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. 2025 Jul 2;51(4):72. doi: 10.1007/s10886-025-01623-0

GC-MS Analysis and Antimicrobial Properties of Defensive Secretions from the Millipede Coxobolellus saratani (Diplopoda: Spirobolida: Pseudospirobolellidae)

Piyatida Pimvichai 1,✉, Warinthan Jumpajan 1, Phikun Buaboon 1, Waraporn Sutthisa 1, Nattawadee Nantarat 2, Thierry Backeljau 3,4
PMCID: PMC12222413  PMID: 40601120

Abstract

The defensive secretions of the millipede, Coxobolellus saratani Pimvichai, Enghoff & Backeljau, 2022 were analyzed by gas chromatography-mass spectrometry to provide the very first data on the composition of the defensive secretions of the family Pseudospirobolellidae (Diplopoda: superorder Juliformia, order Spirobolida). This unveiled at least 12 identifiable compounds, including six quinones, two phenols, and four fatty acid esters. The three most prevalent identifiable compounds were 2,3-dimethoxy-1,4-benzoquinone (25.52%), hexyl pentadecanoate (11.57%) (the first report of a fatty acid ester compound in the order Spirobolida and tentatively indicating that this may be a shared feature of the Juliformia), and 3,4-dimethoxyphenol (10.51%). The antimicrobial activity of the defensive secretions was evaluated against three gram-positive bacteria (Bacillus cereus, Staphylococcus aureus, and S. aureus DMST20654), four gram-negative bacteria (Escherichia coli, E. coli ATCC25922, Pseudomonas aeruginosa, and Salmonella ser. Typhi ATCC16122), and two yeast strains (Candida albicans and C. albicans ATCC10231). The antibiotic kanamycin and the antifungal drug fluconazole were employed as positive controls. Paper disc diffusion assays demonstrated that the fresh, undiluted, secretions inhibited the growth of all tested microorganisms. Furthermore, broth microdilution analysis revealed Minimum Inhibitory Concentrations (MIC) ranging from 40 to 20,000 µg/mL and Minimum Bactericidal/Fungicidal Concentrations (MBC/MFC) ranging from 1,250 to > 20,000 µg/mL. The MIC values indicated that the defensive secretions of C. saratani are notably more efficient than kanamycin and fluconazole in inhibiting the growth of S. aureus DMST20654, E. coli ATCC25922 and C. albicans, but inhibit less effectively the visible growth of the six other microbial taxa tested. Finally, the MBC/MFC values revealed that the secretions of C. saratani may show less potent antimicrobial activity against the nine microbial taxa tested than kanamycin and fluconazole. Nevertheless, these results suggest once more that millipede defensive secretions may not only deter predators, but may also provide millipedes with a chemical defense against pathogens and parasites.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10886-025-01623-0.

Keywords: Antimicrobial activity, Fatty acids, Phenols, Quinones

Introduction

Millipedes (Diplopoda) constitute an old and speciose group of invertebrates (Sierwald and Bond 2007). They are crucial for soil health because they primarily consume leaf litter and decaying plant matter. This feeding activity helps to recycle nutrients in the soil, thus playing a key role in soil replenishment (Smit and Van Aarde 2001). While millipedes may appear rather defenseless, many species are capable of producing secretions (Fig. 1B–C) that deter predators and other disturbing organisms. These defensive secretions are discharged from repugnatorial glands (Fig. 1D) through ozopores (Vujisić et al. 2014; Shear 2015; Morales and Cabrera 2021). The location of these ozopores on the body and the composition of the defensive secretions they discharge vary between millipede taxa (Makarov 2015).

Fig. 1.

Fig. 1

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Coxobolellus saratani from Loei Province, Thailand. A: Living male. B: Living male with discharged defensive secretions. C: Living male with droplets of the defensive secretions leaving the ozopores. D: Location of the repugnatorial glands (arrows) inside a body ring

Millipede defensive secretions contain chemical compounds that inhibit and kill pathogenic microbes, so that these secretions are a promising resource for the development of new antibiotics (Ilić et al. 2019). This is indeed a pressing issue as antibiotic resistance is one of the most important medical problems today, causing several millions of deaths (Murray et al. 2022). Hence, exploring millipede defensive secretions is a much needed line of research to uncover and identify new natural products against pathogenic microbes.

The millipede orders Spirobolida, Julida and Spirostreptida jointly comprise over 3,000 species and form the superorder Juliformia (Enghoff et al. 2015). The defensive secretions of the Juliformia typically contain quinones and phenols (Bodner et al. 2024), while in the Julida and Spirostreptida they regularly also contain fatty acid esters (Shimizu et al. 2012; Stanković et al. 2016; Ilić et al. 2018; Tummanam et al. 2023). However, in at least one julid species, Typhloiulus orpheus Vagalinski, Stoev & Enghoff, 2015, the defensive secretions contain only trace amounts of benzoquinones (< 1%) but consist for up to 99% of methyl N-methylanthranilate, a probable intermediate compound in the biosynthesis of benzoquinones (Bodner et al. 2017). Yet, these observations are based on the screening of the defensive secretions of less than a roughly estimated 3% of Juliformia species. Hence, increasing the taxonomic sampling is needed to better document the diversity and taxonomic distribution of the various compounds in the defensive secretions of this superorder. Moreover, as quinones and phenols may have broad-spectrum antimicrobial effects (Morales et al. 2022a), while fatty acids may enhance the antimicrobial effects of quinones (Vujisić et al. 2014), it is expected that the study of the defensive secretions of juliformian millipedes may have a potential for medicinal applications.

The juliformian family Pseudospirobolellidae (order Spirobolida) comprises small to medium-sized Asian millipede species. To date, only four genera are recognized: Benoitolus Mauriès, 1980 (3 species), Coxobolellus Pimvichai, Enghoff, Panha & Backeljau 2020 (12 species), Pseudospirobolellus Carl, 1912 (3 species), and Siliquobolellus Pimvichai, Enghoff, Panha & Backeljau, 2022 (3 species). However, the taxonomic and phylogenetic investigation of this family has only recently been started (Pimvichai et al. 2020, 2022a, b). Hence, the chemical composition of pseudospirobolellid defensive secretions and their potential antimicrobial properties are not yet documented. The genus Coxobolellus is widely distributed across limestone regions in Thailand. Members of this genus are medium-sized and produce abundant secretions when disturbed.

Against this background, the present paper reports for the first time on the chemical composition and antimicrobial effects of the defensive secretions of a pseudospirobolellid species, viz. the common Thai millipede, Coxobolellus saratani Pimvichai, Enghoff & Backeljau, 2022 (Fig. 1A–D).

Materials and Methods

Specimen Sampling

Coxobolellus saratani is currently known only from the limestone mountains of Loei Province, Thailand, where it lives among leaf litter in moist environments. Morphologically, the species is easily distinguished by its unique gonopodal structure and greenish-grey body colour (Pimvichai et al. 2022a). Specimens of C. saratani (Fig. 1A–C) were collected on 16–17 September 2023 during the rainy season in Phu Pha Lorm, Muang District (17° 33’ 23"N, 101° 52’ 09"E), Loei Province, Thailand. The specimens were euthanized and preserved by placing them in a freezer maintained at -20 °C. This research was conducted under the approval of the Animal Care and Use regulations (numbers U1-07304-2560 and IACUC-MSU-037/2019) of the National Research Council of Thailand.

Collection of Defensive Secretions

The C. saratani secretions were collected by excising the repugnatorial glands (Fig. 1D) from seven females that had been preserved at -20 °C, starting from body ring 6 to the last body ring (51−55 rings). A total of 3.6 mL of secretions were collected and filtered using Whatman® qualitative filter paper. For the GC-MS analyses and microbiological testing the filtered secretions were divided into three 2.0 mL HPLC autosampler vials, with approximately 1.2 mL of secretion in each vial.

GC-MS Analyses

Gas chromatography-mass spectrometry (GC-MS) was done at the Central Lab, Mahasarakham University and analyses were performed on a QP 2010 Shimadzu GC-MS with an Rtx®-5MS column (Restek, 30 m x 0.25 mm ID, 0.25 μm). An injection volume of 1 µL (from dilution factor: 1 µL of secretions + 1 µL methanol) was applied in splitless mode with an injector temperature of 250 °C. The temperature program of the GC-MS followed the protocol outlined by Arab et al. (2003) with a minor adjustment: initially set at 70 ºC, hold for 2 min, then ramped to 250 ºC at a rate of 5 ºC/min, followed by a ramp to 280 ºC at a rate of 5 ºC/min, and finally hold for 16 min. The GC-MS conditions encompassed a full-scan range from m/z 40 to 550 amu, with electron ionization mass spectra generated at 70 eV. To determine the retention index (RI), the n-alkanes standard solution (C7-C30) underwent analysis under identical GC-MS conditions. Compound identification relied on comparing mass spectra with those in the NIST standard reference database, NIST14 libraries and with published data on previously identified compounds (Vujisić et al. 2011; Bodner et al. 2016; Stanković et al. 2016; Tummanam et al. 2023).

Tested Microbial Organisms and Inoculum Preparation

This study used extensively investigated human pathogens as test organisms, so that our results can be compared with previous studies that assessed the antimicrobial activity of certain compounds against these organisms. As such, our microbial test organisms involved: three gram-positive bacteria: Bacillus cereus, Staphylococcus aureus, and Staphylococcus aureus DMST20654; four gram-negative bacteria: Escherichia coli, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, and Salmonella ser. Typhi ATCC16122; and two yeasts: Candida albicans and Candida albicans ATCC10231. All tested microbial organisms used were sourced from the Microbiology Laboratory at the Department of Biology, Faculty of Science, Mahasarakham University. Bacteria were cultured on Mueller Hinton Agar (Himedia, India), and yeasts were grown on Sabouraud Dextrose Agar (SDA) (Himedia, India). Both media were incubated at 37 °C for 24 h. For testing, cell suspensions were prepared to a density of 1.5 × 108 CFU/mL, following the 0.5 McFarland standard.

Paper Disc Diffusion Method

The antimicrobial efficacy of the C. saratani secretion against the microbial organisms was first assessed using the paper disc diffusion method in accordance with the Clinical Laboratory Standard Institute (CLSI) guidelines (CLSI 2012b). Bacterial or yeast suspensions (1.5 × 108 CFU/mL) were evenly spread across Nutrient Agar (NA) medium plates for bacteria and SDA medium plates for yeasts (maximum plate diameters in both cases: 90 mm). Sterile filter paper discs (6 mm in diameter) were saturated with 10 µL of fresh, undiluted secretion and placed onto the plates. Positive controls consisted of 10 µL of 250 µg/mL kanamycin for bacteria and 200 µg/mL fluconazole for yeast, while the negative control consisted of 10 µL of 0.85% normal saline solution. Each treatment was replicated three times. Following a 24 h incubation period at 37 °C, the diameter of the inhibition zone was measured in mm.

Minimum Inhibitory Concentration (MIC)

The MIC was determined in 96-well microtiter plates employing the microdilution method, following the protocols outlined by the CLSI (2012a). Secretions were initially diluted with DMSO to obtain concentrations ranging from 2 to 20,000 µg/mL. These dilutions were subsequently prepared as a two-fold serial dilution series using Mueller Hinton Broth (MHB) for bacterial testing and Sabouraud Dextrose Broth (SDB) for yeast testing. Subsequently, each well was inoculated with 100 µL of 1.5 × 108 CFU/mL of the tested microorganisms. Negative controls consisted of 100 µL of MHB or SDB, while positive controls comprised 100 µL of kanamycin (ranging from 250 to 0.244 µg/mL) for bacteria and fluconazole (ranging from 200 to 0.196 µg/mL) for yeast. Following a 24 h incubation period at 37 °C, optical densities (OD) were measured using a multimode plate reader at a wavelength of 600 nm. All treatments were performed in triplicate to ensure the reliability and reproducibility of the results. The MIC value was defined as the lowest concentration of secretions capable of inhibiting microbial growth.

Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC)

The determination of the MBC and MFC involved serial sub-culturing of wells from the 96-well plate that exhibited no bacterial growth in the MIC test. Each well was subjected to sub-culturing on NA medium for bacteria and SDA medium for yeast using loopfuls of the culture. These sub-cultures underwent incubation at 37 °C for 24 h. Kanamycin served as the positive control for bacteria, while fluconazole served as the positive control for yeast. The MBC or MFC was defined as the lowest concentration at which no colony growth was observed.

Data Analysis

A one-way ANOVA followed by pairwise Least Significant Difference (LSD) testing was performed to analyze the inhibition zone diameter results of the paper disc diffusion method. Statistical significance was determined at the 0.05 level.

Results

Chemical Composition of Defensive Secretions of Coxobolellus saratani

GC-MS analysis of the C. saratani secretions revealed 17 distinct compounds, 12 of which could be identified (Table 1 and S1; Figs. 2 and 3A–L), viz. six quinone compounds, with 2,3-dimethoxy-1,4-benzoquinone (Fig. 3D) as the most prevalent (25.52%), four fatty acid esters, with hexyl pentadecanoate (Fig. 3I) as the most prevalent (11.57%), and two phenols, of which 3,4-dimethoxyphenol (Fig. 3G) was the most prevalent (10.51%).

Table 1.

GC-MS based chemical composition of the defensive secretions of the millipede Coxobolellus saratani

ID No. Compound RI measured*
/literature**		 Relative amount
(%)
1 Not identified 685 0.13
2 Not identified 794 16.16
3 2-Methyl-1,4-benzoquinone (Fig. 3A) 1116/1116 5.54
4 2-Methoxy-3-methyl-1,4-benzoquinone (Fig. 3B) 1189/1189 6.18
5 2-Methoxy-1,4-benzoquinone (Fig. 3C) 1235/1235 2.15
6 3,4-Dimethoxyphenol (Fig. 3G) 1279/1279 10.51
7 2,3-Dimethoxy-1,4-benzoquinone (Fig. 3D) 1324/1324 25.52
8 2-Methoxy-5-methylhydroquinone (Fig. 3E) 1349/1350 2.55
9 2,3-Dimethoxyhydroquinone (Fig. 3F) 1378/1373 0.80
10 2-Methyl-3,4-methylenedioxyphenol (Fig. 3H) 1393/1392 1.75
11 Not identified 1468 3.89
12 Not identified 2061 4.35
13 Hexyl pentadecanoate (Fig. 3I) 2228/2228 11.57
14 Hexyl hexadecanoate (Fig. 3J) 2357/2356 7.39
15 Not identified 2545 0.41
16 Hexyl octadecanoate (Fig. 3K) 2595/2592 0.56
17 Octyl heptadecanoate (Fig. 3L) 2682/2685 0.55
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* Retention index relative to n-alkanes on elite-5MS capillary column ** (Vujisić et al. 2011; Bodner et al. 2016; Stanković et al. 2016; Tummanam et al. 2023).

Fig. 2.

Fig. 2

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GC-MS profiles of secretions of the millipede Coxobolellus saratani with the chemical structure of the three most prevalent identifiable compounds. The Arabic numbers refer to the ID numbers of the compounds in Table 1 and S1

Fig. 3.

Fig. 3

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Chemical structure of the main compounds of the defensive secretions of the millipede Coxobolellus saratani (Table 1). A: 2-Methyl-1,4-benzoquinone. B: 2-Methoxy-3-methyl-1,4-benzoquinone. C: 2-Methoxy-1,4-benzoquinone. D: 2,3-dimethoxy-1,4-benzoquinone. E: 2-Methoxy-5-methylhydroquinone. F: 2,3-Dimethoxyhydroquinone. G: 3,4-dimethoxyphenol. H: 2-Methyl-3,4-methylenedioxyphenol. I: Hexyl pentadecanoate. J: Hexyl hexadecanoate. K: Hexyl octadecanoate. L: Octyl heptadecanoate

Antimicrobial Efficacy Assays by the Paper Disc Diffusion Method

The ANOVA and LSD tests of the mean inhibition zone diameters obtained with the paper disc diffusion assays (Figs. 4 and 5) showed that the fresh, undiluted, C. saratani secretions had a significantly higher antimicrobial activity against 8 out of the 9 tested bacteria and yeasts than the positive controls (kanamycin and fluconazole) (Table 2: row by row lowercase superscripts). Only with Pseudomonas aeruginosa ATCC27853 there was no significant difference between the mean inhibition zone diameters produced by the secretions and kanamycin. More specifically, the secretions completely inhibited the growth of Staphylococcus aureus DMST20654, Candida albicans, and C. albicans ATCC10231 (Fig. 4). The ANOVA and LSD tests also demonstrated that the secretions tend to exhibit a significantly higher antimicrobial activity against gram-positive bacteria than against gram-negative bacteria, except for E. coli in which the secretions produced inhibition zones whose mean diameter did not differ significantly from those in gram-positive bacteria (Table 2: column by column uppercase superscripts).

Fig. 4.

Fig. 4

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Disc diffusion test results for antimicrobial activity against: A: Staphylococcus aureus DMST20654. B: Candida albicans. C: C. albicans ATCC10231. Negative control, top row; Positive control (kanamycin for S. aureus, fluconazole for Candida sp.), middle row; Defensive secretions of the millipede Coxobolellus saratani, bottom row, none of the three tests showed any colony growth on the plates

Fig. 5.

Fig. 5

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Disc diffusion test results for antimicrobial activity against: A: Bacillus cereus. B: Staphylococcus aureusC: Escherichia coli. D: E. coli ATCC25922. E: Salmonella ser. Typhi ATCC16122. F: Pseudomonas aeruginosa. Negative control, top row; Positive control (kanamycin), middle row; Defensive secretions of the millipede Coxobolellus saratani bottom row

Table 2.

Mean diameters of the microbial inhibition zones induced by the defensive secretions of the millipede Coxobolellus saratani using the paper disc diffusion method (Figs. 4 and 5)

Microbial strains Inhibition zone (mm)
Secretions
(undiluted, 10 µL)		 Kanamycin (250 µg/mL, 10 µL)*/
Fluconazole (200 µg/mL, 10 µL)**		 0.85% Normal Saline solution
Gram-positive bacteria Bacillus cereus 66.33 ± 4.93 aAB 12.17 ± 0.79 bBC 0.00 ± 0.00 c
Staphylococcus aureus 44.00 ± 5.30 aAB 21.50 ± 0.87 bAB 0.00 ± 0.00 c
S. aureus DMST20654 90.00 ± 0.00 aA 28.83 ± 1.73 bA 0.00 ± 0.00 c
Gram-negative bacteria Escherichia coli 42.83 ± 3.79 aAB 8.67 ± 3.62 bBC 0.00 ± 0.00 b
E. coli ATCC25922 33.83 ± 3.86 aB 8.17 ± 3.41 bC 0.00 ± 0.00 b
Pseudomonas aeruginosa ATCC27853 16.83 ± 3.25 aB 11.58 ± 1.15 aBC 0.00 ± 0.00 b
Salmonella ser. Typhi ATCC16122 30.50 ± 3.75 aB 10.83 ± 1.08 bBC 0.00 ± 0.00 c
Yeast Candida albicans 90.00 ± 0.00 aA 15.83 ± 0.58 bBC 0.00 ± 0.00 c
C. albicans ATCC10231 90.00 ± 0.00 aA 18.00 ± 1.05 bABC 0.00 ± 0.00 c
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* Kanamycin tested on bacteria, ** Fluconazole tested on yeast.

Values are expressed as mean ± SD of triplicate measurement (n = 3).

Values of 90.00 mm (= diameter of the entire plate) mean complete inhibition.

Mean values followed by the same letters (a, b, c) within rows are not significantly different (ANOVA + LSD at P < 0.05).

Mean values followed by the same letters (A, B, C) within columns are not significantly different (ANOVA + LSD at P < 0.05).

MCI, MBC and MFC

The MIC values of the C. saratani secretions ranged from 40 to 20,000 µg/mL (Table 3). The lowest MIC value, viz. 40 µg/mL, inhibited the growth of S. aureus DMST20654 and C. albicans, followed by 80 µg/mL inhibiting the growth of E. coli ATCC25922. Thus, again the secretions appear to be more effective than fluconazole and kanamycin against these three microbes. Yet, for the six other microbial organisms tested, the MIC values of the C. saratini secretions were substantially higher than those of kanamycin and fluconazole (Table 3). Moreover, the MBC and MFC values of the C. saratani secretions (range: 1,250 to > 20,000 µg/mL) were consistently much higher for all tested microbial organisms than those of kanamycin and fluconazole (resp. 250 and > 200 µg/mL) (Table 4).

Table 3.

Minimum inhibitory concentrations (MIC) of the defensive secretions of the millipede Coxobolellus saratani against the selected microorganims

Microbial strains MIC
Fresh secretions
(µg/mL)		 Kanamycin
(µg/mL)		 Fluconazole (µg/mL)
Gram-positive bacteria Bacillus cereus 5,000.0 125.0 N/A
Staphylococcus aureus 2,500.0 62.5 N/A
S. aureus DMST20654 40.0 62.5 N/A
Gram-negative bacteria Escherichia coli 10,000.0 250.0 N/A
E. coli ATCC25922 80.0 250.0 N/A
Pseudomonas aeruginosa ATCC27853 20,000.0 250.0 N/A
Salmonella ser. Typhi ATCC16122 2,500.0 250.0 N/A
Yeast Candida albicans 40.0 N/A 125.0
C. albicans ATCC10231 630.0 N/A 125.0
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N/A = Not Applicable.

Table 4.

Minimum bactericidal concentration (MBC) and minimum fungicidal concentrations (MFC) of the defensive secretions of the millipede Coxobolellus saratani against the selected microorganisms

Microbial strains MBC/MFC
Fresh secretions (µg/mL) Kanamycin (µg/mL) Fluconazole (µg/mL)
Gram-positive bacteria Bacillus cereus > 20,000 250 N/A
Staphylococcus aureus 5,000 250 N/A
S. aureus DMST20654 1,250 250 N/A
Gram-negative bacteria Escherichia coli 10,000 250 N/A
E. coli ATCC25922 1,250 250 N/A
Pseudomonas aeruginosa ATCC27853 > 20,000 250 N/A
Salmonella ser. Typhi ATCC16122 20,000 250 N/A
Yeast Candida albicans 1,250 N/A > 200
C. albicans ATCC10231 1,250 N/A > 200
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N/A = Not Applicable.

Discussion

Composition of Defensive Secretions of Coxobolellus saratani

This study provides the first detailed analysis of the chemical composition of the defensive secretions produced by C. saratani. These defensive secretions contain at least 12 identifiable compounds, several of which have been previously documented in other juliformian species as well. The main compound in the C. saratani secretions is 2,3-dimethoxy-1,4-benzoquinone (25.52%), which is also the main compound in Anurostreptus sculptus Demange, 1961 (Spirostreptida: Harpagophoridae), where it even shows a still larger peak area of 58.58% (Tummanam et al. 2023). Much earlier, Weatherston and Percy (1969) were the first to report this compound in the defensive secretions of the millipede Uroblaniulus canadensis (Newport, 1844) (Julida: Parajulidae), while subsequently it was found that substantial amounts of this compound are produced by Metiche tanganyicense Kraus, 1958 [now Epibolus pulchripes (Gerstäcker, 1873)] (Spirobolida: Pachybolidae) (Wood et al. 1975). In a majority of Juliformia species, however, 2,3-dimethoxy-1,4-benzoquinone seems far less prevalent (e.g. Deml and Huth 2000; Huth 2000; Kuwahara et al. 2002; Wu et al. 2007; Bodner and Raspotnig 2012; Shimizu et al. 2012; Sekulić et al. 2014; Bodner et al. 2016; Kania et al. 2016; Stanković et al. 2016; Makarov et al. 2017; Bodner et al. 2018; Ilić et al. 2018). Conversely, in most Juliformia species the main (or major) compound of the defensive secretions is 2-methoxy-3-methyl-1,4-benzoquinone, as was first reported by Trave et al. (1959) but later on demonstrated in many other species of Juliformia (e.g. Casnati et al. 1963; Wood et al. 1975; Röper and Heyns 1977; Eisner et al. 1978; Deml and Huth 2000; Huth 2000; Valderrama et al. 2000; Kuwahara et al. 2002; Wu et al. 2007; Vujisić et al. 2011; Bodner and Raspotnig 2012; Shimizu et al. 2012; Sekulić et al. 2014; Vujisić et al. 2014; Shear 2015; Bodner et al. 2016; Kania et al. 2016; Stanković et al. 2016; Makarov et al. 2017; Bodner et al. 2018; Ilić et al. 2018; Medeiros et al. 2020; Morales and Cabrera 2021; Morales et al. 2023).

Less prevalent, quinone compounds in the defensive secretions of C. saratani, such as 2-methyl-1,4-benzoquinone (toluquinone), 2-methoxy-1,4-benzoquinone, 2-methoxy-5-methylhydroquinone, and 2,3-dimethoxyhydroquinone were also reported in other Juliformia species (e.g. Barbier and Lederer 1957; Casnati et al. 1963; Eisner et al. 1978; Wu et al. 2007; Sekulić et al. 2014; Vujisić et al. 2014; Shear 2015; Ilić et al. 2018; Tummanam et al. 2023).

The second most abundant identifiable compound in the secretions of C. saratani is the saturated fatty acid ester hexyl pentadecanoate (11.57%). This compound has been observed in usually far lower concentrations in several species of the order Julida (e.g. Shimizu et al. 2012; Stanković et al. 2016; Bodner et al. 2018; Ilić et al. 2018), while it was for the first time reported in the order Spirostreptida by Tummanam et al. (2023). The present study is the first report of the presence of fatty acid esters in the defensive secretion of a millipede species from the order Spirobolida. It tentatively suggests that the presence of fatty acid esters in the defensive secretions may be another feature that is shared by the Juliformia, next to the shared presence of quinones and phenols. Fatty acid esters, may function as solvents and/or repellents (Shear 2015). In conjunction with benzoquinones, they may form a more potent millipede defense than either compound alone. Additionally, aliphatic fatty acid esters may show antimicrobial activity, suggesting that they play versatile roles across species, contributing to defense, communication, and antimicrobial function (Ilić et al. 2018).

The third most abundant identifiable compound in the secretions of C. saratani is 3,4-dimethoxyphenol, which has also been reported from other species in the orders Spirobolida (Díaz et al. 2009; Morales and Pedroso 2019; Rodríguez-López et al. 2021; Morales et al. 2022b) and Spirostreptida (Tummanam et al. 2023). Yet it remains to be demonstrated in the Julida, which produce other phenol compounds (e.g. Sekulić et al. 2014; Bodner et al. 2016; Makarov et al. 2017; Ilić et al. 2018). Phenols have strong bactericidal and fungicidal properties, making them effective as antiseptics and disinfectants. This is due to their interactions with cell membranes, provoking an increased permeability. This either causes the loss of vital cellular components, the inactivation of essential enzymes, or both (Ilić et al. 2019). The second phenol compound was 2-methyl-3,4-methylenedioxyphenol (IUPAC: 4-methyl-2H-1,3-benzodioxol-5-ol) (Fig. 3H), which in the millipede literature is sometimes listed as a quinone (e.g. Wu et al. 2007; Sekulić et al. 2014; Vujisić et al. 2014; Kania et al. 2016; Stanković et al. 2016; Ilić et al. 2018). However, quinones are generally defined as compounds having a fully conjugated cyclic dione (= diketone = dicarbonyl) structure, such as that of benzoquinones, derived from aromatic compounds by conversion of an even number of -CH = groups into -C(= O)- groups with any necessary rearrangement of double bonds (Moss et al. 1995). In contrast, phenols are defined as compounds having one or more hydroxy groups attached to a benzene or other arene ring (Moss et al. 1995). Obviously, this latter condition applies to 2-methyl-3,4-methylenedioxyphenol, with its hydroxy group on C5 (IUPAC nomenclature) or C1 (non-IUPAC nomenclature) (Fig. 3H). Moreover, the lack of a dione structure in this molecule is incompatible with its interpretation as a quinone. All in all, interpreting 2-methyl-3,4-methylenedioxyphenol as a phenolic compound is well-reflected by its IUPAC name.

The (benzo)quinone compounds, especially 2-methyl-1,4-benzoquinone (toluquinone) and 2-methoxy-3-methyl-1,4-benzoquinone, in the defensive secretions of Juliformia millipedes (including C. saratani) are effective repellents of other organisms (Eisner et al. 1978; Shear 2015). Consequently, some primates and birds rely on this feature to deter mosquitoes and other ectoparasites by rubbing their body with live millipedes that secrete these compounds (e.g. Birkinshaw 1999; Valderrama et al. 2000; Weldon et al. 2003; Carroll et al. 2005; Medeiros et al. 2020; Perin Marcon and Andriola 2023). Conversely, some predators of Juliformia millipedes apply fine-tuned strategies by which they avoid exposure to the (benzo)quinones (i.e. again 2-methyl-1,4-benzoquinone and 2-methoxy-3-methyl-1,4-benzoquinone) in the defensive secretions of their preys (Eisner et al. 1998; Weldon et al. 2006), while for several necrophagous beetles that feed on freshly dead Juliformia millipedes the very same repelling (benzo)quinones act as olfactory attractants that lead the beetles to their food resource (e.g. Krell et al. 1997, 1998; Krell 1999; Brühl and Krell 2003; Krell 2004; Schmitt et al. 2004; Rodríguez-López et al. 2021). On top of their repellent vs. attractant effects, (benzo)quinones and phenols produced by Juliformia millipedes also show strong antibacterial and antifungal activity (e.g. Stanković et al. 2016; Ilić et al. 2018; Lai et al. 2024) (see further below), which perhaps may protect the millipedes against pathogens and various infections such as, for example, parasitic fungi of the ascomycete order Laboulbeniales (Enghoff and Santamaria 2015; Stanković et al. 2016).

Finally, it is hypothesized that the antimicrobial properties of (benzo)quinones may be enhanced by non-quinone compounds, like fatty acid esters (Vujisić et al. 2014), which may serve as solvents or aid in the dispersion of defensive secretions (Ilić et al. 2018). Some of these non-quinone compounds, including fatty acid esters, might also participate in chemical signaling (Stanković et al. 2016; Ilić et al. 2018).

Antimicrobial Efficacy of the Defensive Secretions of Coxobolellus saratani

The paper disc diffusion assays showed that the fresh, undiluted, defensive secretions of C. saratani completely inhibited S. aureus DMST20654, C. albicans and C. albicans ATCC10231 (no colony growth at all). To a lesser degree they also inhibited S. aureus, B. cereus and the tested gram-negative bacteria. In fact, based on the paper disc diffusion assays, the defensive secretions of C. saratani inhibited all tested microbial strains more effectively than the antibiotic kanamycin (more than three times) and the antifungal drug fluconazole (more than five times) in the positive control. This result was likely due to the probably high concentrations of various compounds in the 10 µL of fresh, undiluted defensive secretion that was used for the paper disc diffusion assays. Yet, the MIC values revealed that the defensive secretions of C. saratani were only more effective at inhibiting the visible growth of S. aureus DMST20654, E. coli ATCC25922), and C. albicans than kanamycin and fluconazole, but that for the six other microbial organisms tested kanamycin and fluconazole outperformed the defensive secretions of C. saratani. Finally, with the MBC/MFC values the secretions of C. saratani appeared substantially less potent against the nine microbial organisms tested than kanamycin and fluconazole. As such, the antimicrobial activity of the defensive secretions of C. saratani against S. aureus, E. coli, P. aeruginosa and C. albicans, appears to be similar to that of the Cuban millipede Rhinocricus sp. Karsch, 1881 (Spirobolida: Rhinocricidae) (Morales et al. 2022a), while against P. aeruginosa and S. aureus it is comparable to that of the julid millipede Pachyiulus hungaricus (Karsch, 1881) (Stanković et al. 2016). Against E. coli ATCC25922 the antimicrobial activity of the defensive secretions of C. saratani is more effective (lower MIC values) than that of the julid millipede species Cylindroiulus boleti (C.L. Koch, 1847), Megaphyllum bosniense (Verhoeff, 1897) and M. unilineatum (C.L. Koch, 1838), but against Candida albicans ATCC10231 it is somewhat comparable (MIC and MBC) to that of Megaphyllum bosniense, though less effective than that of M. unilineatum and Cylindroiulus boleti or that of Apfelbeckia insculpta (C.L. Koch, 1867) (Callipodida: Schizopetalidae) (Ilić et al. 2019). The defensive secretion of this latter species is also more effective than that of C. saratani against P. aeruginosa ATCC27853 (Ilić et al. 2019). Finally, both the MIC and MBC/MFC assays indicated that the defensive secretions of C. saratani are less effective against the tested microbial strains than the defensive secretions of the giant millipede Anurostreptus sculptus (Spirostreptida: Harpagophoridae) (Tummanam et al. 2023).

The antimicrobial activity of the secretions of C. saratani is likely due to the induction of oxidative stress and cell membrane disruption by the quinones and phenols in these secretions. Quinones generate reactive oxygen species, such as hydrogen peroxide and superoxide radicals, which cause oxidative damage to microbial cells (Kim et al. 2019). Phenols, with their hydrophobic nature, disrupt cell membrane integrity by integrating into lipid bilayers, leading to membrane permeability changes and protein denaturation (Ilić et al. 2019). In contrast, kanamycin inhibits bacterial protein synthesis (Suzuki et al. 1970), while fluconazole disrupts fungal cell membrane integrity by inhibiting ergosterol biosynthesis (Yang et al. 2018). The fundamentally different mode of antimicrobial action of the scretions of C. saratani (and other Juliformia millipedes) is perhaps clinically advantageous, since the quinones and phenols in the secretions are smaller and more hydrophobic than e.g. kanamycin and fluconazole. As such, it is tentatively expected that quinones and phenols may diffuse better and thus might exert their antimicrobial effects faster and at a wider scale. Yet, this study illustrates that the diameter of inhibition zones and the antimicrobial potency of a compound, as revealed by the paper disc diffusion method and the MIC, MBC or MFC values, do not necessarily correlate well. Moreover, as stated before, quinones and phenols are also toxic, which could impose safety constraints on eventual pharmacological applications. So all in all, while the defensive secretions of C. saratani (and other Juliformia millipedes) may show interesting antimicrobial properties, far more research is needed on their chemical diversity, modes and strength of antimicrobial action, and cytotoxicity, before serious claims can be made about their potential therapeutic and clinical uses.

Electronic Supplementary Material

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Acknowledgements

We are indebted to Prof. Dr. Wanda Guedens (University of Hasselt, Belgium) and Assist. Prof. Dr. Widchaya Radchatawedchakoon (Department of Chemistry, Mahasarakham University) for providing clarifications about the nomenclature and chemical nature of 2-methyl-3,4-methylenedioxyphenol. We are grateful to Sathit Saratan (Sirindhorn Museum, Thailand) for assistance in collecting material, and to Sirinya Pimvichai (Maha Vajiralongkorn Thanyaburi Hospital) for help in editing the references.

Author Contributions

Piyatida Pimvichai: Conceptualization, Funding acquisition, Investigation, Supervision and Writing – original draft; Piyatida Pimvichai, Waraporn Sutthisa, Warinthan Jumpajan, Phikun Buaboon and Nattawadee Nantarat: Resources; Piyatida Pimvichai and Thierry Backeljau: Writing – review and editing.

Funding

This research project was financially supported by Mahasarakham University.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Competing Interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. Arab A, Zacarin GG, Fontanetti CS, Camargo-Mathias MI, dos Santos MG, Cabrera AC (2003) Composition of the defensive secretion of the Neotropical millipede Rhinocricus padbergi Verhoeff 1938 (Diplopoda: Spirobolida: Rhinocricidae). Entomotropica 18:79–82. [Google Scholar]
  2. Barbier M, Lederer E (1957) Sur les benzoquinones du Venin de Trois espèces de Myriapodes. Biokhimiia 22:236–240. [PubMed] [Google Scholar]
  3. Birkinshaw CR (1999) Use of millipedes by black lemurs to anoint their bodies. Folia Primatol 70:170–171. 10.1159/000021691 [DOI] [PubMed] [Google Scholar]
  4. Bodner M, Raspotnig G (2012) Millipedes that smell like bugs: (E)-Alkenals in the defensive secretion of the Julid diplopod Allajulus dicentrus. J Chem Ecol 38:547–556. 10.1007/s10886-012-0127-5 [DOI] [PubMed] [Google Scholar]
  5. Bodner M, Vagalinski B, Makarov SE, Antić D, Vujisić LV, Leis HJ, Raspotnig G (2016) Quinone millipedes reconsidered: evidence for a Mosaic-Like taxonomic distribution of Phenol-Based secretions across the Julidae. J Chem Ecol 42:249–258. 10.1007/s10886-016-0680-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bodner M, Vagalinski B, Makarov SE, Raspotnig G (2017) Methyl N-methylanthranilate: major compound in the defensive secretion of Typhloiulus orpheus (Diplopoda, Julida). Chemoecology 27:171–175. 10.1007/s00049-017-0242-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bodner M, Vagalinski B, Raspotnig G (2018) Chemotaxonomic potential of exocrine alkyl esters in Julid millipedes (Diplopoda: Julidae: Cylindroiulini). Biochem Syst Ecol 81:1–11. 10.1016/j.bse.2018.08.001 [Google Scholar]
  8. Bodner M, Antić D, Jovanović Z, Raspotnig G, Ilić B, Lučić L, Makarov S (2024) Alkaloid chemistry in pill-millipedes: defensive secretion in two species of Typhloglomeris Verhoeff, 1898 (Diplopoda, Glomerida, Glomeridellidae). Chemoecology 34:41–46. 10.1007/s00049-024-00400-x [Google Scholar]
  9. Brühl C, Krell F-T (2003) Finding a rare resource: Bornean Scarabaeoidea (Coleoptera) attracted by defensive secretions of Diplopoda. Coleopt Bull 57:51–55. 10.1649/0010-065X((2003))057[0051:FARRBS]2.0.CO;2 [Google Scholar]
  10. Carroll JF, Kramer M, Weldon PJ, Robbins RG (2005) Anointing chemicals and ectoparasites: effects of benzoquinones from millipedes on the lone star tick, Amblyomma americanum. J Chem Ecol 31:63–75. 10.1007/s10886-005-0974-4 [DOI] [PubMed] [Google Scholar]
  11. Casnati G, Nencini G, Quilico A, Pavan M, Ricca A, Salvatori T (1963) The secretion of the myriapod Polydesmus collaris collaris(Koch). Experientia 19:409–411. [DOI] [PubMed] [Google Scholar]
  12. CLSI (2012a) Methods for Dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved Standard-Ninth edition, vol 32. Clinical Laboratory Standard Institute.
  13. CLSI (2012b) Performance standards for antimicrobial disk susceptibility tests; approved Standard—Eleventh edition, vol 32. Clinical Laboratory Standard Institute.
  14. Deml R, Huth A (2000) Benzoquinones and hydroquinones in defensive secretions of tropical millipedes. Naturwissenschaften 87:80–82. 10.1007/s001140050014 [DOI] [PubMed] [Google Scholar]
  15. Díaz JAM, Morales JET, de Oca Porto RM, Sabater JT, Arteaga RG (2009) Primer reporte de 3, 4-dimetoxifenol, En secreciones defensivas de Milípedos Endémicos Cubanos (Spirobolida, Rhinocricidae, Rhinocricus). Caso de estudio Rhinocricus duvernoyi Karch 1881, población de La Palma. Rev CENIC Cienc Quím 40:27–29. [Google Scholar]
  16. Eisner T, Alsop D, Hicks K, Meinwald J (1978) Defensive secretions of millipeds. In: Bettini S (ed) Arthropod venoms. Springer, Berlin Heidelberg, pp 41–72. 10.1007/978-3-642-45501-8_3 [Google Scholar]
  17. Eisner T, Eisner M, Attygalle AB, Deyrup M, Meinwald J (1998) Rendering the inedible edible: circumvention of a millipede’s chemical defense by a predaceous beetle larva (Phengodidae). Proc Natl Acad Sci USA 95:1108–1113. 10.1073/pnas.95.3.1108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Enghoff H, Santamaria S (2015) Infectious intimacy and contaminated caves—three new species of ectoparasitic fungi (Ascomycota: Laboulbeniales) from blaniulid millipedes (Diplopoda: Julida) and inferences about their transmittal mechanisms. Org Div Evol 15:249–263. 10.1007/s13127-015-0208-8 [Google Scholar]
  19. Enghoff H, Golovatch S, Short M, Stoev P, Wesener T (2015) Treatise on Zoology - Anatomy, taxonomy, biology. In: Minelli A (ed) Treatise on Zoology - Anatomy, taxonomy, biology. The Myriapoda, vol 2. - Diplopoda, Brill, pp 363–453. 10.1163/9789004188273_017 [Google Scholar]
  20. Huth A (2000) Defensive secretions of millipedes: more than just a product of melting point decrease? Fragm Faun 43(Suppl):191–200. [Google Scholar]
  21. Ilić B, Dimkić I, Unković N et al (2018) Millipedes vs. pathogens: defensive secretions of some Julids (Diplopoda: Julida) as potential antimicrobial agents. J Appl Entmol 142:775–791. 10.1111/jen.12526 [Google Scholar]
  22. Ilić B, Unković N, Ćirić A et al (2019) Phenol-based millipede defence: antimicrobial activity of secretions from the Balkan endemic millipede Apfelbeckia insculpta (L. Koch, 1867) (Diplopoda: Callipodida). Sci Nat 106:1–9. 10.1007/s00114-019-1631-z [DOI] [PubMed] [Google Scholar]
  23. Kania G, Kowalski R, Pietraś R (2016) Defensive secretions in millipede species of the order Julida (Diplopoda). Acta Soc Zool Bohem 80:17–20. [Google Scholar]
  24. Kim SY, Park C, Jang HJ et al (2019) Antibacterial strategies inspired by the oxidative stress and response networks. J Microbiol 57:203–212. 10.1007/s12275-019-8711-9 [DOI] [PubMed] [Google Scholar]
  25. Krell F-T (1999) Southern African Dung beetles (Coleoptera: Scarabaeidae) attracted by defensive secretions of Diplopoda. Afr Entomol 7:287–288. https://journals.co.za/doi/pdf/10.10520/AJA10213589_368 [Google Scholar]
  26. Krell F-T (2004) East African Dung beetles (Scarabaeidae) attracted by defensive secretions of millipedes. J E Afr Nat Hist 93:69–73. 10.2982/0012-8317(2004)93[69:EADBSA]2.0.CO;2 [Google Scholar]
  27. Krell F-T, Schmitt T, Linsenmair KE (1997) Diplopod defensive secretions as attractants for necrophagous scarab beetles (Diplopoda; Insecta, Coleoptera: Scarabaeidae). Entomol Scand Suppl 51:281–285. [Google Scholar]
  28. Krell F-T, Schmitt T, Dembele A, Linsenmair K (1998) Repellents as attractants—extreme specialization in Afrotropical Dung beetles (Coleoptera: Scarabaeoidea) as competition avoiding strategy. Zoology 101:12. [Google Scholar]
  29. Kuwahara Y, Noguchi S, Mori N, Higa Y (2002) Identification of benzoquinones and hydroquinones as the secretory compounds from three species of Okinawan millipedes. Jpn J Environ Entomol Zool 13:117–124. 10.11257/jjeez.13.117 [Google Scholar]
  30. Lai J, Peng W, Song S, Jiang J, Liu B (2024) Transcriptome analysis reveals the inhibitory mechanism of 3, 4-Dimethoxyphenol from Streptomyces albidoflavus strain ML27 against Xanthomonas oryzae pv. oryzae. Pestic Biochem Physiol 202:105913. 10.1016/j.pestbp.2024.105913 [DOI] [PubMed] [Google Scholar]
  31. Makarov SE (2015) Diplopoda—integument. In: Minelli A (ed) Treatise on Zoology - Anatomy, taxonomy, biology. The Myriapoda, vol 2. - Diplopoda, Brill, pp 69–99.
  32. Makarov SE, Bodner M, Reineke D et al (2017) Chemical ecology of cave-dwelling millipedes: defensive secretions of the Typhloiulini (Diplopoda, Julida, Julidae). J Chem Ecol 43:317–326. 10.1007/s10886-017-0832-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Medeiros K, Campêlo A, Maia ACD et al (2020) Wild blonde capuchins (Sapajus flavius) perform anointing behaviour using toxic secretions of a millipede (Spirobolida: Rhinocricidae). J Chem Ecol 46:1010–1015. 10.1007/s10886-020-01215-0 [DOI] [PubMed] [Google Scholar]
  34. Morales JET, Cabrera MT (2021) Diplodomica II. Repugnatorial secretion of Ecuadorian endemic millipede Rhinocricus sp. (Diplopoda, Spirobolida, Rhinocricidae) from Orellana Province, Amazonia, Ecuador. Chem Proc 3:90. https://www.mdpi.com/2673-4583/3/1/90 [Google Scholar]
  35. Morales JET, Pedroso MTC (2019) Diplodomica I. Chemical Composition of Repugnatorial Secretions of Cuban Endemic Millipede gen. Rhinocricus sp. Proc (The 23rd International Electronic Conference on Synthetic Organic Chemistry) 41:73. https://www.mdpi.com/2504-3900/41/1/73
  36. Morales JET, Siguenza JC, Canchingre M, Villavicencio CB, Pedroso MTC (2022a) Antimicrobial activity of defensive secretions of terrestrial invertebrates (Diplopoda, Spirobolida, Rhinocricus) from the insular neotropics. Med Sci Forum 2:1–8. 10.3390/eca2022-12729 [Google Scholar]
  37. Morales JET, Siguenza JC, Villavicencio CB, Canchingre M (2022b) Diplodomica III. Chemical ecology of defensive secretions from Neotropical archipelago. Isolation of 3,4-dimethoxyphenol from ejected secretion of endemic Cuban millipedes (Spirobolida, Rhinocricidae, Rhinocricus). Study case Rhinocricus duvernoyi Karch 1881, La Palma population. IECD 2022:1–7. https://sciforum.net/manuscripts/12358/manuscript.pdf [Google Scholar]
  38. Morales JET, Villavicencio CB, Cervantes XLG, Canchingre ME, Pedroso MTC (2023) GC-MS Analysis of Defensive Secretions of a Cuban Endemic Millipede of the Genus Rhinocricus—Chemical Prospecting of Neotropical Biodiversity. Chem Proc 14:53. https://www.mdpi.com/2673-4583/14/1/53
  39. Moss GP, Smith PAS, Tavernier D (1995) Glossary of class names of organic compounds and reactivity intermediates based on structure (IUPAC recommendations 1995). Pure Appl Chem 67:1307–1375. 10.1351/pac199567081307 [Google Scholar]
  40. Murray CJL, Ikuta KS, Sharara F et al (2022) Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399:629–655. 10.1016/S0140-6736(21)02724-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Perin Marcon A, Andriola JVP (2023) Rufous hornero (Furnarius rufus, Furnariidae) anointing with a millipede (Diplopoda, Spirobolidae) in southernmost Brazil. J Field Ornithol 94:13. 10.5751/JFO-00394-940413 [Google Scholar]
  42. Pimvichai P, Enghoff H, Panha S, Backeljau T (2020) Integrative taxonomy of the new millipede genus Coxobolellus, gen. nov. (Diplopoda: Spirobolida: Pseudospirobolellidae), with descriptions of ten new species. Invertbr Syst 34:591–617. 10.1071/IS20031 [Google Scholar]
  43. Pimvichai P, Enghoff H, Backeljau T (2022a) Two new millipede species of the genus Coxobolellus Pimvichai, Enghoff, Panha & Backeljau, 2020 (Diplopoda, Spirobolida, Pseudospirobolellidae). ZooKeys 1128:171–190. 10.3897/zookeys.1128.94242 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pimvichai P, Enghoff H, Panha S, Backeljau T (2022b) A new genus of Pseudospirobolellidae (Diplopoda, Spirobolida) from limestone karst areas in Thailand, with descriptions of three new species. Zoosyst Evol 98:313–326. 10.3897/zse.98.90032 [Google Scholar]
  45. Rodríguez-López M, Gómez B, Bueno-Villegas J, Malo EA (2021) Attraction of Canthon vazquezae (Coleoptera: Scarabaeinae) to volatiles released by Messicobolus magnificus (Diplopoda: Spirobolida). J Insect Behav 34:255–263. 10.1007/s10905-021-09785-x
  46. Röper H, Heyns K (1977) Spurenanalytik von p-Benzochinon-und Hydrochinon-Derivaten mit Gaschromatographie und Gaschromatographie/Massenspektrometrie. Identifizierung von Wehrsekret-Komponenten europäischer juliden / trace analysis of p-Benzoquinone-and Hydroquinone derivatives by Gas-Liquid Chromatography and Gas-Liquid Chromatography/Mass-Spectrometry. Identification of Defensive Secretion Components from European Julids. Z Naturforsch C 32:61–66. 10.1515/znc-1977-1-209 [Google Scholar]
  47. Schmitt T, Krell F-T, Linsenmair KE (2004) Quinone mixture as attractant for necrophagous Dung beetles specialized on dead millipedes. J Chem Ecol 30:731–740. 10.1023/B:JOEC.0000028428.53797.cb [DOI] [PubMed] [Google Scholar]
  48. Sekulić TL, Vujisić LV, Ćurčić B et al (2014) Quinones and non-quinones from the defensive secretion of Unciger transsilvanicus (Verhoeff, 1899) (Diplopoda, Julida, Julidae), from Serbia. Arch Biol Sci 66:385–390. 10.2298/ABS1401385S [Google Scholar]
  49. Shear WA (2015) The chemical defenses of millipedes (Diplopoda): biochemistry, physiology and ecology. Biochem Syst Ecol 61:78–117. 10.1016/j.bse.2015.04.033 [Google Scholar]
  50. Shimizu N, Kuwahara Y, Yakumaru R, Tanabe T (2012) n-Hexyl laurate and fourteen related fatty acid esters: new secretory compounds from the Julid millipede, Anaulaciulus sp. J Chem Ecol 38:23–28. 10.1007/s10886-012-0063-4 [DOI] [PubMed] [Google Scholar]
  51. Sierwald P, Bond JE (2007) Current status of the myriapod class Diplopoda (millipedes): taxonomic diversity and phylogeny. Annu Rev Entomol 52:401–420. https://doi.org/http://dx.doi.org/10.1146/annurev.ento.52.111805.090210 [DOI] [PubMed] [Google Scholar]
  52. Smit AM, Van Aarde RJ (2001) The influence of millipedes on selected soil elements: A microcosm study on three species occurring on coastal sand dunes. Funct Ecol 15:51–59. http://www.jstor.org/stable/826567 [Google Scholar]
  53. Stanković S, Dimkić I, Vujisić L et al (2016) Chemical defence in a millipede: evaluation and characterization of antimicrobial activity of the defensive secretion from Pachyiulus hungaricus (Karsch, 1881) (Diplopoda, Julida, Julidae). PLoS ONE 11:e0167249. 10.1371/journal.pone.0167249 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Suzuki J, Kunimoto T, Hori M (1970) Effects of Kanamycin on protein synthesis: Inhibition of elongation of peptide chains. J Antibiot 23:99–101. 10.7164/antibiotics.23.99 [DOI] [PubMed] [Google Scholar]
  55. Trave R, Garanti L, Pavan M (1959) Ricerche Sulla natura chimica Del Veleno Del Miriapode Archiulus (Schizophyllum) sabulosus L. Chim Ind 41:19–30. [Google Scholar]
  56. Tummanam A, Sutthisa W, Radchatawedchakoon W, Backeljau T, Pimvichai P (2023) Composition and antimicrobial activity of defensive secretions of the giant millipede Anurostreptus sculptus (Diplopoda, Spirostreptida, Harpagophoridae). Trop Nat Hist 23:42–51. https://li01.tci-thaijo.org/index.php/tnh/article/view/258335 [Google Scholar]
  57. Valderrama X, Robinson JG, Attygalle AB, Eisner T (2000) Seasonal anointment with millipedes in a wild primate: a chemical defense against insects? J Chem Ecol 26:2781–2790. 10.1023/A:1026489826714 [Google Scholar]
  58. Vujisić LV, Makarov SE, Ćurčić BP et al (2011) Composition of the defensive secretion in three species of European millipedes. J Chem Ecol 37:1358–1364. 10.1007/s10886-011-0040-3 [DOI] [PubMed] [Google Scholar]
  59. Vujisić LV, Antić DŽ, Vučković IM et al (2014) Chemical defense in millipedes (Myriapoda, Diplopoda): do representatives of the family Blaniulidae belong to the ‘quinone’clade? Chem Biodiv 11:483–490. 10.1002/cbdv.201300355 [DOI] [PubMed] [Google Scholar]
  60. Weatherston J, Percy J (1969) Studies of physiologically active arthropod secretions. III. Chemical, morphological, and histological studies of the defence mechanism of Uroblaniulus canadensis (Say) (Diplopoda: Julida). Can J Zool 47:1389–1394. 10.1139/z69-214 [Google Scholar]
  61. Weldon PJ, Aldrich JR, Klun JA, Oliver JE, Debboun M (2003) Benzoquinones from millipedes deter mosquitoes and elicit self-anointing in capuchin monkeys (Cebus spp). Naturwissenschaften 90:301–304. 10.1007/s00114-003-0427-2 [DOI] [PubMed] [Google Scholar]
  62. Weldon PJ, Cranmore CF, Chatfield JA (2006) Prey-rolling behavior of Coatis (Nasua spp.) is elicited by benzoquinones from millipedes. Naturwissenschaften 93:14–16. 10.1007/s00114-005-0064-z [DOI] [PubMed] [Google Scholar]
  63. Wood WF, Shepherd J, Chong B, Meinwald J (1975) Ubiquinone-O in defensive spray of African millipede. Nature 253:625–626. 10.1038/253625a0 [DOI] [PubMed] [Google Scholar]
  64. Wu X, Buden DW, Attygalle AB (2007) Hydroquinones from defensive secretion of a giant Pacific millipede, Acladocricus setigerus (Diplopoda: Spirobolida). Chemoecology 17:131–138. 10.1007/s00049-007-0372-1 [Google Scholar]
  65. Yang Z, Wang Q, Ma K, Shi P, Liu W, Huang Z (2018) Fluconazole inhibits cellular ergosterol synthesis to confer synergism with Berberine against yeast cells. J Glob Antimicrob Resist 13:125–130. 10.1016/j.jgar.2017.12.011 [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

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Data Availability Statement

No datasets were generated or analysed during the current study.

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