Next Article in Journal
Determination and Pharmacokinetics of Di-(2-ethylhexyl) Phthalate in Rats by Ultra Performance Liquid Chromatography with Tandem Mass Spectrometry
Previous Article in Journal
Oxetane Synthesis through the Paternò-Büchi Reaction
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Harvestman Phenols and Benzoquinones: Characterisation and Biosynthetic Pathway

1
Instituto de Química, Universidade Estadual de Campinas, C.P. 6154, 13083-970 Campinas, SP, Brasil
2
Laboratório de Química Bio-Orgânica, Departamento de Química, Universidade Federal de São Carlos, Caixa Postal 676, 13565-905 São Carlos, SP, Brasil
3
Departamento de Ecologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, trav.14, no. 321, 05508-090 São Paulo, SP, Brasil
*
Author to whom correspondence should be addressed.
Molecules 2013, 18(9), 11429-11451; https://doi.org/10.3390/molecules180911429
Submission received: 19 July 2013 / Revised: 5 September 2013 / Accepted: 6 September 2013 / Published: 16 September 2013
(This article belongs to the Section Natural Products Chemistry)

Abstract

:
Benzoquinones are usually present in arthropod defence exudates. Here, we describe the chemical profiles of 12 harvestman species belonging to the neotropical family Gonyleptidae. Nine of the studied species produced benzoquinones, while three produced alkyl phenols. Two benzoquinones and one phenol exhibited biological activity against bacteria and fungi. We also studied the biosynthesis of 2-ethyl-1,4-benzoquinone by feeding Magnispina neptunus individuals with 13C-labelled precursors; the benzoquinones were biosynthesised through a polyketide pathway using acetate and propionate building blocks.

1. Introduction

Opiliones, which are commonly known as harvestmen or daddy longlegs, compose a large arachnid order with approximately 6,500 species widespread across the World [1]. The large neotropical family Gonyleptidae is chemically and morphologically diverse, comprising nearly 820 species [2]. Gonyleptid scent gland exudates are mainly composed of vinyl ketones and their hetero-Diels-Alder adducts [3,4,5], alkyl phenols [3,6,7,8,9,10] and benzoquinones [3,6,11,12,13,14].
In addition to harvestmen, naturally occurring 1,4-benzoquinones with great structural variety are also found in bacteria, plants and other arthropod orders [15,16]. They are known to be toxic and therefore are employed by beetles [17,18,19,20], earwigs [21], termites [22] and harvestmen [12,13] as a defence against natural predators. Additionally, their antimicrobial activity protects cockchafer larvae [23] and adult harvestmen [11] against pathogens, such as bacteria and fungi. The biosynthesis of 1-hepten-3-one produced by the harvestman Iporangaia pustulosa (Gonyleptidae) was recently described [24] as the condensation of one acetate and two propionate units following a polyketide pathway. However, there is no evidence of polyketide synthases (PKS) in harvestmen or in any other arthropod species studied so far [25]. These results claimed that additional evidence of PKS activities lay in the production of other classes of harvestman metabolites, such as benzoquinones.
We describe here the detailed chemical characterisation of the exudates for 12 gonyleptid species, which all contained mixtures of benzoquinones or alkyl phenols. Additionally, the biosynthetic pathway of the 2-ethyl-1,4-benzoquinone produced by Magnispina neptunus (Gonyleptidae) was investigatedusing 13C-labelled precursors.

2. Results and Discussion

2.1. Chemical Profile of Gonyleptid Exudates

The chemical compositions of 12 harvestman species—Bourguyia trochanteralis (Bourguyiinae), Chavesincola inexpectabilis, Magnispina neptunus (both Heteropachylinae), Discocyrtus oliverioi, Pachylus paessleri (both Pachylinae), Liogonyleptoides tetracanthus, Mischonyx cuspidatus (both Gonyleptinae), Metarthrodes longipes (Caelopyginae), Mitopernoides variabilis, Progonyleptoidellus striatus (both Progonyleptoidellinae), Multumbo terrenus (Hernandariinae) and Pachylospeleus strinati (Pachylospeleinae)—were investigated (Table 1). Nine of these species contained mainly mixtures of 1,4-benzoquinones in their scent gland exudate, while the other three produced alkyl phenols in higher abundance (Figure 1). All of the compounds have been characterised by mass spectrometry. Additionally, 2-methylbenzoquinone (6), 2-ethylbenzoquinone (7), 2,5-dimethylphenol (18) and 2,3,6-trimethylphenol (20) were fully characterised by 1H- and 13C-NMR spectroscopy. Co-elutions with synthetic standards were used to confirm the presence of 4-methyl-1-hepten-3-one (1) [5], 1-methyl-1,4-benzoquinone (6) and 2,5-dimethyl-1,4-benzoquinone (8). Additionally, 2,5-dimethyl-phenol (18) was compared to a previously characterised natural sample.

2.1.1. Benzoquinones Identification

The benzoquinones’ (6 to 13) mass spectra have intense molecular ions and characteristic fragmentation patterns, which feature CO and/or C2H2 loss (Figure 2) [26].
Table 1. Compounds detected in defensive secretion of gonyleptid harvestmen, ordered by retention index (RI).
Table 1. Compounds detected in defensive secretion of gonyleptid harvestmen, ordered by retention index (RI).
StructureRICharacteristic ions [m/z (abundance)]SpeciesRelative abundance
Molecules 18 11429 i001831112(M+,15), 97(12), 84(35), 83(12), 69(12), 58(28), 56(23), 55(100), 41(29) Pachylus paessleri0.9%
Molecules 18 11429 i002835114(M+,14), 85(10), 57(100), 41(14)Mischonyx cuspidatus7.0%
Molecules 18 11429 i003845 *72(64), 71(19), 70(17), 57(19), 55(14), 43(100), 41(21)Mischonyx cuspidatus0.2%
Molecules 18 11429 i004929128(M+, 3), 99(11), 86(60), 71(82), 57(100), 55(13), 43(64), 41(16)Mischonyx cuspidatus2.8%
Molecules 18 11429 i005956 *126(M+,26), 111(49), 97(100), 83(21), 69(43), 67(21), 56(26), 55(63), 43(73), 41(78)Pachylus paessleri0.1%
Molecules 18 11429 i0061010124(45), 123(27), 122(M+,100), 94(64), 82(55), 68(31), 66(46), 54(55), 40(24)Chavesincola inexpectabilis
Magnispina neptunus
10.1%
9.2%
Molecules 18 11429 i0071103136(M+,67), 123(16), 108(100), 107(42), 82(42), 80(18), 79(73), 77(15), 54(52), 53(30)Magnispina neptunus
Chavesincola inexpectabilis
90.8%
80.2%
Molecules 18 11429 i0081104138(14), 137(13), 136(M+,100), 108(25), 96(20), 80(19), 79(37), 68(67) Multumbo terrenus
Pachylus paessleri
Mischonyx cuspidatus
24.2
%10.3%
8.7%
Molecules 18 11429 i0091119136(M+,100), 108(47), 107(47), 82(39), 80(17), 79(41), 54(37), 53(16)Mischonyx cuspidatus
Bourguyia trochanteralis
Pachylospeleus strinati
Liogonyleptoides tetracanthus
Discocyrtus oliverioi
Pachylus paessleri
Chavesincola inexpectabilis
68.3%
65.0%
60.3%
58.6%
57.4%
53.2%
2.4%
Molecules 18 11429 i0101182150(M+,100), 135(10), 122(31), 121(16), 107(69), 82(20), 79(32), 77(16), 67(10), 54(18), 53(11)Discocyrtus oliverioi
Liogonyleptoides tetracanthus
Bourguyia trochanteralis
Pachylus paessleri
Chavesincola inexpectabilis
Mischonyx cuspidatus
Multumbo terrenus
41.4%
39.9%
17.1%
4.6%
2.7%
1.3%
0.9%
Molecules 18 11429 i0111197150(M+,100), 137(14), 122(45), 121(14), 107(41), 82(13), 79(54), 77(17), 68(24), 54(13), 53(19)Multumbo terrenus
Mischonyx cuspidatus
Pachylus paessleri
38.4%
9.3%
0.6%
Molecules 18 11429 i0121216150(M+,100), 122(35), 121(19), 107(55), 96(11), 79(39), 77(13), 68(29), 54(16), 53(14)Pachylospeleus strinati
Multumbo terrenus
Pachylus paessleri
Bourguyia trochanteralis
Mischonyx cuspidatus
Liogonyleptoides tetracanthus
39.7%
27.6%
27.2%
15.8%
1.5%
0.1%
Molecules 18 11429 i0131280164(M+,100), 136(23), 135(13), 121(82), 93(24), 91(15), 77(13), 68(18), 67(13)Multumbo terrenus
Pachylus paessleri
Bourguyia trochanteralis
8.9%
2.0%
1.3%
Molecules 18 11429 i0141409 *138(M+,58), 123(100), 107(4), 95(6), 67(10)Chavesincola inexpectabilis4.6%
Molecules 18 11429 i0151433 *138(M+,100), 137(29), 123(50), 95(12), 91(13)Pachylus paessleri
Bourguyia trochanteralis
Liogonyleptoides tetracanthus
Mischonyx cuspidatus
Discocyrtus oliverioi
1.1%
0.8%
0.8%
0.9%
0.5%
Molecules 18 11429 i0161467 *150(M+,100), 149(21), 121(17), 107(37), 77(15)Discocyrtus oliverioi
Liogonyleptoides tetracanthus
0.4%
0.1%
Molecules 18 11429 i0171487 *152(M+,53), 151(11), 138(9), 137(100), 107(8), 79(10), 77(8)Liogonyleptoides tetracanthus
Discocyrtus oliverioi
0.5%
0.3%
Molecules 18 11429 i0181138122(M+,100), 121(42), 107(96), 91(21), 79(15), 77(27)Progonyleptoidellus striatus
Mitopernoides variabilis
67.4%
24.5%
Molecules 18 11429 i0191172 *122(M+,91), 121(32), 107(100), 91(19), 79(16), 77(29)Progonyleptoidellus striatus1.3%
Molecules 18 11429 i0201220136(M+,81), 135(20), 121(100), 91(26), 77(13)Metarthrodes longipes
Progonyleptoidellus striatus
97.1%
31.3%
Molecules 18 11429 i0211230136(M+,43), 121(100), 91(15), 77(13)Mitopernoides variabilis75.5%
(C4H9)-phenol (22)1315 *150(M+,48), 135(100)Metarthrodes longipes2.9%
* RI calculated from linear regression from data from the other compounds (see experimental details).
Figure 1. Chromatograms of the harvestman exudates.
Figure 1. Chromatograms of the harvestman exudates.
Molecules 18 11429 g001
Figure 2. High-resolution mass spectra of benzoquinones from harvestman exudates.
Figure 2. High-resolution mass spectra of benzoquinones from harvestman exudates.
Molecules 18 11429 g002
The exudate of Ma. neptunus was analysed by CG/TOF-MS; benzoquinones 6 and 7 displayed molecular ions at m/z 122 and 136, respectively (Figure 2a,b). The presence of a radical ion at m/z 54 (54.0055 for 6 and 54.0078 for 7) is characteristic of a cyclopropenone radical ion 22 (calculated mass 54.0100), signalling the presence of an asymmetric benzoquinone with substituents on one side of the ring. The benzoquinone present in Ps. strinati exudate (Figure 2c) has a molecular ion at m/z 136 and could possess either structure 9 or 7. However, the presence of the two fragments at m/z 54.0067 (22) and at m/z 54.0427 (23) (calculated mass 54.0470) indicate that two methyl substituents are on the same side of the benzoquinone, confirming structure 9. Benzoquinone 12 (Figure 2d), however, has fragments at m/z 54.0430 (23), indicating the presence of two methyl groups on C-2 and C-3, and at m/z 68.0229 (24) (calculated mass 68.0257), indicating the presence of a methyl group at C-5. This rationale was utilised to suggest benzoquinone structures 8, 11 and 13, which also have methyl substituents at C-5.
The 1H-NMR analyses of Ma. neptunus exudate revealed signals consistent with benzoquinones 6 and 7 (Figure 3); the methyl hydrogens at 2.07 ppm corresponded to minor benzoquinone 6 and the triplet at 1.15 ppm, when combined with the quartet at 2.47 ppm, was typical of the ethyl substituent assigned to major benzoquinone 7 [13]. The 13C-NMR spectra completed the spectroscopic characterisations of 6 and 7, except for the assignments of the carbons at the positions C-1/C-4 and C-5/C-6 of 7. Full structural assignment required 2D NMR experiments, such as HSQC and HMBC (Table 2 and Supporting Information). These data were essential for the biosynthetic experiment described below.
Figure 3. 1H-NMR spectrum (400.13 MHz, CDCl3, TMS) of Magnispina neptunus exudate containing benzoquinones 6 and 7 as major components: a-signals refer to benzoquinone 6 and b-signals refer to benzoquinone 7.
Figure 3. 1H-NMR spectrum (400.13 MHz, CDCl3, TMS) of Magnispina neptunus exudate containing benzoquinones 6 and 7 as major components: a-signals refer to benzoquinone 6 and b-signals refer to benzoquinone 7.
Molecules 18 11429 g003
Table 2. NMR assignments for 2-methyl-1,4-benzoquinone (6) and 2-ethyl-1,4-benzo-quinone (7) (CDCl3, TMS, 400.13 MHz for 1H-NMR and 100.61 MHz for 13C-NMR).
Table 2. NMR assignments for 2-methyl-1,4-benzoquinone (6) and 2-ethyl-1,4-benzo-quinone (7) (CDCl3, TMS, 400.13 MHz for 1H-NMR and 100.61 MHz for 13C-NMR).
Molecules 18 11429 i022 Molecules 18 11429 i023
CδHδCaδHδCb
1-n.d. c (C)-187.5 (C)
2-n.d. c (C)-150.9 (C)
36.62 (1H, m)133.4 (CH)6.57 (1H, m)131.7 (CH)
4-n.d. c (C)-187.9 (C)
56.71 (1H, dd, 3J = 10 Hz; 4J = 2.25 Hz)136.5 (CH)6.71 (1H, dd, 3J = 10 Hz; 4J = 2.25 Hz)136.3 (CH)
66.77 (1H, d, 3J = 10 Hz)136.6 (CH)6.77 (1H, d, 3J = 10 Hz)136.8 (CH)
72.07 (3H, d, 4J = 1,5 Hz)15.8 (CH3)2.47 (2H, qd, 3J = 7.5 Hz; 4J = 1.5 Hz)22.1 (CH2)
8--1.15 (3H, t, 3J = 7.5 Hz)11.6 (CH3)
a The results from 13C-NMR (fully decoupled, DEPT-90 and DEPT-135); b The results from 13C-NMR (fully decoupled, DEPT-90 and DEPT-135), 2D NMR gCOSY (1H-1H) and gHSQC (1H-13C 1J) experiments; c not detected due to low abundance.
The 1,4-hydroquinones 1417 were detected in low abundance and identified by mass spectrometry using the peaks at m/z 77 and 91, which are typical of alkyl-substituted phenyl derivatives. Additionally, their intense molecular ions possessed two additional mass units when compared with the other corresponding major benzoquinones present in the same exudate (Figure 4). Some 1,4-hydroquinones are also reported for other gonyleptid species alongside their respective benzoquinones [3].
Figure 4. Benzoquinones and corresponding hydroquinones found in gonyleptid exudates.
Figure 4. Benzoquinones and corresponding hydroquinones found in gonyleptid exudates.
Molecules 18 11429 g004
The exudate of Mi. cuspidatus has already been studied [3] and the composition was reported to be benzoquinone 9 (46.8%) and its respective hydroquinone 15 (41.5%). In the specimens we studied, the exudate did contain benzoquinone 9, but only 0.9% of 15 was found. The exudate of Pa. paessleri has also been previously studied [14], and the reported composition was similar to ours, except for the presence of the ketone 1 (0.1%) and the hydroquinone 15 (1.1%), reported here for the first time.

2.1.2. Phenols Identification

Phenol derivatives were detected in Me. longipes, Mp. variabilis and Pr. striatus. The exudates of Pr. striatus were clean enough to be analysed by mass spectrometry and NMR spectroscopy. The mass fragmentation of 18 and 20 revealed intense molecular ions at m/z 122 and 136 and fragments at m/z 77 and 91, which are typical of substituted aromatic rings (Figure 5).
Figure 5. Mass spectra of the alkyl phenols 18 and 20 from Progonyleptoidellus striatus.
Figure 5. Mass spectra of the alkyl phenols 18 and 20 from Progonyleptoidellus striatus.
Molecules 18 11429 g005
The NMR analysis of Pr. striatus revealed the expected methyl substituents and the substitution pattern of 18 (Figure 6a, Table 3). However, the structure of 20 could either be 2,3,6-trimethylphenol or 2,3,4-trimethylphenol. Differential Nuclear Overhauser Effect (NOE) experiments irradiating the aromatic hydrogens at 6.66 and 6.86 ppm enhanced the neighbouring methyl signals 8b (δ2.24) and 9b (δ2.22), respectively (Figure 6b, Table 3). Therefore, the structure of 2,3,6-trimethylphenol, in which the aromatic hydrogens are spatially next to both methyl groups, was assigned.
Figure 6. (a) 1H-NMR spectrum (499.89 MHz, CDCl3, TMS) of the harvestman Progonyleptoidellus striatus exudate containing phenols 18 and 20 as major components; (b) Differential NOE NMR experiments of Pr. striatus exudate.
Figure 6. (a) 1H-NMR spectrum (499.89 MHz, CDCl3, TMS) of the harvestman Progonyleptoidellus striatus exudate containing phenols 18 and 20 as major components; (b) Differential NOE NMR experiments of Pr. striatus exudate.
Molecules 18 11429 g006
Table 3. NMR assignments of 2,5-dimethylphenol (18) and 2,3,6-trimethylphenol (20) (CDCl3, TMS, 499.89 MHz for 1H-NMR and 125.71 MHz for 13C-NMR).
Table 3. NMR assignments of 2,5-dimethylphenol (18) and 2,3,6-trimethylphenol (20) (CDCl3, TMS, 499.89 MHz for 1H-NMR and 125.71 MHz for 13C-NMR).
Molecules 18 11429 i024 Molecules 18 11429 i025
ХδHδCaδHδCa
1-153.8 (C)-152.1 (C)
2-120.6 (C)-n.d. b
36.99 (1H, d, 3J = 7.6 Hz)131.0 (CH)-n.d. b
46.66 (1H, d, 3J = 7.6 Hz)121.6 (CH)6.66 (1H, d, 3J = 7.6 Hz)121.9 (CH)
5-137.3 (C)6.86 (1H, d, 3J = 7.6 Hz)127.6 (CH)
66.60 (1H, s)115.8 (CH)-n.d. b
72.20 (3H, s)15.5 (CH3)2.16 (3H, s)11.9 (CH3)
82.27 (3H, s)21.2 (CH3)2.24 (3H, s)20.2 (CH3)
9--2.22 (3H, s)16.1 (CH3)
a The results from 13C-NMR (fully decoupled, DEPT-90 and DEPT-135); b not detected due to the low abundance of this minor compound.
The mass spectrum of 2-methyl-4-phenol (21) has an intense fragment at m/z 121, corresponding to a methyl loss, which is typical from an ethyl substituent. This identity was confirmed by co-elution with the exudate of the harvestman Hoplobunus mexicanus (Stygnopsidae), which was fully characterised by NMR spectroscopy [10].
Within the gonyleptid family, the occurrence of additional phenol derivatives has already been reported for some species, including Pachyloidellys goliath [8], Daguerreia inermis (both Pachylinae) [3], Camarana flavipalpi [9] and Pseudopachylus longipes (both Tricommatinae) [3]. Alkyl phenols are also found in species belonging to other families, such as Cynorta astora (Cosmetidae), which produces 19 and 21 [6], and Hoplobunus mexicanus (Stygnopsidae), which produces 18 and 21 [10].

2.2. Antimicrobial Activity of Harvestman Benzoquinones and Phenols

Benzoquinones 6 (from C. inexpectabilis and Ma. neptunus), 8 (from Mu. terrenus, Pa. paessleri and Mi. cuspidatus) and phenol 18 (from Ps. striatus and Mp. variabilis) were tested against representative pathogenic microorganisms: a Gram-positive bacterium (Bacillus pumilus), a Gram-negative bacterium (Pseudomonas aeruginosa) and yeasts (Candida albicans and Rhodotorula glutinis). The disc diffusion method was applied to obtain the average inhibitory concentrations (data not shown), which were further evaluated in microtiter plates; MIC values are reported in Table 4 (see plates pictured in the Supporting Information).
Table 4. Minimal inhibitory concentration values (MIC) for harvestman scent gland components.
Table 4. Minimal inhibitory concentration values (MIC) for harvestman scent gland components.
MicroorganismMIC (µg/mL)
Molecules 18 11429 i026 Molecules 18 11429 i027 Molecules 18 11429 i028
Bacillus pumilus<125<125250
Pseudomonas aeruginosa<125<1251000
Candida albicans125<82.5>500
Rhodotorula glutinis<82.5<82.5<82.5
Phenol 18 has lower biological activity than benzoquinones 6 and 8 against all tested microorganisms, except for R. glutinis. The biological activities of benzoquinones 6 and 8 were very similar against all tested microorganisms, except for C. albicans. The biological activity of the benzoquinones has been assigned to the electrophilic character of the conjugated double bond [27].
Benzoquinone 6 and its corresponding hydroquinone were tested together with other benzoquinone derivatives against Bacillus subtilis, Micrococcus luteus, and C. albicans,showing moderated antimicrobial activity [28]. Ruther and coworkers [23] also found 6 had antimicrobial activity against Escherichia coli, Saccharomyces cerevisiae, and the entomopathogenic fungi Metarhizium anisopliae and Beauveria brongniartii.
The antimicrobial activity of phenolic compounds was attributed to membrane permeabilisation, followed by cellular damage [29,30]. A set alkyl phenols were tested against oral bacteria revealing that the ortho-substituents on the phenolic groups decreased the biological activity [31]. In fact, o-phenol 18 displayed higher MIC values (Table 4). The dramatic MIC difference displayed by 18 against Gram-positive and Gram-negative bacteria is caused by the lipophilicity of this compound. Therefore, Gram-negative bacteria such as P. aeruginosa, have up to 25% of lipidic content and can retain the liposoluble phenols, while Gram-positive species, such as B. pumilus have 0% to 3%, therefore allowing the phenol to cause cellular membrane damage [31].

2.3. Biosynthetic Study of Magnispina neptunus Benzoquinone

In a previous report, we described the polyketide pathway for a harvestman vinyl ketone [24], in which the polyketide chain was composed of Pr+Ac+Pr units. To investigate whether this biosynthetic pathway was also present in the quinone producing harvestmen, we selected Ma. neptunus as a model species. We added [1-13C]acetate and [4-13C]methylmalonate as precursors incorporated into the diet of the individuals, and the labelling of 7 was monitored by 13C-NMR spectroscopy. The observed enrichment was typical of an aromatic polyketide pathway [32], yielding 13C labelling at C-2, C-4, and C-6 of 7, which are alternating carbons on the benzoquinone ring (Figure 7, Scheme 1).
Figure 7. 13C-NMR spectra (CDCl3) of 2-ethyl-1,4-benzoquinone (7) after the feeding experiment with Magnispina neptunus individuals. Black arrow: control signal; blue arrow: enriched positions. Balls on the structures indicate enriched positions.
Figure 7. 13C-NMR spectra (CDCl3) of 2-ethyl-1,4-benzoquinone (7) after the feeding experiment with Magnispina neptunus individuals. Black arrow: control signal; blue arrow: enriched positions. Balls on the structures indicate enriched positions.
Molecules 18 11429 g007
Feeding the individuals [4-13C]methylmalonate enriched 7 only at C-8 (Figure 7), which is consistent with the incorporation of a propionyl-CoA starter unit (Scheme 1). This labelling pattern also indicates that the alternative catabolism of propionate to acetate via 3-hydroxypropionate [33] does not occur in Ma. neptunus because the positions corresponding to acetate units were not enriched. The same was effect was observed for the vinyl ketone pathway in the harvestman Iporangaia pustulosa [24].
[1-13C]acetate incorporation enriched C-2, C-4 and C-6 of 7. Positions C-4 and C-6 are clearly labelled due to malonate incorporation. The third extender unit loses its labelled carbon, leaving only the non-labelled carbon at C-3. The unexpected enrichment at C-2 is consistent with the incorporation of a propionate unit and the conversion of [1-13C]acetate into [1-13C]propionate via succinyl-CoA with a methylmalonyl-CoA mutase, as reported for the harvestman I. pustulosa [24]. However, the C3 label scrambling observed in I. pustulosa was not present in biosynthetic pathway of 7 for Ma. neptunus, suggesting a simpler propionate metabolism for this species.
Scheme 1. (a) Biosynthetic route for 7 in the harvestman Magnispina neptunus; (b) Labelling pattern of 7 after feeding a labelled precursor to Ma. neptunus individuals. Black balls indicate the enriched positions, and the red ball indicates unexpected enrichment.
Scheme 1. (a) Biosynthetic route for 7 in the harvestman Magnispina neptunus; (b) Labelling pattern of 7 after feeding a labelled precursor to Ma. neptunus individuals. Black balls indicate the enriched positions, and the red ball indicates unexpected enrichment.
Molecules 18 11429 g008
The labelling at C-5 by [1-13C] acetate was also unexpected (Scheme 1) because it corresponds to the C-2 of the incorporated malonate unit. The enrichments at C-4 and C-5, which belong to the same acetate unit, are identical (1.3%), excluding the possibility of a measurement error. On the other hand, C-2 and C-6 belong to two C2 independent units with 1.6% and 2.2% of enrichment, respectively. Considering that the three C2 extender units are malonate, their metabolism should be similar. Therefore, this scrambled labelling suggests that the malonate biosynthesis has more than one route to produce and incorporate this unit onto a hypothetic domain of the PKS. Analogous results were reported for the I. pustulosa vinyl ketone biosynthesis [24].
Benzoquinone 6 is the minor constituent of the Ma. neptunus exudate and was not detected in the NMR spectra because it had a very low relative abundance. However, its biosynthetic pathway may be similar to 7, with an acetate starter unit rather than a propionate. The same rationale may be applied to the biosynthesis of the benzoquinones detected in the nine species described in this study, in which changes to the starter and extender units’ assembly provide a different polyketide chain, and therefore generate a set of benzoquinones with different substitution patterns (Scheme 2).
Meinwald and coworkers reported that both aromatic amino acids and carboxylic acids are precursors for unsubstituted 1,4-benzoquinones in the beetle Eleodes longicollis [34]. However, the incorporation data for the substituted benzoquinones, such as 6 and 7, are better accommodated in a polyketide pathway due to the exclusive incorporation of acetate and propionate as starter and extender units, respectively [34]. Sun and Toia studied the biosynthesis of 2,4-dihydroxyacetophenone in the ant Rhytidoponera chalybaea revealing the polyketide origin of this aromatic compound in insects [35].
The proposed benzoquinone biosynthetic routes in harvestmen occur via carbonyl enolysation of the cyclised polyketide chain, followed by a decarboxylation (Scheme 1), which is the final step for phenols 1821 from Me. longipes, Mp. variabilis and Pr. striatus (Scheme 2). Eisner and co-workers reported that benzoquinone 9 and phenol 21 from Zygopachylus albimarginis (Manaosbiidae) [6] had the same biosynthetic origin for both components. The production of benzoquinones, hydroquinones and phenols by the forked fungus beetle Bolitotherus cornutus also suggests that the alkyl benzoquinones and alkyl phenols might share a similar biosynthetic origin [36].
Scheme 2. Proposed biosynthetic routes for harvestman benzoquinones and phenols. Black balls indicate a broken acetate unit caused by benzoquinone decarboxylation.
Scheme 2. Proposed biosynthetic routes for harvestman benzoquinones and phenols. Black balls indicate a broken acetate unit caused by benzoquinone decarboxylation.
Molecules 18 11429 g009
In five of the 12 studied harvestman exudates, the benzoquinone and its corresponding 1,4-hydroquinone occur together (Figure 4). This feature provides additional evidence for the proposed biosynthetic route of phenol p-oxidation to provide 1,4-benzoquinones. A classic example of this mechanism is the bombardier beetle (Carabidae), which enzymatically oxidises 1,4-hydroquinones with a catalase producing 1,4-benzoquinones, water and heat [17,18,37]. Beetles of the family Tenebrionidae also produce 1,4-benzoquinones from 1,4-hydroquinone oxidation [15,19,20].
The alkyl and methoxybenzoquinones are also often components of millipede defensive secretions alongside their corresponding hydroquinones [38,39,40,41]. Some millipede species in the families Spirostreptidae and Harpagophoridae secrete benzoquinones 6 and 7, the hydroquinone 15 and several methoxy substituted benzo- and hydroquinones. According to our hypothesis, the putative biosynthetic route for these compounds is the p-oxidation of hydroquinones; however, there have been no labelling experiments using millipedes [39].
Based on these results, as well as on our previous report [24], it can be inferred that harvestmen catabolise propionate to form acetate via succinyl-CoA, followed by the TCA cycle, while insects oxidise the propionate to form acetate via 3-hydroxypropionate [33,42]. The biosynthetic pathway observed in harvestmen most likely relies on the participation of a methylmalonyl-CoA mutase, which is an enzyme exclusive to non-insect arthropods [42,43,44,45]. This feature appears to indicate a key metabolic difference between insects and other arthropods, such as arachnids. The labelling pattern found in the I. pustulosa biosynthetic study revealed that this species possesses a complex propionate metabolism, in which the labelling scrambling indicates different loadings of C3 starter and extender units [24]. The scrambling was also present in Ma. neptunus, but it was observed only for C2 extender unit incorporation.

3. Experimental

3.1. Chemical Profile of Harvestman Exudates

3.1.1. General Methods

The NMR spectra were acquired with either an 11 T Varian Inova instrument, operating at 499.88 MHz for 1H-NMR and 125.71 MHz for 13C-NMR, or a 5.87 T Bruker Avance DPX, at 250.13 MHz for 1H-NMR and 62.89 MHz for 13C-NMR. The solvent was CDCl3 and tetramethylsylane (TMS) was an internal reference (0.0 ppm). The chemical shifts (δ) are reported in ppm and coupling constants J are reported in Hz. The GC-MS analyses were performed using an Agilent 6890-5973 system with a DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 µm). The EIMS were recorded at 70 eV using 3.54 scans·s−1 from m/z 40 to 400. The oven temperature ranged from 50 to 200 °C at 10 °C·min−1 and subsequently to 290 °C at 16 °C·min−1. The natural samples were injected in splitless mode, while the synthetic samples were injected in split 1:10 mode. The injector temperature was 250 °C and the detector was maintained at 280 °C; helium was used as the carrier gas. The retention index (RI) [46] was determined using splitless injection mode and temperatures ranging from 50 to 290 °C at a rate of 4 °C·min−1 and 7.62 psi; an alkane standard solution C8-C20 (Fluka) was injected using the same program. The HREIMS were acquired using a Waters GCT premier at 20 scans·s−1, at a resolution of 7,000 FWHM, with sub-5 ppm RMS with an internal lock mass correction and electron impact (EI) at 70 eV. The Agilent 7683 operated with oven temperature ranging from 50 to 250 °C at 10 °C·min−1 and HP5-MS column with 30 m × 0.25 mm × 0.25 µm for GC analysis. The injection volume was 1 μL in splitless mode. The injector temperature was 270 °C and the detector was kept at 250 °C while using helium as the carrier gas.

3.1.2. Collection of Individuals

The individuals were collected in different places, most of them in the Atlantic Forest in southeastern (SE) Brazil (Table 5) during the wet and warm season (October to March), when the individuals of many gonyleptid species are more active. Individuals of the studied species were taken to the laboratory and kept alive in plastic vials containing a piece of wet cotton to maintain moisture. The scent gland exudates were collected by pressing the gland openings with cotton wool cleaned with bidistilled EtOAc. The liquid absorbed in the cotton wool was washed off with CDCl3 (2 mL) for NMR analyses before being eluted with EtOAc (2 mL) for GC-MS analyses. All solvents were of high analytical grade and were doubly distilled before use.
Table 5. Identity of the gonyleptid species used in this study. The column “Locality” indicates the places where the individuals were collected in the field and the column “Number of individuals” indicates the sample size used in the chemical analyses.
Table 5. Identity of the gonyleptid species used in this study. The column “Locality” indicates the places where the individuals were collected in the field and the column “Number of individuals” indicates the sample size used in the chemical analyses.
SpeciesLocalityNumber of individuals
BOURGOUYINAE
Bourguyia trochanteralisCananéia, São Paulo, SE Brazil22
CAELOPYGINAE
Metarthrodes longipesUbatuba, São Paulo, SE Brazil3
GONYLEPTINAE
Liogonyleptoides tetracanthusLinhares, Espírito Santo, SE Brazil9
Mischonyx cuspidatusCampinas, São Paulo, SE Brazil29
HERNANDARIINAE
Multumbo terrenusTeresópolis, Rio de Janeiro, SE Brazil30
HETEROPACHYLINAE
Chavesincola inexpectabilisSanta Tereza, Espírito Santo, SE Brazil31
Magnispina neptunusArraial D’Ajuda, Bahia, NE Brazil20
PACHYLINAE
Discocyrtus oliverioiCampinas, São Paulo, SE Brazil11
Pachylus paessleriSan Carlos de Apoquindo, Santiago, Chile24
PACHYLOSPELEINAE
Pachylospeleus strinatiIporanga, São Paulo, SE Brazil34
PROGONYLEPTOIDELINAE
Mitopernoides variabilisUbatuba, São Paulo, SE Brazil9
Progonyleptoidellus striatusSanto André, São Paulo, SE Brazil10

3.2. Antimicrobial Activity

Bacillus pumillus (LaBioSin collection) and Pseudomonas aeruginosa (CCT 1987) were cultured in Nutrient Broth (NB) (peptone 10 g, glucose 40 g and agar 15 g, and the volume completed to 1 L with distilled water). Candida albicans (CCT 0776) and Rhodotorula glutinis (CCT 0783) were cultured in yeast-malt extract (YM) Merck (yeast extract 3 g, malt-extract 3 g, peptone 5 g, glucose 10 g and agar 20 g, and the volume completed to 1 L with distilled water). The microorganisms were cultured in 10 mL of the medium for 24 h before the MIC experiment. Aqueous microorganism suspensions (100 µL, 1.5∙× 107 cells·mL−1) were added to the wells of a 96 titer plate. The bioactive compounds 6, 8 and 18 (100 µL) in final concentrations of 1,000, 500, 250 and 125 µg·mL−1 for bacteria and 500, 250, 125 and 82.5 µg·mL−1 for yeast, diluted in H2O/DMSO 95:5 (v/v) were added in the wells. Positive controls were prepared by substituting the test compounds by either chloramphenicol (4 mg·mL−1) for bacteria and ciclopiroxolamine (10 mg·mL−1) for the yeast. Negative controls were prepared using only the aqueous DMSO plus inoculum. The plates were incubated at 30 °C for 24 h. Aliquots of 20 µL of aqueous MTT (1 mg·mL−1) were added to the wells, and the reduction of the terazolium salt (yellow) to formazan (blue) by living cells was observed within 1 h. All of the tests were run in triplicates.

3.3. Biosynthetic Study of Magnispina neptunus

The individuals used in the biosynthetic study were collected at Arraial D’Ajuda, state of Bahia, northeastern Brazil. Before beginning the experiment, a dorso-ventral pressure was applied to all of the individuals to empty their gland sacs. The individuals were divided into two groups and fed with canned dog food containing 5% w/w of the labelled precursors: [1-13C]sodium acetate (Cambridge Isotope Laboratories, CIL, Tewksbury, MA, USA) (n = 40 individuals) and [4-13C]sodium methylmalonate (for synthetic procedure see [17]) (n = 33 individuals). The control group was the exudate extracted before initiating each experiment (n = 68 individuals). The experiment was set up over a period of 60 days, and the food was renewed every 48 h. The exudates were collected with dewaxed cotton wool and extracted from the cotton wool with CDCl3. 13C-NMR spectra of 7 were acquired with a Bruker Avance III 11 tesla operating at 125.75 MHz, 25 °C, acquisition time 1.1 s, 30° pulse, and approximately 40,000 scans, using equal scan numbers for samples within the same experiment (sample and control) [47,48].

4. Conclusions

The chemical characterisation of the 12 harvestman exudates provides important information related to the chemotaxonomy of the gonyleptid harvestmen. For three species studied here (L. tetracanthus, Me. longipes and Mp. variabilis), there was no previous chemical characterisation in the literature. The data for these three species, which were not included in the recent phylogeny of the Gonyleptidae, support the notion that the production of benzoquinones is plesiomorphic in the family [49]. Additionally, the production of alkyl-phenols evolved several times independently from the ancestral states of production of benzoquinones and vinyl ketones [49]. These frequent evolutionary transitions agree with the proposed biosynthetic route for benzoquinones, phenols and ketones, all of which begin from acetate and propionate units in a common polyketide pathway. Specifically, our studies with I. pustulosa [24] and Ma. neptunus indicate that these phylogenetically distant species partially share the biosynthetic pathway for vinyl ketones and benzoquinones, respectively (Scheme 3). Additionally, the scrambled labelling at specific positions of the polyketide chain also indicates that the biosynthetic routes are complex, offering several opportunities for the diversification of the molecules produced within both species.
Scheme 3. Biosynthetic pathway for vinyl ketone and benzoquinone in harvestmen.
Scheme 3. Biosynthetic pathway for vinyl ketone and benzoquinone in harvestmen.
Molecules 18 11429 g010
Most species that produce benzoquinones live on the ground, taking shelter under rocks or rotten logs [49]. In these types of habitats, individuals are likely to be in direct contact with many pathogenic microorganisms [50]. However, most species living on low and high vegetation produce phenols and ketones as the main constituents of their defensive exudates [24]. The results from our experiments concerning antimicrobial activity revealed that the minimal inhibitory concentration values for benzoquinones are consistently lower than for phenols, suggesting that the benzoquinones are more effective at deterring microorganisms. Therefore, the diversification of the chemical compounds in the defensive exudates of gonyleptid harvestmen may be at least partially explained by the differences in habitat-related uses among the species of different subfamilies.

Supplementary Materials

Supplementary materials can be accessed at: https://www.mdpi.com/1420-3049/18/9/11429/s1.

Acknowledgments

We acknowledge D. Caetano, B. Buzatto, R. Pinto-da-Rocha, C. Bragagnollo, R. Werneck, G. S. Requena, T. M. Nazareth and A. L. Guil for helping to collect the individuals, and CNPq, Petrobrás, and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-GM 08/06604-7) and FAPESP-VALE (AJM 10/51278-0) for financial support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Machado, G.; Pinto-da-Rocha, R.; Giribet, G. What are Harvestmen. In Harvestmen: The Biology of Opiliones; Pinto-Da-Rocha, R., Machado, G., Giribet, G., Eds.; Harvard University Press: Cambridge, MA, USA, 2007. [Google Scholar]
  2. Raspotnig, G. Scent gland chemistry and chemosystematics in harvestmen. Biol. Serbica 2012, 34, 5–18. [Google Scholar]
  3. Hara, M.R.; Cavalheiro, A.J.; Gnaspini, P.; Santos, D.Y.A.C. A comparative analysis of the chemical nature of defensive secretions of Gonyleptidae (Arachnida: Opiliones: Laniatores). Biochem. Syst. Ecol. 2005, 33, 1210–1225. [Google Scholar] [CrossRef]
  4. Rocha, D.F.O.; Hamilton, K.; Gonçalves, C.C.S.; Machado, G.; Marsaioli, A.J. 6-Alkyl-3,4-dihydro-2H-pyrans: Chemical secretion compounds in neotropical harvestmen. J. Nat. Prod. 2011, 74, 658–663. [Google Scholar] [CrossRef]
  5. Wouters, F.C.; Rocha, D.F.O.; Gonçalves, C.C.S.; Machado, G.; Marsaioli, A.J. Additional vinyl ketones and their pyranyl ketones in gonyleptid harvestmen (Arachnida: Opiliones) suggest that the hetero-Diels-Alder reaction is widespread in this family. J. Nat. Prod. 2013. [Google Scholar] [CrossRef]
  6. Eisner, T.; Jones, T.H.; Hicks, K.; Silberglied, R.E.; Meinwald, J. Quinones and phenols in the defensive secretions of neotropical opilionids. J. Chem. Ecol. 1977, 3, 321–329. [Google Scholar] [CrossRef]
  7. Duffield, R.M.; Olubajo, O.; Wheeler, J.W.; Shear, W.A. Alkylphenols in the defensive secretion of the nearctic opilionid, Stygnomma spinifera (Arachnida: Opiliones). J. Chem. Ecol. 1981, 7, 445–452. [Google Scholar] [CrossRef]
  8. Acosta, L.E.; Poretti, T.I.; Mascarelli, P.E. The defensive secretions of Pachyloidellus goliath (Opiliones, Laniatores, Gonyleptidae). Bonn. Zool. Beitr. 1993, 44, 19–31. [Google Scholar]
  9. Machado, G.; Pomini, A.M. Chemical and behavioral defenses of the neotropical harvestman Camarana flavipalpi (Arachnida: Opiliones). Biochem. Syst. Ecol. 2008, 36, 369–376. [Google Scholar] [CrossRef]
  10. Pomini, A.M.; Machado, G.; Pinto-da-Rocha, R.; Macías-Ordóñez, R.; Marsaioli, A.J. Lines of defense in the harvestman Hoplobunus mexicanus (Arachnida: Opiliones): Aposematism, stridulation, thanatosis, and irritant chemicals. Biochem. Syst. Ecol. 2010, 38, 300–308. [Google Scholar] [CrossRef]
  11. Estable, C.; Ardao, M.I.; Brasil, N.P.; Fieser, L.F. Gonyleptidine. J. Am. Chem. Soc. 1955, 77, 4942. [Google Scholar]
  12. Eisner, T.; Rossini, C.; Gonzalez, A.; Eisner, M. Chemical defense of an opilionid (Acanthopachylus aculeatus). J. Exp. Biol. 2004, 207, 1313–1321. [Google Scholar] [CrossRef]
  13. Machado, G.; Carrera, P.C.; Pomini, A.M.; Marsaioli, A.J. Chemical defense in harvestmen (Arachnida, Opiliones): Do benzoquinone secretions deter invertebrate and vertebrate predators? J. Chem. Ecol. 2005, 31, 2519–2539. [Google Scholar] [CrossRef]
  14. Föttinger, P.; Acosta, L.E.; Leis, H.; Raspotnig, G. Benzoquinone-rich exudates from the harvestman Pachylus paessleri (Opiliones: Gonyleptidae: Pachylinae). J. Arachnol. 2010, 38, 584–587. [Google Scholar] [CrossRef]
  15. Blum, M.S. Biosynthesis of arthropods exocrine compounds. Ann. Rev. Entomol. 1987, 32, 381–413. [Google Scholar] [CrossRef]
  16. Abraham, I.; Joshi, R.; Pardasani, P.; Pardasani, R.T. Recent advances in 1,4-benzoquinone chemistry. J. Braz. Chem. Soc. 2011, 22, 385–421. [Google Scholar] [CrossRef]
  17. Schildknecht, H.; Holoubek, K. Die bombardierkafer und ihre explosionschemie. Angew. Chem. 1961, 73, 1–7. [Google Scholar] [CrossRef]
  18. Eisner, T.; Jones, T.H.; Aneshansley, D.J.; Tschinkel, V.R.; Silberglied, R.E.; Meinwald, J. Chemistry of defensive secretions of bombardier beetles (Brachinini, Metriini, Ozaenini, Paussini). J. Insect Physiol. 1977, 23, 1383–1386. [Google Scholar] [CrossRef]
  19. Happ, G.M. Quinone and hydrocarbon production in the defensive glands of Eleodes longicollis and Tribolium castaneum (Coleoptera, Tenebrioidae). J. Insect Physiol. 1968, 14, 1821–1837. [Google Scholar] [CrossRef]
  20. Ikanl, R.; Cohen, E.; Shulov, A. Benzo- and hydroquinones in the defense secretions of Blaps sulcata and Blaps wiedemanni. J. Insect Physiol. 1970, 16, 2201–2206. [Google Scholar] [CrossRef]
  21. Eisner, T.; Rossini, C.; Eisner, M. Chemical defense of an earwig (Doru taeniatum). Chemoecology 2000, 10, 81–87. [Google Scholar] [CrossRef]
  22. Olagbemiro, T.O.; Lajide, L.; Sani, K.M.; Staddon, B.W. 2-Hydroxy-5-methyl-l,4-benzoquinone from the salivary gland of the soldier termites Odontotermes magdalenae. Experientia 1988, 44, 1022–1024. [Google Scholar] [CrossRef]
  23. Ruther, J.; Podsiadlowski, L.; Hilker, M. Quinones in cockchafers: Additional function of a sex attractant as an antimicrobial agent. Chemoecology 2001, 11, 225–229. [Google Scholar] [CrossRef]
  24. Rocha, D.F.O.; Wouters, F.C.; Machado, G.; Marsaioli, A.J. Alternative sources of propionate and methylmalonate in the biosynthesis of a vinyl ketone in the defensive secretion of an arachnid. Sci. Reports 2013. submitted for publication. [Google Scholar]
  25. Pankewitz, F.; Hilker, M. Polyketides in insects: Ecological role of these widespread chemicals and evolutionary aspects of their biogenesis. Biol. Rev. 2008, 83, 209–226. [Google Scholar] [CrossRef]
  26. Gross, J.H. Mass Spectrometry—A Textbook, 2nd ed.; Springer-Verlag: Heidelberg, Germany, 2011. [Google Scholar]
  27. El-Najar, N.; Gali-Muhtasib, H.; Ketola, R.A.; Vuorela, P.; Urtti, A.; Vuorela, H. The chemical and biological activities of quinones: Overview and implications in analytical detection. Phytochem. Rev. 2011, 10, 353–370. [Google Scholar] [CrossRef]
  28. Cole, L.K.; Blum, M.S.; Roncadori, R.W. Antifungal properties of the insect alarm pheromones, citral, 2-heptanone, and 4-methyl-3-heptanone. Mycologia 1975, 67, 701–708. [Google Scholar]
  29. Shapiro, S.; Guggenheim, B. The action of thymol on oral bacteria. Oral Microbiol. Immunol. 1995, 10, 241–246. [Google Scholar] [CrossRef]
  30. Tortora, G.J.; Funke, B.R.; Case, C.L. Microbiology: An Introduction, 10th ed.; Pearson Benjamin Cummings: San Francisco, CA, USA, 2010. [Google Scholar]
  31. Greenberg, M.; Dodds, M.; Tian, M. Naturally occurring phenolic antibacterial compounds show effectiveness against oral bacteria by a quantitative structure-activity relationship study. J. Agric. Food Chem. 2008, 56, 11151–11156. [Google Scholar] [CrossRef]
  32. Morgan, E.D. Biosynthesis in Insects, Advanced ed.; RSC: Cambridge, UK, 2010. [Google Scholar]
  33. Halarnkar, P.P.; Chambers, J.D.; Blomquist, G.J. Metabolism of propionate to acetate in nine insect species. Comp. Biochem. Physiol. 1986, 84, 469–472. [Google Scholar]
  34. Meinwald, J.; Koch, K.F.; Rogers, J.E., Jr.; Eisner, T. Biosynthesis of arthropod secretions. III. Synthesis of simple p-benzoquinones in a beetle (Eleodes 1ongicollis). J. Am. Chem. Soc 1996, 88, 1590–1592. [Google Scholar]
  35. Sun, C.M.; Toia, R.F. Biosynthetic studies on ant metabolites of Mellein and 2,4-dihydroxyacetophenone from [1,2–13C2] acetate. J. Nat. Prod. 1993, 56, 953–956. [Google Scholar] [CrossRef]
  36. Holliday, A.E.; Walker, F.M.; Brodie, E.D., III; Formica, V.A. Differences in defensive volatiles of the forked fungus beetle, Bolitotherus cornutus, living on two species of fungus. J. Chem. Ecol. 2009, 35, 1302–1308. [Google Scholar]
  37. Eisner, T. The protective role of the spray mechanism of the Bombardier beetle. J. Insect Physiol. 1958, 2, 215–220. [Google Scholar] [CrossRef]
  38. Eisner, T.; Alsop, D.; Hicks, K.; Meinwald, J. Defensive Secretions of Millipeds. In Handbook of Experimental Pharmacology; Bettini, S., Ed.; Springer: Berlin, Germany, 1978; Volume 48, pp. 41–72. [Google Scholar]
  39. Deml, R.; Huth, A. Benzoquinones and hydroquinones in defensive secretions of tropical millipedes. Naturwissenschaften 2000, 87, 80–82. [Google Scholar] [CrossRef]
  40. Wu, X.; Buden, D.W.; Attygalle, A.B. Hydroquinones from defensive secretion of a giant Pacific millipede, Acladocricus setigerus (Diplopoda: Spirobolida). Chemoecology 2007, 17, 131–138. [Google Scholar] [CrossRef]
  41. Vujisić, L.V.; Makarov, S.E.; Ćurčić, B.P.M.; Ilić, B.S.; Tešević, V.V.; Gođevac, D.M.; Vučković, I.M.; Ćurčić, S.B.; Mitić, B.M. Composition of the defensive secretion in three species of european millipedes. J. Chem. Ecol. 2011, 37, 1358–1364. [Google Scholar]
  42. Halarnkar, P.P.; Chambers, J.D.; Wakayama, E.J.; Blomquist, G.J. Vitamin B12 levels and propionate metabolism in selected non-insect arthropods and other invertebrates. Comp. Biochem. Physiol. 1987, 88, 869–873. [Google Scholar]
  43. Chu, A.J.; Blomquist, G.J. Biosynthesis of hydrocarbons in insects: Succinate is a precursor of the methyl branched alkanes. Arch. Biochem. Biophys. 1980, 201, 304–312. [Google Scholar]
  44. Wakayama, E.D.; Dillwith, J.W.; Howard, R.W.; Blomquist, G.J. Vitamin B12 levels in selected insects. Insect Biochem. 1984, 14, 175–179. [Google Scholar]
  45. Halarnkar, P.P.; Blomquist, G.J. Comparative aspects of propionate metabolism. Comp. Biochem. Physiol. 1989, 92B, 227–231. [Google Scholar]
  46. Van den Dool, H.; Kratz, P.D. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr. A 1963, 11, 463–471. [Google Scholar]
  47. Schneider, B. Nuclear magnetic resonance spectroscopy in biosynthetic studies. Prog. Nucl. Magn. Reson. Spectrosc. 2007, 51, 155–198. [Google Scholar]
  48. Maier, W.; Shneider, B.; Strack, D. Biosynthesis of sesquiterpenoid cyclohexenone derivatives in mycorrhizal barley roots proceeds via glyceraldehyde 3-phosphate/pyruvate pathway. Tetrahedron Lett. 1998, 39, 521–524. [Google Scholar] [CrossRef]
  49. Caetano, D.S.; Machado, G. The ecological tale of Gonyleptidae (Arachnida, Opiliones) evolution: Phylogeny of a Neotropical lineage of armoured harvestmen using ecological, behavioural and chemical characters. Cladistics 2013. [Google Scholar] [CrossRef]
  50. Cokendolpher, J.C.; Mitov, P.G. Natural Enemies. In Harvestmen: The Biology of Opiliones; Pinto-Da-Rocha, R., Machado, G., Giribet, G., Eds.; Harvard University Press: Cambridge, MA, USA, 2007. [Google Scholar]
  • Sample Availability: Samples of the compounds 6 and 8 are available from the authors.

Share and Cite

MDPI and ACS Style

Rocha, D.F.O.; Wouters, F.C.; Zampieri, D.S.; Brocksom, T.J.; Machado, G.; Marsaioli, A.J. Harvestman Phenols and Benzoquinones: Characterisation and Biosynthetic Pathway. Molecules 2013, 18, 11429-11451. https://doi.org/10.3390/molecules180911429

AMA Style

Rocha DFO, Wouters FC, Zampieri DS, Brocksom TJ, Machado G, Marsaioli AJ. Harvestman Phenols and Benzoquinones: Characterisation and Biosynthetic Pathway. Molecules. 2013; 18(9):11429-11451. https://doi.org/10.3390/molecules180911429

Chicago/Turabian Style

Rocha, Daniele F. O., Felipe C. Wouters, Dávila S. Zampieri, Timothy J. Brocksom, Glauco Machado, and Anita J. Marsaioli. 2013. "Harvestman Phenols and Benzoquinones: Characterisation and Biosynthetic Pathway" Molecules 18, no. 9: 11429-11451. https://doi.org/10.3390/molecules180911429

Article Metrics

Back to TopTop