DMDS derivative were interpreted according to Carlson et al. (1989): In a nutshell, DMDS diadducts of alkadienes theoretically show (1) a molecular ion at 188 amu higher than
in the original compound (= plus 4 × SCH3); (2) fragments arising
from the loss of HSCH3 (− 48 amu), CH3SSCH3 (− 94 amu), and
SCH3 + CH3SSCH3 (− 141 amu) from the molecular ion; (2) two
complementary pairs of fragments ([A]+ and [B]+, [C]+ and [D]+),
arising from cleavage of C–C bonds between the SCH3-substituents
(= at the sites of double bonds in the original molecule, allowing to
locate the regarding positions); and 4) loss of HSCH3 (- 48 amu) and
CH3SSCH3 (- 94 amu) from fragments ([B]+ and [C]+) that bear three
SCH3-groups
(M+—94) and m/z 339 (M+—141). The fragments from
cleavage of C–C bonds between the outer SCH3-bearing
groups were found at m/z 61 (= [A]+) and 173 (= [D]+),
indicating one double bond in position 1 (61—SCH3 = 14;
i.e., only one CH 2 unit in this fragment), the other in
position 9 from the other end of the molecule (173—
SCH3 = 126: i.e., 9 CH2-units in this fragment). Position
9 from the other end of a heineicosadiene corresponds
to position 12 in regular direction (21Cs in total minus
a 9 C-fragment = position 12). The remaining two fragments from cleavage of C–C bonds between the inner
SCH 3-bearing groups bear three SCH 3-groups (= [B] +
and [C] +) and represent the complementary fragments
to [A]+ and [D]+. Thus, fragment [A]+ plus [B]+ should
give the whole molecule (= M+ at m/z 480), and [D] + and
[C] + should also give 480. Fragment [B]+ was expected at
m/z 419 (480—61), and [C]+ at m/z 307 (480–173). Such
fragments generally show further losses of 48 and 94 mass
units (= one and two SCH3-groups, respectively), facilitating their detection. In our case, fragment [B]+ itself was
not observed, but ions [B—48]+ (at m/z 371) and [B—94]+
(at m/z 325) were visible. On the other hand, fragment
[C]+ was observed at m/z 307, along with [C—48]+ (at m/z
259) and [C—94]+ (at m/z 213). This data together clearly
indicated a doubly unsaturated C21-alkadiene with double
bonds in positions 1 and 12 (i.e., a 1,12-heneicosadiene)
(Fig. 4, Table 3).
A further (but minor) hydrocarbon, obviously a tripleunsaturated analog (peak IV: M+ at m/z 290, heneicosatriene), accompanied 1,12-heneicosadiene. Due to the low
13
After chemo‑metamorphosis: p‑menthane monoterpenoids characterize the oil gland secretion…
quantity of the compound, it could not be recovered after
derivatization.
Adult extracts exhibited a number of additional, minor
peaks, all of which showed mass spectra of p-menthanes or
menthane derivatives. All these minor compounds together
amounted for less than 10% of the total peak area of the
extracts. Five consistently occurring minor components
(peaks I–V) were further investigated (Table 1), though
straightforward identifications via MS were hampered by
weak molecular ions. Peaks I and II, however, showed
intense molecular ions m/z at 134 and fragments at m/z
119 and 91. A library search pointed to compounds, such
as dimethyl-octatetraenes, cymenes, and menthatrienes.
For well-documented p-menthatrienes (such as 1,3,8- and
1,5,8-p-methatriene) and 2,6-dimethyl-1,3,5,7-octatetraene,
the observed retention indices were too low (see discussion).
For p-, o-, and m-cymenes, reported mass spectra and indices fairly fitted (e.g., Adams et al. 2012), but a direct comparison to authentic standards did not show full correspondence. However, based on the prevalence of p-menthanes in
the secretion (e.g., peaks B, C, III), we tentatively assume
compounds I and II to be isomeric menthatrienes.
Peak III appeared to be a mono-oxygenated p-menthane monoterpenoid of formula C 10 H 16 O, exhibiting a visible molecular ion at m/z 152, a base ion at m/z
137 (M+-CH3), as well as elimination of water from M+
(m/z 134). We assume that peak III represented the corresponding alcohol to the p-menthane formates B and
C, i.e., 6-isopropenyl-3-methyl-cyclohex-3-en-1-ol
(= p-1,8-menthadien-5-ol = limonen-5-ol).
Peaks IV and V also showed the mass spectra of isomeric p-menthane monoterpenoids. The molecular ions
were weak, and well-visible fragments of highest mass
were recorded at m/z 137, with a base ion at m/z 134. We
thus expected a molecular ion at m/z 152 (again indicating
a menthadienol), but then found weak fragments at m/z 180
and 165. Proposing a molecular mass of 180 amu, the molecules theoretically contained one or two oxygens and thus
corresponded to a molecular formula of either C12H20O or
C11H16O2, respectively.
By contrast, extracts of all juvenile stages (i.e., larvae,
proto-, deuto-, and tritonymphs) were predominated by one
single peak, geranial (M+ at m/z 152), as already reported
by Shimano et al. (2002).
Discussion
p‑Mentha‑1,8‑dien‑5‑yl‑compounds in Nothrus
versus p‑mentha‑1,8‑dien‑3‑yl‑compounds
in Astigmata
Menthane monoterpenoids represent a chemical class
new to the oil gland secretions of Oribatida. The
menthanes found in N. palustris contain one ring and two
or three double bonds, and are based on/derived from a
limonene backbone (= 4-isopropenyl-1-methyl-cyclohexene = p-mentha-1,8-diene). Oxygen-containing substituents, if present, preferably occur in position 5, suggesting
p-mentha-1,8-dien-5-ols as leading structures (Fig. 5).
Intriguingly, from the oil glands of Astigmata, several
similar p-menthane monoterpenoids have been reported:
Isopiperitenone, a p-mentha-1,8-dien-3-one, was found
in the oil glands of species of Tyrophagus (Oudemans
1924a), Tyroborus (Oudemans 1924b), and Schwiebea
Oudemans 1916. Further p-menthane derivatives, such as
robinal and isorobinal, were described from Rhizoglyphus,
Tyroborus, and Schwiebea (Kuwahara et al. 1987; Leal
et al. 1990; Tarui et al. 2002; Tomita et al. 2003; Mizoguchi et al. 2005; Maruno et al. 2012). Kuwahara (2004)
reviewed astigmatid oil gland constituents and additionally listed limonene (p-mentha-1,8-diene) and isopiperitenol (p-mentha-1,8-dien-3-ol). All these compounds from
astigmatid mite oil glands share the p-mentha-1,8-dienebackbone with Nothrus, but differ in the position of the
oxygen-containing moiety attached to the ring (which is in
position 3 in Astigmata). Thus, oxygenated menthanes in
Astigmata represent p-mentha-1,8-dien-3-ols and derivatives of these (Fig. 5).
The major components in the secretion of N. palustris
were identified as two isomeric p-1,8-menthadien-5-yl
formates. These findings shed new light on the initial
study on Nothrus: Shimano et al. (2002) did not mention
a p-menthane predominated secretion but show a figure
(Fig. 1/4 in Shimano et al. 2002) of an adult chromatogram
with a few unidentified peaks in characteristic positions
(apart from dehydrocineole and heneicosadiene). We tentatively assume that these peaks, with much lower abundance in the Japanese population, might correspond to the
herein described p-menthanes.
p‑Menthane subclasses in Nothrus
The Nothrus-menthanes may be classed into three groups,
according to their molecular mass and degree of oxygenation, respectively. There are (i) tentatively identified
p-menthane-hydrocarbon monoterpenes, (ii) such with
one oxygen atom, and (iii) compounds with two oxygens.
Diagnostic ions were given by the fragment m/z 134,
which is a pure hydrocarbon fragment (C10H14+); whereas
the fragment at m/z 137 contained one oxygen (C9H13O+).
Thus, molecular ions at m/z 134, as present in compounds
I and II, probably indicated hydrocarbon p-menthanes.
For compounds I and II, there are assumed to be menthatrienes, but reported retention indices for several menthatrienes (e.g., Adams et al. 2012: p-1,3,8-menthatriene;
13
G. Raspotnig et al.
7
(1)
(1‘)
(2)
(2‘)
(3)
1
2
6
(or)
3
5
H3C
8
10
(4)
H3C
(or)
4
CH2
H3C
9
(5)
CH2
H3C
CH2
H3C
(6)
CH2
H3C
CH2
(7)
CH2
H2C
H3C
CH2
H3 C
CH2
(8)
CH3
H3C
CH2
Fig. 5 p-Menthanes in the oil glands of Oribatida and Astigmata.
(1–3) p-Menthanes and substitution patterns in the secretion of adult
Nothrus palustris: p-1,8-menthadien-5-yl-compounds. p-1,8-Menthadien-5-yl formates (1, 2) are the major p-menthanes in N. palustris,
showing a formyl group in position 5 (following the p-1,8-menthadien-nomenclature). By NMR, the absolute stereochemistry of the
formates (1 or 1’; 2 or 2’) remained open. p-1,8-Menthadien-5-ol
(3) was tentatively identified on the basis of its mass spectrum and
proposed to represent the corresponding alcohol to the p-1,8-men-
thadien-5-yl formates. All compounds rely on a p-1,8-menthadien5-yl-structure. 4–8) p-Menthanes and derivatives in the Astigmata:
p-1,8-menthadien-3-yl-compounds. Limonene* (p-1,8-menthadiene), isopiperitenol (p-1,8-menthadien-3-ol), (S)-isopiperitenone
(p-1,8-menthadien-3-one) (4), robinal, and isorobinal have been
reported as astigmatid oil gland constituents (see Kuwahara 2004 and
text). Apart from limonene, all compounds rely on a p-1,8-menthadien-3-yl-structure. *Limonene is the backbone-structure for both,
p-1,8-menthadien-3-yl- and p-1,8-menthadien-5-yl-compounds
p-1,5,8-menthatriene) did not fit to our compounds. For
p-1,4,8-menthatriene, we found a reference spectrum, but
no retention index (Thomas and Bucher 1970). Since all
the prominent ions in the spectra of compounds I and II (at
m/z 134, 119, and 91) are rather unspecific hydrocarbon
ions, arising from M+ (C 10H14+), M-CH 3 (C 9H11+), and
M-C3H7 (C7H7+), this pattern is also found in the other
components, such as cymols and in oxygenated compounds as carenol.
On the other hand, menthanes with M+ at m/z 152, as in
compound III, generally possess one oxygen, and likely represent p-menthadienols of formula C10H16O. Our assumption for the compound III was p-mentha-1,8-dien-5-ol
(= 6-isopropenyl-3-methyl-cyclohex-3-en-1-ol), for which
no viable references are available. This molecule consists
of a limonene backbone with a hydroxy group in position
5, analogously to the formates B and C. We, nevertheless,
compared published spectra and retention indices for various
other p-1,8-menthadienols to our compound III, not finding
correspondence. Reference data for p-1,8-menthadienols
were from the NIST webbook (including p-1,8-menthadien4-ol; p-1,8-menthadien-6-ol = carveol; p-1,8-menthadien7-ol; p-menthadien-9-ol) as well as from literature sources
(e.g., cis- and trans carveol: Garneau et al. 1997; p-1,8-menthadien-3-ol = isopiperitenol: Lücker et al. 2004). Generally, p-1,8-menthadien-5-ol appears to be a rare compound
in nature: we only found one old report on its occurrence,
namely in the oil of a particular population of lemongrass
Cymbobogon flexuosus (Naves 1961). A rudimentary mass
spectrum for trans- and cis-p-1,8-menthandien-5-ol (showing the three most prominent ions only and not sufficient for
a comparison) is given by Brenna et al. (2006).
The remaining menthane compounds in N. palustris, i.e.,
those with M+ at m/z 180 (compounds IV and V), may contain one or two oxygen atoms. Regarding the fragmentation
of the p-mentha-1,8-dien-5-yl formates B and C, the neutral
loss of formic acid from the molecular ion at m/z 180 leads
to fragment m/z 134 (M+—HCOOH) and indicates two oxygens present in the molecule. Compounds IV and V showed
spectra very similar to compounds B and C, possibly—but
not indicatively—representing formates as well but with the
formyl group in a different position. Their amounts were
too low for a final identification by NMR. Compounds IV
and V again exhibited intense ions at m/z 134 (C10H14+),
but additional ions at m/z 137 (C9H13O+) as well. A loss of
CH2COH from a molecular ion at m/z 180 (M-43) theoretically leads to m/z 137 (indicating two oxygens in the original
molecule), whereas a loss of C3H7 would lead to m/z 137 as
well (indicating one oxygen in the original molecule).
13
Menthane functions, occurrence, and biosynthesis
Generally, p-menthanes rarely predominate in arthropod
exocrine secretions, but some compounds of this group such
as limonene, terpinene, and carvone are found in several
insects, arachnids, and some millipedes (e.g., Blum 1981).
After chemo‑metamorphosis: p‑menthane monoterpenoids characterize the oil gland secretion…
While mostly acting as repellents, some p-menthanes in the
secretions of Astigmata show pheromonal properties (Kuwahara et al. 1987; Mizoguchi et al. 2005). The biological functions of menthanes in Nothrus remain unstudied for the time
being. On the other hand, p-menthanes in plants are frequent
and abundant, characterizing the oils of plants of different
families, such as Lamiaceae, Apiaceae, Rutaceae, and Eucalyptae (e.g., Lange 2015; Bergmann and Phillips 2021).
Geranial, the major component in the juvenile stages of
N. palustris, is considered to arise via the mevalonate pathway that is well described for terpene-producing Oribatida
(Brückner et al. 2022). In this pathway, geranyl pyrophosphate (GPP) represents a central intermediate from which
various terpenes can be built. Regarding geranial, GPP has
to be hydrolyzed into geraniol and pyrophosphate, and geranial may be produced by enzymatic oxidation of geraniol.
In case of menthanes, it is generally limonene that is produced from GPP by limonene synthetases (e.g., Bergmann
and Phillips 2021). For oxygenated p-menthanes, a hydroxy
group is introduced into limonene, and subsequently, p-menthadienols and also p-menthadienyl formates can be built.
Schempp et al. (2021), for instance, describe a limonene5-hydroxylase that adds a hydroxy group to (S)-limonene,
thus leading to the formation of trans-p-1,8-menthadien5-ol. In adult N. palustris, p-menthane monoterpenoids most
likely also originate from the mevalonate pathway. Depending on the expression of appropriate sets of enzymes in juveniles and adults, we assume a split of the biosynthetic route
at the level of geranyl pyrophosphate, which leads to geranial in juveniles but to p-menthanes in adults, and finally to
juvenile–adult polymorphism of oil gland secretions in this
particular species. We here call the phenomenon of changes
in the composition of an exocrine secretion during ontogeny
“chemo-metamorphosis”.
Juvenile–adult polymorphism:
chemo‑metamorphosis
Geranial is a monoterpene compound frequently found in
the oil glands of middle-derivative Oribatida and Astigmata.
Besides neral, neryl formate, γ-acaridial, and 2,6-HMBD,
it constitutes one of the so-called “astigmatid compounds”
(e.g., Sakata and Norton 2001; Raspotnig 2010) that are
characteristic of the oil gland secretions of middle-derivative Oribatida and Astigmata. No chemo-metamorphosis
has been described for these groups, with juveniles basically
producing the same secretions as adults (see introduction).
Some derivative mixonomatans, such as some Euphthiracaroidea, already show an expanded, partly renewed compound-repertoire, including various iridoid monoterpenes.
Interestingly, also these newly added compounds equally
occur in both juveniles and adults (Raspotnig et al. 2008).
For some Desmonomata, however, and especially for latederivative oribatid groups such as Brachypylina, an increasing number of examples for chemo-metamorphosis has been
reported. N. palustris is only one of these examples, and possibly one of the rather early derivative examples of chemometamorphosis in the Oribatida. According to the oribatid
catalog of Subias (2004; updated 2022), the Nothridae comprises about 100 species in three genera, with 87 species
described for Nothrus. Notably, our preliminary data indicate that chemo-metamorphosis does not affect all species of
genus Nothrus neither all Nothridae: initial analyses of the
oil glands of several other Austrian species of Nothrus show
a widespread production of geranial irrespective of ontogenetic state, i.e., including adults (Raspotnig, unpublished).
This makes a generalized phenomenon of chemo-metamorphosis in the Nothridae unlikely. Chemo-metamorphosis
also occurs in the Hermanniidae and later on in a number of
late-derivative Brachypylina. In Hermannia convexa (C.L.
Koch 1839), for instance, chemo-metamorphosis is present
in an extreme form, even affecting the morphology of oil
glands: juveniles produce astigmatid compounds from large
oil glands, whereas the glands of adults become inactive
and degenerate (Raspotnig et al. 2005a). The currently most
striking example for chemo-metamorphosis may be found in
species of Scheloribates (Berlese 1908) (Brachypylina, i.e.,
late-derivative Oribatida) which produce geranial as juveniles, but alkaloids as adults (Takada et al. 2005). In terms of
Ernst Haeckel’s biogenetic law and his theory of recapitulation, respectively (Haeckel 1866: “ontogeny recapitulates
phylogeny”), Scheloribates-juveniles might be considered
to express phylogenetically ancient oil gland secretion characters which are replaced by a completely novel chemistry
in adults. A comparable situation of chemo-metamorphosis
may be true for many or even all alkaloid-producing taxa,
thus for many Oripodoidea (e.g., Saporito et al. 2015).
Generally, the phenomenon of juvenile–adult polymorphism of exocrine secretions is not rare in arthropods, but
currently available data are biased: irrespective of taxonomic
group, mostly adult individuals have been investigated, and
the ontogeny of secretion chemistry is only known for a
minority of species. Chemo-metamorphosis, however, is
known to occur in a number of insects (e.g., in bugs, see
Aldrich 1988 for an overview), some polydesmidan millipedes (Kuwahara et al. 2015, 2019), exceptionally in
Julida (Bodner and Raspotnig 2012), as well as in a few
arachnids. In harvestmen, for instance, a first instance of
juvenile–adult polymorphism of defensive secretions has
recently been published (Raspotnig et al. 2022). Up to now,
only a few species of late-derivative oribatid groups have
been chemically investigated, and apart from Scheloribates,
Hermannia Nicolet 1855, and Nothrus, investigations exclusively included adult individuals (e.g., Liacaridae: Raspotnig
and Leis 2009; Brückner et al. 2015; Oripodoidea: Saporito
13
G. Raspotnig et al.
et al. 2015; Brückner et al. 2017; Euphthiracaridae: Heethoff
et al. 2018). We here hypothesize that chemo-metamorphosis is an adaptive trait in Oribatida that evolved somewhen in
derivative Desmonomata, possibly to accommodate different
ecological requirements of adults.
Funding Open access funding provided by Austrian Science Fund
(FWF). This work was supported by the Austrian Science Fund
(FWF), under Project Nos. P33629-BBL and P33840-B. MB was partly
financed by the Styrian government, under Grant No. PN 37.
Declarations
Conflict of interest The authors have no financial interests. GR designed the study, collected individuals, evaluated the data, and wrote
the manuscript. Further collections, material preparation such as extraction of individuals for GC–MS, and NMR were performed by MB
and DF; DF also prepared the DMDS derivatives. JB synthesized relevant reference compounds. OK conducted and evaluated the NMR
analyses and wrote the corresponding paragraphs. MB prepared the
specimens for SEM, and ES and MB took the scanning electron micrographs. All authors read and approved the final manuscript. The
authors have no competing interests to declare that are relevant to the
content of this article.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
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