Phenol-based millipede defence: antimicrobial activity of secretions from the Balkan endemic millipede Apfelbeckia insculpta (L. Koch, 1867) (Diplopoda: Callipodida).

Millipedes use an array of chemical compounds to defend themselves from predator attack. These chemical substances can have additional roles, i.e. defence against various pathogens. We evaluated the efficacy of the defensive secretion of Apfelbeckia insculpta (L. Koch, 1867) against bacteria, yeasts, and filamentous fungi. The tested secretion consisted of two compounds, p-cresol and phenol, and showed antibacterial, antibiofilm, and antifungal potential against all selected microorganisms. The most sensitive bacterium in our study was Pseudomonas aeruginosa, while the tested defensive secretion manifested the lowest activity against Escherichia coli. The defensive secretion of A. insculpta also showed an ability, albeit mild, to suppress biofilm formation by P. aeruginosa. Among the tested yeasts, Candida albicans and C. krusei were the most susceptible and most resistant species, respectively. Finally, the concentration of extracts obtained from the tested defensive secretion needed to achieve an antifungal effect was lowest in the case of Cladosporium cladosporioides. Fusarium verticillioides and Penicillium rubens were the micromycetes most resistant to the tested secretion. Our results indicate that antibacterial activity of the defensive secretion of A. insculpta is similar to or slightly weaker than that of streptomycin, while comparison with antimycotics showed that the tested millipede secretion has stronger activity than fluconazole, but weaker activity than nystatin and ketoconazole. The present study corroborates previous findings indicating that the defensive secretions of millipedes can have different roles apart from antipredator protection and are effective against pathogenic microorganisms.

Chemical Ecology of Cave-Dwelling Millipedes: Defensive Secretions of the Typhloiulini (Diplopoda, Julida, Julidae).

Cave animals live under highly constant ecological conditions and in permanent darkness, and many evolutionary adaptations of cave-dwellers have been triggered by their specific environment. A similar "cave effect" leading to pronounced chemical interactions under such conditions may be assumed, but the chemoecology of troglobionts is mostly unknown. We investigated the defensive chemistry of a largely cave-dwelling julid group, the controversial tribe "Typhloiulini", and we included some cave-dwelling and some endogean representatives. While chemical defense in juliform diplopods is known to be highly uniform, and mainly based on methyl- and methoxy-substituted benzoquinones, the defensive secretions of typhloiulines contained ethyl-benzoquinones and related compounds. Interestingly, ethyl-benzoquinones were found in some, but not all cave-dwelling typhloiulines, and some non-cave dwellers also contained these compounds. On the other hand, ethyl-benzoquinones were not detected in troglobiont nor in endogean typhloiuline outgroups. In order to explain the taxonomic pattern of ethyl-benzoquinone occurrence, and to unravel whether a cave-effect triggered ethyl-benzoquinone evolution, we classed the "Typhloiulini" investigated here within a phylogenetic framework of julid taxa, and traced the evolutionary history of ethyl-benzoquinones in typhloiulines in relation to cave-dwelling. The results indicated a cave-independent evolution of ethyl-substituted benzoquinones, indicating the absence of a "cave effect" on the secretions of troglobiont Typhloiulini. Ethyl-benzoquinones probably evolved early in an epi- or endogean ancestor of a clade including several, but not all Typhloiulus (basically comprising a taxonomic entity known as "Typhloiulus sensu stricto") and Serboiulus. Ethyl-benzoquinones are proposed as novel and valuable chemical characters for julid systematics.

"Quinone Millipedes" Reconsidered: Evidence for a Mosaic-Like Taxonomic Distribution of Phenol-Based Secretions across the Julidae.

The defensive chemistry of juliformian millipedes is characterized mainly by benzoquinones ("quinone millipedes"), whereas the secretions of the putative close outgroup Callipodida are considered to be exclusively phenolic. We conducted a chemical screening of julid secretions for phenolic content. Most species from tribes Cylindroiulini (15 species examined), Brachyiulini (5 species examined), Leptoiulini (15 species examined), Uncigerini (2 species examined), Pachyiulini (3 species examined), and Ommatoiulini (2 species examined) had non-phenolic, in most cases exclusively benzoquinonic secretions. In contrast, tribes Cylindroiulini, Brachyiulini, and Leptoiulini also contained representatives with predominantly phenol-based exudates. In detail, p-cresol was a major compound in the secretions of the cylindroiulines Styrioiulus pelidnus and S. styricus (p-cresol content 93 %) and an undetermined Cylindroiulus species (p-cresol content 51 %), in the brachyiulines Brachyiulus lusitanus (p-cresol content 21 %) and Megaphyllum fagorum (p-cresol content 92 %), as well as in an undescribed Typhloiulus species (p-cresol content 32 %, Leptoiulini). In all species, p-cresol was accompanied by small amounts of phenol. The secretion of M. fagorum was exclusively phenolic, whereas phenols were accompanied by benzoquinones in all other species. This is the first incidence of clearly phenol-dominated secretions in the Julidae. We hypothesize a shared biosynthetic route to phenols and benzoquinones, with benzoquinones being produced from phenolic precursors. The patchy taxonomic distribution of phenols documented herein supports multiple independent regression events in a common pathway of benzoquinone synthesis rather than multiple independent incidences of phenol biosynthesis.

'Does my Diet Affect my Perfume?' Identification and Quantification of Cuticular Compounds in Five Drosophila melanogaster Strains Maintained over 300 Generations on Different Diets.

Cuticular hydrocarbons (CHCs) in Drosophila melanogaster represent the basis of chemical communication being involved in many important biological functions. The aim of this study was to characterize chemical composition and variation of cuticular profiles in five D. melanogaster strains. These strains were reared for approximately 300 generations on five diets: standard cornmeal medium and substrates prepared with apple, banana, tomato, and carrot. Differences in quantity and/or quality in CHCs were assumed as a result of activation of different metabolic pathways involved in food digestion and adaptations to the particular diet type. In total, independently of sex and strain, 66 chemical compounds were identified. In females of all strains, 60 compounds were identified, while, in males, 47 compounds were extracted. Certain new chemical compounds for D. melanogaster were found. MANOVA confirmed that CHC amounts significantly depend on sex and substrates, as well as on their interactions. Discriminant analysis revealed that flies belonging to 'apple' and 'carrot' strains exhibited the most noticeable differences in CHC repertoires. A non-hydrocarbon pheromone, cis-vaccenyl acetate (cVA) also contributed to the variation in the pheromone bouquet among the strains. Variability detected in CHCs and cVA may be used in the explanation of differences in mating behaviour previously determined in analyzed fly strains.

Molecular Diversity of Compounds from Pygidial Gland Secretions of Cave-Dwelling Ground Beetles: The First Evidence.

Three adult cave-dwelling ground beetle species were induced to discharge secretions of their pygidial glands into vials. Dichloromethane extraction was used to obtain the secretions. In total, 42 compounds were identified by GC/MS analysis. Pheggomisetes ninae contained 32 glandular compounds, Laemostenus (Pristonychus) punctatus 13, whereas Duvalius (Paraduvalius) milutini had nine compounds. Caproic, oleic, palmitic, and stearic acids were present in the samples of all analyzed species. Undecane was predominant in the extract of L. punctatus. Palmitic acid was the major component in the secretion of D. milutini. Finally, the most abundant compounds in P. ninae secretion were heptacosene and nonacosadienes. Herein, we present the first data on the identification of pygidial gland secretion components in both troglophilous and troglobite cave-dwelling ground beetles. Some compounds are reported for the first time in the secretions of ground beetles and other higher or lower taxa. The adaptation to underground life has not led to a reduction or changes in the chemical defense mechanism in the analyzed troglophilous and troglobitic Platyninae and Trechinae taxa.

Chemical defense in millipedes (Myriapoda, Diplopoda): do representatives of the family Blaniulidae belong to the 'quinone' clade?

The defensive secretions of two blaniulid millipedes, Nopoiulus kochii and Cibiniulus phlepsii, were characterized by GC-FID and GC/MS analyses, which showed the presence of a complex mixture of benzoquinones, hydroquinones, and oleates. Altogether, 13 compounds were identified. The major compound in the secretions of both analyzed species was 2-methyl-1,4-benzoquinone (toluquinone). The second major constituent in the N. kochii secretion was 2-methyl-3,4-(methylenedioxy)phenol, while in that of C. phlepsii, it was 2-methoxy-3-methyl-1,4-benzoquinone. The defensive secretion of N. kochii also showed a high content of hydroquinones (13.5%) in comparison to that of C. phlepsii (0.8%). Hexyl oleate and octyl oleate were detected for the first time in defensive millipede fluids. The chemical composition of the defensive secretions supports the chemotaxonomic position of the family Blaniulidae in the 'quinone' millipede clade.

A new dwarf schendylid centipede (Chilopoda: Geophilomorpha: Schendylidae) with a low number of legs from Serbia, Balkan Peninsula.

Schendyla antici sp. nov., a dwarf geophilomorph from the Mt. Medvednik (Western Serbia, Balkan Peninsula), is described and illustrated based on the specimens extracted from the soil samples. A detailed comparison with all species within the genus is provided. The new species has the lowest number of leg-bearing segments within the genus Schendyla Bergse & Meinert, 1866, and one of the lowest in the order Geophilomorpha in general.

Chemistry of the sternal gland secretion of the Mediterranean centipede Himantarium gabrielis (Linnaeus, 1767) (Chilopoda: Geophilomorpha: Himantariidae).

The geophilomorph centipede, Himantarium gabrielis, when disturbed, discharges a viscous and proteinaceous secretion from the sternal glands. This exudate was found by gas chromatography-mass spectrometry, liquid chromatography-high resolution mass spectrometry, liquid chromatography-mass spectrometry-mass spectrometry and NMR analyses to be composed of hydrogen cyanide, benzaldehyde, benzoyl nitrile, benzyl nitrile, mandelonitrile, mandelonitrile benzoate, 3,7,6O-trimethylguanine (himantarine), farnesyl 2,3-dihydrofarnesoate and farnesyl farnesoate. This is the first report on the presence of benzyl nitrile and mandelonitrile benzoate in secreted substances from centipedes. Farnesyl 2,3-dihydrofarnesoate is a new compound, while himantarine and farnesyl farnesoate were not known as natural products. A post-secretion release of hydrogen cyanide by reaction of mandelonitrile and benzoyl nitrile was observed by NMR, and hydrogen cyanide signals were completely assigned. In addition, a protein component of the secretion was analysed by electrophoresis which revealed the presence of a major 55 kDa protein. Analyses of the defensive exudates of other geophilomorph families should produce further chemical surprises.

Dazbogosoma mokoshae, a new cavernicolous species of the previously monospecific genus Dazbogosoma Makarov amp; uri, 2012, from Serbia (Diplopoda: Chordeumatida: Anthroleucosomatidae).

A new troglobiotic species, Dazbogosoma mokoshae sp. nov., is described from a cave in the eastern Serbia. This is the second species from the previously monospecific genus Dazbogosoma Makarov uri, 2012. An updated diagnosis of the genus, as well as a diagnosis, detailed description, illustrations and in situ photographs of a new species, based on both males and females, are presented. The distribution and relationships within the Bulgarosoma complex comprising D. mokoshae sp. nov. are briefly discussed.

Composition of the defensive secretion in three species of European millipedes.

Three European julid species, Cylindroiulus boleti, Leptoiulus trilineatus, and Megaphyllum bosniense, secrete mixtures of up to 12 different quinones. The major components in these species are 2-methoxy-3-methyl-1,4-benzoquinone and 2-methyl-1,4-benzoquinone. 2-Methoxy-5-methylhydroquinone is detected for the first time in the Class Diplopoda. 2-Hydroxy-3-methyl-1,4-benzoquinone, 2,3-dimethoxyhydroquinone, 2-methyl-3,4-methylendioxyphenol, and 2,3-dimethoxy-5-methylhydroquinone are registered for the first time in representatives of the family Julidae. The similar chemical composition of defense secretions in all analyzed European julids and Pacific spirobolids supports the idea of the chemical consistency of defensive compounds in juliform millipedes.

Defensive secretions in Callipodella fasciata (Latzel, 1882; Diplopoda, Callipodida, Schizopetalidae).

The small millipede Callipodella fasciata secretes an earthy smell when disturbed. This secretion was obtained by CH(2) Cl(2) extraction from specimens of both sexes and was identified by GC/MS analyses to be composed of p-cresol (96.5%), phenol (3.5%), and p-ethylphenol (traces). This is the first identification of these compounds in an epigean European callipodidan species and the first report of intergeneric differences in the chemical composition of defensive secretions in callipodidans. These compounds have repellent, antimicrobial, and antifungal properties.

Macedomeris, a new monotypic doderiinine genus (Diplopoda, Glomerida) from a cave in North Macedonia, Balkan Peninsula.

Here we describe a new monotypic glomerid genus, Macedomeris gen. nov., with Macedomeris ivoi sp. nov. (a presumed troglobiont) as its type species. The new genus clearly differs from all other genera within the order Glomerida by the combination of several morphological characters: the presence of a characteristic deep lateral pit ("Ohrgrube") on both sides of the thoracic shield, the absence of striking ornamentation on tergites, the fusion of tergite 11 with the anal shield, and the lack of a large medial hump. In addition, Macedomeris gen. nov. differs from other members of the Glomerida by its general appearance, the presence of 2 or 3 vestigial ommatidia and a very wide syncoxite of a 2-segmented leg-pair 17 in the male. Notes on its ecology, troglomorphic features, and relationships with similar members of the tribe Doderiini are also given.

Morphological integration of the head capsule in the millipede Megaphyllum unilineatum (C. L. Koch, 1838) (Diplopoda: Julida): can different modules be recognized?

Covariation of multiple morphological traits and modularity have been widely studied in the field of evolutionary developmental biology. Subunits of a morphological structure can evolve separately from each other in a modular fashion. The aims of our study therefore were: i) to test the hypothesis of modularity in the dorsal part of the head capsule and the gnathochilarium separately during late postembryogenesis in the julidan millipede Megaphyllum unilineatum (C. L. Koch, 1838) using geometric morphometrics; and ii) to investigate the influence of allometry on overall morphological integration in the dorsal part of the head capsule and the gnathochilarium in the mentioned species. Individuals from different ontogenetic stadia (stadium VI - stadium XI) were included in the analyses. Significant influence of fluctuating asymmetry on the dorsal part of the head capsule shape was detected by Procrustes ANOVA. Regressions were significant for the symmetric component of both analysed morphological traits, while non-significant regression was detected for the asymmetric component of the head capsule's dorsal part. Hypotheses of modularity for the dorsal part of the head capsule and the gnathochilarium are rejected because our results indicate that a small proportion of alternate partitions has higher covariation between subsets of structure than between the hypothesized modules. Contrary to our expectations, results of the present study show that allometry does not increase the level of morphological integration in the dorsal part of the head capsule and the gnathochilarium in M. unilineatum. Based on the obtained results, we conclude that the dorsal part of the head capsule and the gnathochilarium are not composed of independent modules and that in the case of the capsule's dorsal part, developmental processes affect morphological integration in different ways at different levels of shape variation.

Defensive secretions in three species of polydesmids (Diplopoda, Polydesmida, Polydesmidae).

Nine compounds were detected in three different millipede species: Polydesmus complanatus (L.), Brachydesmus (Stylobrachydesmus) avalae Curcic & Makarov, and Brachydesmus (Stylobrachydesmus) dadayi Verhoeff. Benzaldehyde, benzyl alcohol, benzoylnitrile, benzyl methyl ketone, benzoic acid, benzyl ethyl ketone, mandelonitrile, and mandelonitrile benzoate were identified by GC-FID and GC-MS analyses. Hydrogen cyanide was detected qualitatively by the picric acid test. Benzyl ethyl ketone, benzyl methyl ketone, and benzyl alcohol were detected for the first time in polydesmidan millipedes. Benzoylnitrile was the major component in all three hexane extracts. These compounds are suspected to be active in the defensive secretions of these millipede species.

Sexual size and shape dimorphism in Brachydesmus troglobius Daday, 1889 (Diplopoda, Polydesmida).

Until now, morphological trait variation has been investigated in several millipede species using geometric morphometrics. The present study is the first attempt to explore sexual shape and size dimorphism (SShD and SSD) of morphological structures in Polydesmida. We here analyse antennal, head, and leg SShD and SSD in Brachydesmus troglobius Daday, 1889. Our results show that SSD exists in all of the analysed structures, while SShD is present only in the legs. In comparison with females, males possess longer and wider legs, as well as longer antennae and a shorter head. Contrary to previous findings in some Julida, in B. troglobiusSSD of the antennae and legs varies more than SShD in these morphological structures.

Life after the mother's hug: Late post-embryonic development of Cryptops parisi (Chilopoda: Scolopendromorpha: Cryptopidae).

Here we describe in detail the late post-embryonic development of the common European scolopendromorph centipede Cryptops parisi. Canonical variate analyses of two groups of external morphological characters, viz., cephalic capsule characters (head length, length of the anterior and posterior paramedian cephalic sutures) and coxopleuron surface characters (number of pores in the coxal pore-field, number of setae on the posterior coxopleuron edge, their number on the coxal pore-field, and their number posterior to the coxal pore-field) were conducted on a large sample of specimens collected from two localities in Serbia. Ten free-living stages are recognized: three pre-adult stages (adolescens I, II, and III) and seven adult stages (one maturus junior stage, four maturus, and two maturus senior stages). The fourth late post-embryonic stage is the first mature stage in both sexes. Sexual dimorphism in the aforementioned characters was not observed. Morphological variation of coxopleuron characters was more informative for the discrimination of developmental stages in Cryptops than the morphological variation of cephalic capsule characters.

Identification of secretory compounds from the European callipodidan species Apfelbeckia insculpta.

Defensive secretions of the callipodidan species Apfelbeckia insculpta contain a p-cresol as the main component and phenol in traces. This is the first identification of these compounds in a European callipodidan species. The repugnatory glands of A. insculpta are of the spirobolid type and consist of a spherical reservoir, a simple duct, and a valvular cuticular infolding that opens onto the lateral surface of the millipede via a pore.

Multifaceted activity of millipede secretions: Antioxidant, antineurodegenerative, and anti-Fusarium effects of the defensive secretions of Pachyiulus hungaricus (Karsch, 1881) and Megaphyllum unilineatum (C. L. Koch, 1838) (Diplopoda: Julida).

Members of the millipede order Julida rely on dominantly quinonic defensive secretions with several minor, non-quinonic components. The free radical-scavenging activities of ethanol, methanol, hexane, and dichloromethane extracts of defensive secretions emitted by Pachyiulus hungaricus (Karsch, 1881) and Megaphyllum unilineatum (C. L. Koch, 1838) were investigated using the ABTS, DPPH, and total reducing power (TRP) tests. The obtained extracts were also tested for inhibition of acetylcholinesterase and tyrosinase activity. Finally, the antifungal potential of both julid extracts was evaluated against seven Fusarium species. Secretions of both species showed activity against free radicals, acetylcholinesterase, tyrosinase, and all of the selected fungal species. The secretions of P. hungaricus exhibited a more potent antioxidative effect than did those of M. unilineatum, while there were no significant differences of antiacetylcholinesterase activity between the tested extracts. Only the hexane extract of M. unilineatum showed an effect on tyrosinase activity stronger than that of P. hungaricus. Fusarium sporotrichioides, F. graminearum, and F. verticillioides were the fungi most resistant to secretions of both julids. The Fusarium species most susceptible to the secretion of P. hungaricus was F. avenaceum, while the concentrations of M. unilienatum extracts needed to inhibit and completely suppress fungal growth were lowest in the case of their action on F. lateritium. Our data support previous findings that julid defensive secretions possess an antimicrobial potential and reveal their antioxidative and antineurodegenrative properties. Bearing in mind the chemical complexity of the tested defensive secretions, we presume that they can also exhibit other biological activities.

Geophilus serbicus sp. nov., a new species from the Balkan Peninsula (Chilopoda: Geophilomorpha: Geophilidae).

Geophilus serbicus sp. nov., a new epigeic centipede species, is described and illustrated based on specimens collected from the Stara Planina Mountains (the Balkan Mountain Range), Eastern Serbia, Balkan Peninsula. Considerations on the taxonomic relationships with some similar Geophilus species are briefly presented, and the distribution of the new species is mapped.

Rhabditophanes schneideri (Rhabditida) phoretic on a cave pseudoscorpion.

Information of phoretic nematode-pseudoscorpion associations and cases of parasitism on five European species of pseudoscorpions was summarized by Curcic et al. [Curcic, B.P.M., Dimitrijevic, R.N., Makarov, S.E., Lucic, L.R., Curcic, S.B., 1996. Further report on nematode-pseudoscorpion associations. Acta arachnol. 45, 43-46; Curcic, B.P.M., Sudhaus, W., Dimitrijevic, R.N., Tomic, V.T., Curcic, S.B., 2004. Phoresy of Rhabditophanes schneideri (Bütschli) (Rhabditida: Alloionematidae) on pseudoscorpions (Arachnida: Pseudoscorpiones). Nematology 6 (3), 313-317]. An examination of a sample of the cavernicolous pseudoscorpion Neobisium rajkodimitrijevici (Curcic and Tomic, 2006) (comprising a holotype male and a paratype tritonymph) from a cave in Eastern Serbia revealed this false scorpion to be a nematode carrier; the present paper reports this finding and extends our knowledge of phoresy of Rhabditophanes on pseudoscorpions. This is the first time that the rhabditid R. schneideri (Bütschli, 1873) has been noted in association with a cavernicolous pseudoscorpion. There must be some patchily distributed micro-habitats in caves where saprobiotic nematodes and small arthropods can complete their life-cycles, for example something like deposits of bat guano. The transportation of Rhabditophanes J3 by pseudoscorpions indicate that Neobisium specimens often visit these micro-habitats to find their prey.