Sexual selection can be a major driving force that favours morphological evolution at the intraspecific level. According to the sexual selection theory, morphological variation may accompany non-random mating or fertilization. Here both variation of linear measurements and variation in the shape of certain structures can significantly influence mate choice in different organisms. In the present work, we quantified sexual behaviour of the millipede Megaphyllum bosniense (Verhoeff, 1897) as characterized by several sequences. These are: mating latency, duration of copulation, contact to copulation time, duration of contact without copulation, time from entrance (time-point when individuals were placed in boxes in which tests occurred) to contact with copulation, and time from entrance to contact without copulation. Further, we analysed the influence of morphological variation (both variation of linear measurements and variation in the shape of several structures) on mating success. Variation of body length, antennal length, length of the walking legs, trunk width, and trunk height was analysed by traditional morphometrics, while variation in size and shape of the antennae, walking legs, head, and gonopods (promeres, opisthomeres) was analysed using geometric morphometrics. More than half of all physical contacts detected among the millipedes resulted in copulation. Based on the value of sexual selection coefficients, preferences toward the previous partner were found to be prevalent in both female and male choice tests. Individuals with different mating status significantly differed in some morphological traits (body mass, head centroid size, head shape, and promere shape). Our study yielded new information about the sexual behaviour of millipedes and variation of morphological traits as a potential basis for mate preferences.

The chemical defence of the millipede Pachyiulus hungaricus is reported in the present paper, in which a chemical characterization is given and antimicrobial activity is determined. In total, independently of sex, 44 compounds were identified. All compounds belong to two groups: quinones and pentyl and hexyl esters of long-chain fatty acids. The relative abundances of quinones and non-quinones were 94.7% vs. 5.3% (males) and 87.3% vs. 12.7% (females), respectively. The two dominant quinones in both sexes were 2-methyl-1,4,-benzoquinone and 2-methoxy-3-methyl-1,4-benzoquinone. Antibacterial and antifungal activity of the defensive secretion was evaluated in vitro against seven bacterial strains and eight fungal species. With the aid of a dilution technique, the antimicrobial potential of the secretion and high sensitivity of all tested strains were confirmed. The lowest minimum concentrations of these compounds (0.20–0.25 mg/mL) were sufficient for inhibition of Aeromonas hydrophila, Listeria monocytogenes and Methicillin resistant Staphylococcus aureus (MRSA). The growth of eight tested fungal species was inhibited by slightly lower concentrations of the secretion, with Fusarium equiseti as the most sensitive fungus and Aspergillus flavus as the most resistant. Values of MIC and MFC in the employed microdilution assay ranged from 0.10 to above 0.35 mg/mL. The given extract contains antimicrobial components potentially useful as therapeutic agents in the pharmaceutical and agricultural industries.

Although millipedes (Diplopoda) represent one of the most diverse classes of arthropods, data concerning details of their mating behaviour are very scarce. In this work, we explored mating behaviour of the European millipede Pachyiulus hungaricus under laboratory conditions, and its relationship with the size and shape of certain morphological traits. We conducted 3 types of behavioural tests: a mating arena test, a female choice test, and a male choice test. Premating behaviour was "sequenced" in 5 behavioural steps and, together with the duration of copulation, scored in all mating assays. Males were the more active sex in searching for mates, while females were the "choosier" sex. Furthermore, in the choice tests, previous mating partners had significantly more copulations than new ones, thus raising questions about postcopulatory sexual selection in this species. On the other hand, our results indicate that size and/or shape of the tested morphological traits, except for the shape of the male walking legs, were not subject to precopulatory sexual selection. Other sensory domains known to influence courtship behaviour need to be investigated in this regard in P. hungaricus.

Sexual size and shape dimorphism (SSD and SShD) are understudied phenomena in millipedes. In the present study, we investigated both kinds of sexual dimorphism in some morphological traits, as well as whether shape of the morphological traits varied more than their size. Three julidan species, viz., Pachyiulus hungaricus (Karsch, 1881), Megaphyllum unilineatum (C.L. Koch, 1838), and M. bosniense (Verhoeff, 1897), were used for these purposes. By means of traditional and geometric morphometrics, we found that SSD exists in linear measurements of the tested morphological traits, as well as SShD of the legs in all analysed species. Also, SSD of antennal centroid size was detected in P. hungaricus and M. unilineatum, in addition to SShD of antennae in P. hungaricus and M. bosniense. Our results indicate that morphological intersexual differences are species-specific and that the shape of some morphological traits varies more than the size of centroids of the same structures.

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.