After chemo-metamorphosis: p-menthane monoterpenoids characterize the oil gland secretion of adults of the oribatid mite, Nothrus palustris

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.

The secretions of oil glands have mainly been studied in early-and middle-derivative Oribatida (e.g., Norton 2001, 2003;Raspotnig et al. 2001Raspotnig et al. , 2004Raspotnig et al. , 2005aRaspotnig et al. , b, 2008Heethoff 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; Communicated by Marko Rohlfs. 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. 2004Heethoff 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(Kuwahara , 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 C 21 -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 (CDCl 3 ) 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 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 1 H, 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 investigated with a Hitachi FlexSEM1000 (Tokyo, Japan) using 20 kV acceleration voltage in the high-vacuum mode.

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-dehydro-1,8-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.
We indeed observed a molecular ion an m/z 480 (= 292 plus 4 × SCH 3 ) along with characteristic losses of SCH 3 -groups from M + at m/z 432 (M + -48), m/z 386   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 CH 2 -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 ( . This data together clearly indicated a doubly unsaturated C 21 -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 Table 3 Localization of double-bond position: diagnostic ions in the DMDS derivative of heneicosadiene (peak D) Diagnostic fragments were interpreted following Carlson et al. (1988), as outlined in detail in the text and shown in Fig. 4  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.
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 C 12 H 20 O or C 11 H 16 O 2 , respectively.
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 (C 10 H 14 + ); whereas the fragment at m/z 137 contained one oxygen (C 9 H 13 O + ). 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;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 10 H 14 + ), M-CH 3 (C 9 H 11 + ), and M-C 3 H 7 (C 7 H 7 + ), this pattern is also found in the other components, such as cymols and in oxygenated compounds as carenol.
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 (C 10 H 14 + ), but additional ions at m/z 137 (C 9 H 13 O + ) as well. A loss of CH 2 COH 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 C 3 H 7 would lead to m/z 137 as well (indicating one oxygen in the original molecule).

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).
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 limonene-5-hydroxylase that adds a hydroxy group to (S)-limonene, thus leading to the formation of trans-p-1,8-menthadien-5-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(Kuwahara et al. , 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 ). 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: Brückner et al. 2015;Oripodoidea: Saporito 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.

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.
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