Arthropoda Selecta 29(4): 413–418 © ARTHROPODA SELECTA, 2020 Assessment of p-cresol and phenol antifungal interactions in an arthropod defensive secretion: the case of an endemic Balkan millipede, Apfelbeckia insculpta (L. Koch, 1867) (Diplopoda: Callipodida) Îöåíêà ôóíãèöèäíûõ âçàèìîäåéñòâèé p-êðåçîëà è ôåíîëà â çàùèòíîì ñåêðåòå ÷ëåíèñòîíîãîãî: ïðèìåð ýíäåìè÷íîé áàëêàíñêîé äèïëîïîäû Apfelbeckia insculpta (L. Koch, 1867) (Diplopoda: Callipodida) Bojan Ilić*, Nikola Unković, Danica Ćoćić, Jelena Vukojević, Milica Ljaljević Grbić, Slobodan Makarov Áîÿí Èëè÷*, Íèêîëà Óíêîâè÷, Äàíèöà ×î÷è÷, Åëåíà Âóêîéåâè÷, Ìèëèöà Ëüÿëüåâè÷ Ãðáè÷, Ñëîáîäàí Ìàêàðîâ University of Belgrade, Faculty of Biology, Studentski Trg 16, 11000 Belgrade, Serbia. Email: [email protected] KEY WORDS: allomones, antimycotics, checkerboard method, millipede, phenolics. КЛЮЧЕВЫЕ СЛОВА: алломоны, антимикотики, шахматный метод, двупарноногие многоножки, фе- нолики. ABSTRACT. Millipedes (Diplopoda) are a group interactions, i.e., synergism, additivism and indiffer- of arthropods that produce and deploy chemically di- ence, were observed. No antagonism between com- verse exudates from defensive glands in the event of pounds was documented in any of the tested combina- predator attack. These exocrine secretions are also po- tions. Meyerozyma guilliermondii was the only tested tent antimicrobials. In view of the fact that the defen- fungus where no interactions could be determined (MIC sive secretion of the endemic Balkan millipede, Apfel- >1.0 mg mL–1). beckia insculpta (L. Koch, 1867) consists of only two How to cite this article: Ilić B., Unković N., Ćoćić compounds with known antifungal properties (p-cresol D., Vukojević J., Ljaljević Grbić M., Makarov S. 2020. and phenol), it represents an ideal model for studying Assessment of p-cresol and phenol antifungal interac- the contribution of individual compounds to the overall tions in an arthropod defensive secretion: the case of antifungal activity of this natural product, thereby en- an endemic Balkan millipede, Apfelbeckia insculpta abling us to define the nature and type of interactions (L. Koch, 1867) (Diplopoda: Callipodida) // Arthropo- between the main compound (p-cresol) and the trace da Selecta. Vol.29. No.4. P.413–418. doi: 10.15298/ compound (phenol). Twenty-five combinations of con- arthsel. 29.4.03 centrations, ranging from 0.1 to 1.0 mg mL–1, were tested on 14 filamentous fungi belonging to the genera РЕЗЮМЕ. Двупарноногие многоножки (Diplo- Aspergillus, Cladosporium, Fusarium, Penicillium and poda) — это группа членистоногих, производящая Trichoderma; two yeasts (Sporobolomyces roseus and и выделяющая разнообразные химические эксуда- Meyerozyma guilliermondii); and one yeast-like fila- ты из защитных желез в случае атаки хищника. Эти mentous fungus (Aureobasidium pullulans var. melan- экзокринные выделения одновременно являются и ogenum) using the checkerboard method. Among the потенциальными антимикробными агентами. По- 29 interactions observed, the tested combinations of скольку защитный секрет эндемичного балканско- concentrations showed mainly additive (16 instances) го вида Apfelbeckia insculpta (L. Koch, 1867) состо- and, to a lesser extent, indifferent (12 instances) prop- ит лишь из двух компонент с известными противо- erties, with notably 2–8 times lower concentrations of грибковыми свойствами (р-крезол и фенол), он compounds needed to suppress fungal growth than those представляет собой идеальную модель для изу- recorded for the individual compounds. A synergistic чения вклада отдельных компонент в общую фун- effect was observed only for Aspergillus niger when гицидную активность этого натурального продук- 0.1 mg mL–1 of p-cresol was supplemented with 0.2 mg та, тем самым позволяя нам определить природу и mL–1 of phenol. Furthermore, A. niger was the only тип взаимодействий между главной (р-крезол) и fungus where all three types of documented antifungal следовой компонентами (фенол). В 25 комбинаци- 414 B. Ilić et al. Figure. Habitus of Apfelbeckia insculpta (L. Koch, 1867). Рисунок. Общий вид Apfelbeckia insculpta (L. Koch, 1867). ях их концентраций, от 0,1 до 1,0 мг/мл, шахмат- ical defence, millipedes (Diplopoda) are regarded as ным методом было проверено действие секрета на particularly “accomplished chemists” [Makarov, 2015; 14 видов мицелиальных грибков родов Aspergillus, Shear, 2015; Ilić et al., 2018]. Cladosporium, Fusarium, Penicillium и Trichoderma, Besides the thick cuticle hardened by deposits of два вида дрожжей (Sporobolomyces roseus и Meyero- calcium [Hopkin, Read, 1992; Makarov, 2015], the zyma guilliermondii) и один вид похожего на дрож- majority of millipedes possess a serially arranged sys- жи мицелиального грибка (Aureobasidium pullulans tem of defensive glands (ozadenes), starting from the var. melanogenum). Среди 29 наблюдавшихся взаи- third or fifth trunk segment [Sierwald, Bond, 2007; модействий проверяемые комбинации концентра- Koch, 2015; Makarov, 2015; Shear, 2015]. Millipede ций, в основном, оказывали аддитивное действие defensive glands are places of production and storage (16 случаев) и в меньшей мере были нейтральны of a wide range of chemical compounds — heterocy- (12 случаев) в концентрациях даже в 2–8 раз ниже clic nitrogen-containing compounds, terpenes, benzo- требуемых, чтобы подавить рост грибков, чем для quinones and hydroquinones, fatty acid esters, various отдельных компонент. Синэргический эффект на- aliphatic compounds, phenolics and cyanogenic com- блюдался только для Aspergillus niger при концент- pounds [Makarov, 2015; Shear, 2015]. Among the Hel- рации 0,1 мг/мл р-крезола с добавкой 0,2 мг/мл minthomorpha, it is presumed that the phenolic defen- фенола. Более того, A. niger был единственным из sive system is the oldest one [Rodriguez et al., 2018]. грибков, где проявились все три типа документи- Although members of the order Callipodida are regard- рованных фунгицидных взаимодействий, т.е. синэ- ed as “phenolic” millipedes (sensu Eisner et al., 1978) ригизм, аддитивизм и нейтральность. Антагонизм because these millipedes rely exclusively on phenolic не был выявлен ни в одной из тестируемых комби- allomones [Ilić et al., 2019 and references therein], наций компонент. Вид Meyerozyma guilliermondii phenolics can also be found in the defensive secretions был единственным тестируемым грибком, где не of members of other millipede orders, e.g., Polydesmi- выявлено никаких взаимодействий (MIC >1,0 мг/ da and Julida [Makarov, 2015; Shear, 2015]. мл). Three phenolic compounds have been so far re- corded in callipodidan defensive secretions: p-cresol, Introduction phenol and p-ethylphenol [Ćurčić et al., 2009; Shear et al., 2010; Makarov et al., 2011; Ilić et al., 2019]. A In contrast to faster moving and much more aggres- common feature of all screened callipodidan defensive sive predators, organisms with a limited range of move- secretions is that p-cresol is the only or dominant com- ment or limited control over their movements are usu- pound. For example, p-cresol constitutes 99% of the ally well defended chemically [Berenbaum, 1995]. defensive secretion in Apfelbeckia insculpta (L. Koch, Many arthropod groups are included in the ranks of 1867) (Figure) [Ilić et al., 2019], while phenol and p- these organisms. Among arthropods that rely on chem- ethylphenol can be present either as minor or trace Antifungal interactions in a millipede defensive secretion 415 compounds in other callipodidan defensive gland exu- final concentration of suspensions was adjusted to 1.0 dates [Ćurčić et al., 2009; Makarov et al., 2011; Ilić et × 105 CFU mL–1. Before use, a measured volume (10 al., 2019]. µL) of suspensions was cultured on MEA plates to Millipede defensive secretions are in most cases bi-, check validity of the inocula and verify the absence of oligo- or multicomponent mixtures. This fact, i.e., the contamination. existence of at least two chemical compounds in such gland exudates, is a precondition for different interac- Determination of interaction type tions (e.g. synergism or antagonism) between these The type of interaction between compounds of the chemicals. Both compounds that are recorded in the defensive secretion of A. insculpta was assessed by the defensive secretion of A. insculpta (p-cresol and phe- checkerboard method [Schwalbe et al., 2007]. Malt nol) are known to possess high bactericidal and fungi- extract broth medium (of the following composition, cidal activity [Murakami et al., 2014; Sabbineni, 2016] per litre of deionized water: malt extract, 40 g; pH 6.8) and are responsible for the antimicrobial potency of was distributed into each well of 96-well microplates this secretion, which was proven for the first time by (F-bottom, Ratiolab). Two-fold serial dilutions of p- Ilić et al. [2019] on a number of pathogenic bacteria, cresol and phenol were made individually, on two sep- yeasts and filamentous fungi. As this defensive secre- arate plates, with concentrations in the range of from tion consists of only two compounds, it represents an 0.1 to 1.0 mg mL–1. Next, well contents were mixed on ideal model on which to study the contribution of indi- a single plate and 25 combinations of concentrations vidual compounds to the overall antifungal activity of were tested. Suspensions of tested fungi (10 µL) were this potent natural product. Here we report for the first added to each well reaching a final volume of 100 µL time the nature and type of antifungal interactions be- per well. After 72 h of incubation at 25 ± 2°C (UE 500, tween