Društvo biologov Slovenije 2023 Vol. 66 | Št. 2 2 Acta Biologica Slovenica, 2023, 66 (2) Acta Biologica Slovenica, 2023, 66 (2) Založila/Published by Založba Univerze v Ljubljani / University in Ljubljana Press Društvo biologov Slovenije / Slovenian biological society Za založbo/For the publisher Gregor Majdič, rektor Univerze v Ljubljani / the Rector of the University of Ljubljana Anita Jemec Kokalj, predsednica Društva biologov Slovenije / Chairman of Slovenian Biological Society Izdala/Issued by Univerza v Ljubljani, Biotehniška fakulteta, Oddelek za biologijo / University of Ljubljana, Biotehnical Faculty, Department of Biology Za izdajatelja/For the Issuer Marina Pintar, dekanja Biotehniške fakultete UL / Dean of Biotechnical Faculty Naslov uredništva/Editorial Office Address Univerza v Ljubljani, Biotehniška fakulteta, Acta Biologica Slovenica, Večna pot 111, 1000 Ljubljana, Slovenija Glavni urednik/Editor-in-chief Matevž Likar, Slovenija / Slovenia, matevz.likar@bf.uni-lj.si Odgovorna urednica/Managing editor Anita Jemec Kokalj, Slovenija / Slovenia, anita.jemec@bf.uni-lj.si Uredniški odbor/Editorial Board Gregor Belušič (SLO), Univerza v Ljubljani, Biotehniška fakulteta Tina Eleršek (SLO), Nacionalni inštitut za biologijo Alenka Gaberščik (SLO), Univerza v Ljubljani, Biotehniška fakulteta Király Gergely (HU), University of Sopron, Faculty of Forestry Georg A. Janauer (A), University of Vienna Simona Strgulc Krajšek (SLO), Univerza v Ljubljani, Biotehniška fakulteta Vida Jojić (SRB), Univerzitet u Beogradu, Institut za biološka istraživanja „Siniša Stanković” Tina Klenovšek (SLO), Univerza v Mariboru, Fakulteta za naravoslovje in matematiko Alenka Malej (SLO), Nacionalni inštitut za biologijo Maria Mueller (A), University of Salzburg Siniša Ozimec (HR), Univerza Josipa Juraja Strossmayerja Hubert Potočnik (SLO), Univerza v Ljubljani, Biotehniška fakulteta Mihael Jožef Toman (SLO), Univerza v Ljubljani, Biotehniška fakulteta Božo Frajman (A), Univerza v Innsbrucku Gordana Glavan (SLO), Univerza v Ljubljani, Biotehniška fakulteta Nataša Mori (SLO), Nacionalni inštitut za biologijo Polona Mrak (SLO), Univerza v Ljubljani, Biotehniška fakulteta Oblikovanje/Design Ajda Fortuna Naslovnica/Cover page Navadni prašiček (Porcellio scaber), avtor: Teo Delič To delo je ponujeno pod licenco Creative Commons Priznanje avtorstva-Deljenje pod enakimi pogoji 4.0 Mednarodna licenca (izjema so fotografije). / This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License (except photographies). Izdajanje revije sofinancira Javna agencija za znanstvenoraziskovalno in inovacijsko dejavnost Republike Slovenije (ARIS) The journal is co-financed by Slovenian Research and Innovation Agency (ARIS) Publication is free of charge. ISSN 1854-3073 (spletna verzija/online version) UDK 57(497.4) DOI: 10.14720/abs.66.2 http://journals.uni-lj.si/abs/ Acta Biologica Slovenica je indeksirana v – is indexed in: CAB Abstracts, Web of Science Clarivate, Ebsco, Scopus 3 Acta Biologica Slovenica, 2023, 66 (2) Original Research Paper 4 Vpliv simptomatske bakterijske okužbe na izražanje genov, povezanih z imunostjo v hemocitih kopenskega raka Porcellio scaber / Effect of symptomatic bacterial infection on the expression of immune-related genes in haemocytes of the terrestrial crustacean Porcellio scaber Andraž Dolar, Jernej Ogorevc, Anita Jemec Kokalj 16 Contribution of neutral processes to the assembly of microbial communities on Phragmites australis leaf litter / Pomen nevtralnih procesov pri oblikovanju mikrobnih skupnosti na listnem opadu vrste Phragmites australis Matevž Likar, Mateja Grašič, Alenka Gaberščik News 26 In memoriam: prof. Dr. Dušan Zavodnik (1934–2023) dr. Andrej Jaklin Table of Contents 4 1 Oddelek za biologijo, Biotehniška fakulteta, Univerza v Ljubljani, Jamnikarjeva 101, 1000 Ljubljana 2 Oddelek za zootehniko, Biotehniška fakulteta, Univerza v Ljubljani, Jamnikarjeva 101, 1000 Ljubljana * Corresponding author: E-mail address: andraz.dolar@bf.uni-lj.si Citation: Dolar, A., Ogorevc, J., Kokalj, A. J. (2023). Vpliv simptomatske bakterijske okužbe na izražanje genov, povezanih z imunostjo v hemocitih kopenskega raka Porcellio scaber. Acta Biologica Slovenica 66 (2) https://doi.org/10.14720/abs.66.2.14428 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Original Research Article Vpliv simptomatske bakterijske okužbe na izražanje genov, povezanih z imunostjo v hemocitih kopenskega raka Porcellio scaber Andraž Dolar 1,*, Jernej Ogorevc 2, Anita Jemec Kokalj 1 Izvleček Kopenski enakonožci vrste Porcellio scaber so v naravnem okolju izpostavljeni številnim patogenom in parazitom, ki lahko pri gostitelju povzročijo poškodbe tkiv ter vplivajo na imunokompetenco in fitnes organizma. Bakterijska okužba povzroči aktivacijo mehanizmov prirojene imunosti, kot so fagocitoza, tvorba reaktivnih kisikovih in dušikovih zvrsti, aktivnost antioksidativnih encimov, nodulacija ter proces melanizacije. Molekularni vzorci patogenov oziroma mikrobov ter s patogenezo povezane poškodbe pri gostitelju sprožijo prepisovanje genov v celicah hemolimfe, tj. hemocitih, ki opravljajo pomembno funkcijo mediatorjev imunskega odgovora. V aktualni raziskavi smo preučevali spremembe v izražanju genov ob simptomatski bakterijski okužbi z Rhabochlamydia porcellionis ter jih primerjali z asimptomatskimi oziroma zdravimi P. scaber. Iz hemolimfe (hemocitov) asimptomatskih in simptomatskih živali smo izolirali celokupno RNA, jo prepisali v cDNA ter z metodo RT-qPCR določili relativno izražanje izbranih genov, povezanih z imunostjo (Toll4, Dscam, MyD88, Cat, MnSod, CypG, A2m, Atg5 in Nos). Ugotovili smo značilne spremembe v izražanju izbranih genov, kar kaže na njihovo vlogo v imunskem odgovoru P. scaber v primeru bakterijske okužbe, dodatno pa smo z biokemijskimi metodami dokazali povečano aktivnost encima alfa-2-makroglobulin ter mejno značilno povišanje encima katalaza. Na podlagi rezultatov lahko zaključimo, da preučevani geni predstavljajo molekularne označevalce za imunski odziv, ki jih je moč uporabiti v različnih okoljskih raziskavah. Ključne besede bakterijska okužba, imunski odziv, izražanje genov, kopenski enakonožec Porcellio scaber, Rhabdochlamydia porcellionis 5 Acta Biologica Slovenica, 2023, 66 (2) Effect of symptomatic bacterial infection on the expression of immune-related genes in haemocytes of the terrestrial crustacean Porcellio scaber Abstract Terrestrial isopods Porcellio scaber are exposed to many pathogens and parasites in their natural environment, which can cause tissue damage in the host and affect the immunocompetence and fitness of the organism. Bacterial infection leads to activation of innate immunity mechanisms, such as phagocytosis, formation of reactive oxygen and nitrogen species, activity of antioxidant enzymes, nodule formation, and the process of melanization. Molecular patterns of pathogens or microbes and pathogenesis-induced injury in the host trigger the transcription of genes in haemolymph cells, i.e., haemocytes, which have an important function as mediators of the immune response. In the current study, we examined changes in gene expression during symptomatic bacterial infection with Rhabochlamydia porcellionis and compared them with asymptomatic or healthy P. scaber. We isolated total RNA from the haemolymph (haemocytes) of asymptomatic and symptomatic animals, transcribed it into cDNA, and determined the relative expression of selected immune-related genes (Toll4, Dscam, MyD88, Ppae2a, Cat, MnSod, CypG, A2m, Atg5, and Nos). We found characteristic changes in the expression of selected genes confirming their role in the immune response of P. scaber in case of bacterial infection, and, in addition, biochemical methods showed increased activity of the enzyme alpha-2-macroglobulin and a borderline characteristic increase in the enzyme catalase. Based on the results, we can conclude that the studied genes represent molecular markers of immune response that can be used in various environmental studies. Keywords bacterial infection, gene expression, immune response, terrestrial isopod Porcellio scaber, Rhabdochlamydia porcellionis okužbo, ki je izražena pri 15-20 % osebkov naravne pop- ulacije (Cole in Morris, 1980), medtem ko je prevalenca okužbe z bakterijo Rhabdochlamydia porcellionis še višja, in sicer dosega 27 % (Kostanjšek in Pirc Marolt, 2015). Bakterijo R. porcellionis (družina Rabdoklamidij) uvrščamo v skupino patogenih, obligatno znotrajceličnih bakterij, imenovanih klamidije, s širokim naborom gostiteljev, tako med vretenčarskimi kot tudi nevretenčarskimi organizmi (Kostanjšek in sod., 2004; Halter in sod., 2022). Pri P. scaber je okužba z R. porcellionis primarno omejena na celice prebavne žleze hepatopankreas, na površini katere v simptomatski fazi okužbe opazimo bele lise, od tod pa se okužba lahko razširi tudi na druga tkiva, med drugim tudi v hematopoetsko tkivo in pa celice hemolimfe, tj. hemocite (Kostanjšek in Pirc Marolt, 2015). V hemolimfi P. scaber s simptomatsko R. porcellionis okužbo pride do oblikovanja izrazitega imunskega odgovora, kar se kaže v drastični spremembi vrednosti imunskih parametrov hemolimfe, v primerjavi z asimptomatskimi živalmi (Kostanjšek in Pirc Marolt, 2015; Dolar in sod., 2020). Gostiteljski organizmi se Uvod Navadni prašiček Porcellio scaber (Latreille, 1804) je pred- stavnik enakonožnih rakov, ki poseljujejo naravne habitate centralne in zahodne Evrope, hkrati pa so izrazito sinantro- pna vrsta, kar pomeni, da so tesno povezani s človeškimi bivališči in njegovo dejavnostjo. Znotraj skupine rakov so enakonožci najuspešnejši kolonizatorji kopenskega okolja, ki so tekom prilagajanja na kopensko življenje razvili številne vedenjske, fiziološke in strukturne lastnosti (Hornung, 2011). V ekosistemu opravljajo pomembno vlogo razkrojevalcev odmrlega organskega materiala, s čimer prispevajo h kroženju snovi v naravi in so tako nepogrešljiv člen talnega ekosistema (Hornung in sod., 1998; van Gestel in sod., 2018). V okolju so kopenski enakonožci, podobno kot tudi ostali organizmi, podvrženi različnim nevarnostim, kot so patogeni in paraziti. Znano je, da virusne in bak- terijske okužbe pogosto prizadenejo naravne populacije višjih rakov (Wang, 2011). V naravni populaciji kopenskih rakov P. scaber pogosto zaznamo Iridovirusno IIV-31 6 Acta Biologica Slovenica, 2023, 66 (2) na vdor patogenov, parazitov in na poškodbo odzovejo z aktivacijo nabora mehanizmov prirojene imunosti, katerih glavni namen je ponovna vzpostavitev stabilnega notran- jega ravnovesja (tj. homeostaza) ter preprečitev nadaljnjih poškodb oziroma smrti organizma (Wang in Wang, 2013; Mengal in sod., 2023). V tem pogledu simptomatska bakterijska okužba, ki je prisotna v naravni populaciji P. scaber predstavlja dostopen in enostaven model za študije posameznih komponent in mehanizmov prirojene imunosti v luči odkrivanja novih bioloških označevalcev, ki jih je moč prenesti v okoljske raziskave z namenom ocene stanja organizma po izpostavitvi različnim okoljskim onesnažilom, kot so recimo kemikalije in umetnimi delci, npr. nano- in mikroplastika (Dolar in sod., 2021, 2022b,c; Jemec Kokalj in sod., 2021, 2022). Prirojen imunski sistem kopenskega enakonožca P. scaber je odgovoren za prepoznavanje in odzivanje na raznovrstne zunanje in notranje »izzivalce« (npr. patogene, parazite, poškodbe, okoljske spremembe), ki lahko resno ogrozijo zdravje in v skrajnem primeru povzročijo tudi smrt organizma (Dolar in sod., 2020, 2022a,b,c). Imunski odgovor gostitelja na mikrobno okužbo temelji na evolucijsko ohranjenih efektorskih mehanizmih celične in humoralne komponente prirojene imunosti, kot so fagocitoza, nodulacija in enkapsulacija, proces melanizacije in produkcija ter sproščanje drugih humor- alnih molekul, npr. reaktivnih kisikovih (ROS) in dušikovih spojin (RNS) ter antimikrobnih peptidov (Jiravanichpaisal in sod., 2006; Söderhäll, 2016). V hemolimfi P. scaber so prisotni trije glavni tipi celic hemolimfe oziroma hemociti, tj. semigranularni, granularni in hialini, ki opravljajo specifične naloge tekom imunskega odgovora, pri čemer so hialinociti v splošnem odgovorni za fagocitozo, delno tudi semigranulociti, medtem ko pa so semigranulociti in večinsko granulociti odgovorni za produkcijo in sekrecijo različnih humoralnih molekul v hemolimfo in oblikovanje humoralnega imunskega odgovora (Tassanakajon in sod., 2013, 2018). Bakterijska okužba pri rakih izzove značilne spremembe v celokupnem (angl. total haemocyte count) kot tudi diferencialnem številu (angl. differential haemo- cyte count) hemocitov (Dolar in sod., 2020). Hkrati lahko bakterijski toksini, kot je endotoksin lipopolisaharid, poškodujejo hemocite in povzročijo njihov propad, kar se odrazi v zmanjšani viabilnosti hemocitov (Dolar in sod., 2022a). Slednji pojav je povezan tudi s povečano fagoci- totsko aktivnostjo hemocitov (hialinih in semigranularnih) in tvorbo ROS ter RNS (npr. dušikov oksid; NO) z namenom razgradnje fagocitiranih tujkov (Raman in sod., 2008; Sánchez-Salgado in sod., 2019). Povečana proizvodnja reaktivnih spojin v primeru mikrobne okužbe v hemolimfi rakov posledično izzove tudi povečano aktivnost encimov superoksid dismutaze in katalaze, ki sta odgovorna za odstranjevanje presežka reaktivnih spojin (Gopalakrishnan in sod., 2011; Liu in sod., 2013). Histopatološke poškodbe, ki jih povzročajo patogene bakterije, lahko prizadenejo tudi ostala tkiva in organe gostitelja, med drugim tudi eksoskelet, kar še dodatno poveča tveganje za vdor novih patogenov v organizem (Esteve in Herrera, 2000; Chevalier in sod., 2011; Wang, 2011; Kostanjšek in Pirc Marolt, 2015). Poškodba tkiva sproži aktivacijo sistema profenoloksidaze (proPO), kaskado dogodkov in komponent, med katerimi ima terminalno vlogo encim fenoloksidaza (PO), ki katal- izira proces sinteze rdeče-rjavega pigmenta melanina, ki skupaj s hemociti fizično omeji tujke v telesu gostitelja (t. i., nodulacija ali enkapsulacija), medtem ko stranski produkti melanizacije (kinoni, ROS, RNS) delujejo toksično in povzročijo lizo mikrobnih celic (Amparyup in sod., 2013). Pomembna komponenta proPO sistema je tudi serinski proteinazni inhibitor alfa-2 makroglobulin, ki se aktivira ob prisotnosti mikroorganizmov ter regulira fenoloksidazno aktivnost, poleg tega pa je vpleten tudi v proces strjevanja hemolimfe ter fagocitoze (Ponprateep in sod., 2017). Vdor patogena v telo oziroma poškodba gostitelja v prvi vrsti povzroči aktivacijo receptorskih molekul, ki se nahajajo v plazmi hemolimfe oziroma na površini hemoc- itov, sledi signalna transdukcija, tj. kaskada dogodkov, ki privede do sprememb v profilu izražanja genov, ki so posredno ali neposredno povezani z imunostjo in drugimi procesi, odgovornimi za uravnavanje homeostaze (Sán- chez-Salgado in sod., 2021; Liu in sod., 2022). V fizioloških in funkcionalnih raziskavah genov in molekul, ki so odgov- orni za imunske mehanizme v primeru mikrobne okužbe gostitelja se uporablja napredne omske metode, te metode pa v okoljskih raziskavah odpirajo povsem novo ero v raziskovanju vplivov različnih onesnažil in okoljskih sprememb na poskusne organizme (Lou in sod., 2022; Sun in sod., 2022; Mengal in sod., 2023). Namen študije je bil preučiti izražanje izbranih genov, povezanih z imunostjo, v hemocitih P. scaber s simptom- atsko okužbo z Rhabdochlamydia porcellionis. Izražanje genov smo primerjali z asimptomatskimi osebki P. scaber. Poleg tega nas je zanimala tudi razlika v aktivnosti encimov alfa-2-makroglobulin ter katalaza v primeru simptomatskih in asimptomatskih živali. 7 Acta Biologica Slovenica, 2023, 66 (2) Material in metode Poskusni organizem Poskusni organizmi P. scaber so bili izbrani iz labora- torijske kulture, ki smo jo gojili v steklenih terarijih pri konstantnih pogojih, tj. temperaturi 20 ± 2 °C, visoki vlažnosti in dnevno nočnem režimu (16/8 ur : svetloba/ tema). Stekleni terarij je bil napolnjen z ne-kontaminirano zemljo in debelo plastjo posušenih listov navadne leske (Corylus avellana), ki so bili predhodno sterilizirani. Za analizo izražanja genov so bili odbrani poskusni organizmi z izraženimi simptomatskimi belimi lisami na prebavnih žlezah, ki jih je mogoče opazovati neposredno skozi ventralno stran telesa, kot je opisano v Kostanjšek in Pirc Marolt (2015) ter Dolar in sod. (2020). Odbrane živali so imele prisotne značilne simptome bakterijske okužbe z Rhabdochlamydia porcellionis (Sl. 1). Živali brez izraženih simptomov okužbe z R. porcellionis (asimptomatske živali) smo uporabili kot kontrolo, kot je opisano v Dolar in sod. (2020). Izolacija hemolimfe Hemolimfo smo izolirali iz posameznih P. scaber v skladu s protokolom opisanim v Dolar in sod. (2020). Na kratko, s sterilno siringo smo prebodli integument med 5. in 6. dor- zalnim segmentom P. scaber in z rahlim stiskanjem živali pridobili kapljico hemolimfe, ki smo jo posesali s stekleno mikrokapilarno pipeto (Brand). Hemolimfo za meritev aktivnosti encimov katalaze in alfa-2-makroglobulina smo pridobili iz posamezne živali in jo nemudoma razredčili v fosfatnem pufru Dulbecco (DPBS; pH 7,1-7,5), medtem ko smo vzorec hemolimfe za izolacijo RNA pridobili z združevanjem hemolimfe iz 30 živali na skupino, tj. asimp- tomatskih in simptomatskih P. scaber. Vzorce hemolimfe smo do analize hranili na ledu. Aktivnost encima katalaza Aktivnost encima katalaza (CAT) smo v hemolimfi P. scaber določili fotometrično z meritvijo razgradnje vodik- ovega peroksida (H2O2). Izolirano hemolimfo (5 µL) smo nemudoma razredčili v 65 µL 50 mM kalij-fosfatnega pufra (KP, pH= 7) z dodano 5 mM EDTA. Za meritev smo uporabili UV mikrotiterske plošče s 96 vdolbinicami. V vdolbinico na plošči smo odpipetirali 20 µL redčene hemolimfe, 30 µL KP pufra s 5 mM EDTA in nemudoma pred meritvijo dodali še 100 µL 15,18 mM H2O2, pripravljenega v 50 mM KP pufru (pH = 7) z dodano 5 mM EDTA. Reakcijski volumen smo pred meritvijo 5-krat premešali s pipeto. Absorbanco smo merili pri 240 nm in 25 °C, 3 minute s 30-sekundnim intervalom med zaporednimi meritvami, na mikročitalcu Cytation 3 imaging reader (Biotek, ZDA). Kot negativno kontrolo smo uporabili 50 mM KP pufer (pH= 7) s 5 mM EDTA. Aktivnost CAT smo izračunali kot spremembo absorbance, merjene pri 240 nm na minuto na mg proteinov (ΔA240nm min-1 mg proteinov-1). Slika 1. (A) Simptomatska bakterijska okužba z R. porcellionis v prebavni žlezi P. scaber, razvidna z ventralne strani skozi sternite organizma. (B, C) Simptomi okužbe, tj. značilne drobne bele pike (rdeča puščica) v celicah prebavne žleze hepatopankreas. Figure 1. (A) Symptomatic bacterial R. porcellionis infection in the digestive gland of P. scaber seen from the ventral side through the sternite of the organism. (B, C) Symptoms of infection, i.e., characteristic small white dots (red arrow) in the cells of the digestive gland hepatopancreas. 8 Acta Biologica Slovenica, 2023, 66 (2) Aktivnost encima alfa-2-makroglobulin Aktivnost encima alfa-2-makroglobulin (A2M) smo v vzorcih hemolimfe P. scaber določili fotometrično z meritvijo absorbance pri 405 nm. Izolirano hemolimfo (5 µL) smo redčili v razmerju 1 : 15 (v/v) s pufrom DPBS (pH 7,1–7,5), dobro premešali in do uporabe shranili na ledu. K 50 µL razredčenega vzorca hemolimfe smo dodali 50 µL tripsina (1 mg/mL DPBS, Sigma) in inkubiral 10 min pri 37 °C na toplotnem mešalu Thermomixer compact (Eppendorf). K reakcijski mešanici smo dodali 20 µL sojinega tripsinskega inhibitorja (2 mg/mL DPBS, Sigma) in znova inkubirali 15 min pri 37 °C. Nato smo dodali še 250 µL N-benzoil-DL-arginin-pnitroanilida (BAPNA; 0,5 mg/L DPBS, Sigma), ki smo ga pripravili s 100-kratnim redčenjem založne raztopine BAPNA (50 mg/mL dimetil sulfokida; DMSO) v pufru DPBS. Sto µL reakcijske mešanice smo v treh ponovitvah prenesli na mikrotitersko ploščo s 96 vdolbinicami in 30 min merili absorbanco z mikročitalcem Cytation 3 imaging reader (Biotek, ZDA) pri 405 nm in 37 °C. Aktivnost A2M v vzorcu hemolimfe smo merili posredno preko detekcije aktivnosti tripsina. Aktivnost A2M smo izrazili kot spremembo absorbance pri 405 nm na minuto na mg proteinov (ΔA405nm min-1 mg proteinov-1). Koncentracija proteinov v hemolimfi Vsebnost proteinov v hemolimfi smo določili z uporabo komercialnega kita BCATM (Pierce, Rockford, IL, ZDA). Reagenta A in B smo pripravili v razmerju 50 : 1 (v/v) in 190 µL odpipetirali k 10 µL vzorca hemolimfe, kar je povzročilo barvno reakcijo, sorazmerno količini proteinov v vzorcu. Reakcijsko mešanico smo v treh ponovitvah prenesli na mikrotitersko ploščo s 96 vdolbinicami, inkubirali 30 min pri 37 °C, nato pa absorbanco pri 562 nm z mikročitalcem Cytation 3 imaging reader (Biotek, USA). Koncentracijo proteinov v vzorcu smo izračunali glede na umeritveno krivuljo za goveji serumski albumin (BSA; 25–2000 µM). Za negativno kontrolo smo uporabili pufer DPBS (pH= 7,1–7,5). Izolacija RNA in prepis v cDNA Celokupno RNA smo izolirali iz hemocitov v skladu z mod- ificiranim protokolom z uporabo kita RNeasy Plus Micro (Qiagen, Nemčija). Za simptomatsko in asimptomatsko skupino smo izolirali skupno 3 neodvisne vzorce RNA, pri čemer je vsak vzorec predstavljal biološko ponovitev. Združeni vzorec hemolimfe smo homogenizirali v 600 µL pufra RTL Plus in 30 sekund močno stresali, nato pa ga prenesli na gDNA odstranjevalno kolono, ki smo jo postavili v 2 mL zbiralno plastično epruveto, in centrifu- girali 15 sekund pri 12.000 obratih na minuto (Eppendorf Minispin centrifuga). Eluatu smo dodali enak volumen 70 % etilnega alkohola (EtOH) in vse skupaj prenesli v RNeasy MinElute kolono, nameščeno v 2 mL zbiralno plastično epruveto, ter centrifugirali 15 sekund pri 12.000 obratih na minuto. Vsi nadaljnji koraki centrifugiranja, vključno s prejšnjimi, so bili izvedeni pri sobni temperaturi (20–25 °C) in pri 12.000 obratih na minuto. Eluat smo zavrgli in nadal- jevali s čiščenjem vzorca z dodajanjem pufrov RW1 in RPE ter 15 sekundnim centrifugiranjem, oziroma dodatkom 80-odstotnega EtOH in 2 minutnim centrifugiranjem. V nadaljevanju smo zbiralno plastično epruveto zamenjali z novo in kolono ponovno centrifugirali, tokrat 5 minut. Celokupno RNA smo izolirali iz RNeasy MinElute kolone z dodajanjem 14 µL vode brez RNaz in centrifugiranjem 1 minuto v novo sterilno mikrocentrifugirko. Izolirano RNA smo do uporabe hranili (na hladnem) pri -70 °C. Količino in kakovost izolirane RNA smo preverili s fluorometrom Qubit 2.0 (Thermo Fisher Scientific) z uporabo kita RNA HS Assay (Qiagen, Nemčija) in Tape Station 4100 (Agilent technologies, ZDA). Iz 35 ng izolirane RNA smo pripravili komplementarno cDNA z uporabo kompleta reagentov za reverzno transkripcijo (A3500; Promega, Wisconsin, ZDA), v skladu z navodili proizvajalca (Sl. 2). Skupni reakcijski volumen sintetizirane cDNA je bil 20 µL. Do uporabe smo cDNA hranili (na hladnem) pri -20 °C. Izbor kandidatnih genov, povezanih z imunostjo ter oblikovanje začetnih oligonukleotidov Kandidatni geni, ki igrajo vlogo v imunskem sistemu rakov (Cat, katalaza; MnSod, mangan superoksid dismutaza; Nos, sintaza dušikovega oksida; CypG, ciklofilin G; Dscam, celična adhezijska molekula Downovega sindroma; Toll4, toll-u podobni receptor 4; MyD88, mieloidni diferencia- cijski faktor 88; Ppae2a, profenoloksidaza aktivacijski encim 2a; A2m, alpha-2-makroglobulin; Atg5, avtofagni protein 5) so bili izbrani zaradi njihove nepogrešljive vloge pri delovanju prirojenega imunskega sistema rakov oziroma drugih nevretenčarjev in so pogosto preučevani 9 Acta Biologica Slovenica, 2023, 66 (2) in diferenčno izraženi v primeru izpostavitve različnim stresorjem (Chevalier in sod., 2012; Tassanakajon in sod., 2013; Clark in Greenwood, 2016; Sun in sod., 2020). Poleg genov povezanih z imunostjo smo uporabili tudi dva referenčna gena, tj. faktor raztezka 2; Ef2 in beta aktin; β-Act. Zaporedja genov (kandidatnih in referenčnih) smo pridobili iz podatkovne zbirke NCBI za druge skupine rakov. Z uporabo orodja BLAST (Discontiguous Megablast in BLAST; E-vrednost < 1e-5) za prepoznavanje podobnih zaporedij smo jih primerjali s transkriptomom P. scaber. V ta namen smo uporabili transkriptom kopenskega raka P. scaber (Dolar in sod., 2022d), kot tudi dodatni transkriptom, ki je prosto dostopen v podatkovni zbirki NCBI (referenčna številka: SRX2600493; Becking in sod., 2017). Zaporedja začetnih oligonukleotidov za referenčne gene in gene, povezane z imunostjo, so bila zasnovana s programsko opremo Geneious Prime 2022.0.1 (Biomat- ters, Nova Zelandija) in sintetizirana pri komercialnem ponudniku IDT (Coralville, ZDA) (Tab. 1). Analiza izražanja genov Reakcija RT-qPCR je bila izvedena v volumnu 10 µL z uporabo GoTaq qPCR Master Mix (Promega, ZDA). V reakcijski volumen smo dodali 5 µL reagenta GoTaq® qPCR Master Mix, 1,25 µL F začetnega oligonukleotida (5 µM) in 1,25 µL R začetnega oligonukleotida (5 µM) in 2,5 µL vzorca cDNA (5-krat razredčenega v vodi brez nukleaz). Reakcije RT-qPCR so bile izvedene na plošči s 384 vdolbinicami (MicroAmp™ Optical 384-Well Reaction Plate, Applied Biosystems™), z uporabo sistema PCR v realnem času ViiA 7 (Applied Biosystems, ZDA) z nasled- njim profilom cikla: 3 minute pri 95 °C in 40 ciklov (15 s pri 95 °C , 25 s pri 60 °C in 35 s pri 72 °C), čemur je sledila določitev (talilne) krivulje. Vse reakcije kot tudi negativna kontrola brez dodane matrice (cDNA) so bile izvedene v treh tehničnih ponovitvah. Izražanje genov smo normali- zirali z uporabo dveh referenčnih genov, tj. Ef2 in β-Act, ki imata stabilno izražanje in sta bila uporabljena v podobnih Slika 2. Shema analize izražanja genov, povezanih z imunostjo, v hemocitih P. scaber. Figure 2. Scheme of analysis of immune-related gene expression in P. scaber haemocytes. 10 Acta Biologica Slovenica, 2023, 66 (2) študijah pri drugih rakih (Chevalier in sod., 2012; Xu in sod., 2020). Učinkovitost pomnoževanja začetnih oligo- nukleotidov kandidatnih genov smo določili s pripravo redčitvene vrste cDNA in validacijske krivulje. Relativno izražanje kandidatnih genov je bilo izračunano po metodi 2–ΔΔCt, ki temelji na qPCR učinkovitosti za izbrane začetne oligonukleotide, ki je znašala med 90 % in 110 % (Pfaffl, 2001). Rezultati, ki prikazujejo stopnjo izražanja genov, povezanih z imunostjo, v asimptomatski in simptomatski skupini so podani kot relativne vrednosti. Statistična analiza Statistično obdelavo in prikaz podatkov smo izvedli v programu OriginPro 2022v (Origin Lab). Porazdelitev podatkov smo preverili s Kolmogorov-Smirnovim testom. V primeru simetrične porazdelitve podatkov smo uporabili T-test za neodvisne vzorce, pred tem pa preverili enakost varianc. V primeru asimetrične porazdelitve ali neenakosti varianc podatkov smo aplicirali Mann-Whitney test. Z zvezdicami nad stolpci na grafu smo označili statistično značilne razlike med testiranimi skupinami (*, p < 0,05; **, p < 0,01; ***, p < 0,001), medtem ko lojtra (#) označuje mejno značilne spremembe (0,05 < p < 0,1). Rezultati Spremembe aktivnosti encimov katalaza in alfa-2-makrogobulin v hemolimfi P. scaber z bakterijsko okužbo V hemolimfi P. scaber s simptomatsko bakterijsko okužbo smo opazili mejno značilno povečanje aktivnosti encima katalaza (Sl. 3A), v primerjavi s kontrolo. Nasprotno pa je bila aktivnost encima alfa-2-makroglobulin značilno povečana (p < 0,01; Sl. 3B), v primerjavi z asimptomatskimi organizmi. Gen Oligonukleotidni začetnik (5′ –> 3′) Velikost produkta (bp) Referenčna št. β-Act F: CGGACGTACCACTGGTATCG R: GAGGAGGCTGCAGTTGTCAT 264 KY780298.1 Ef2 F: CGACAAAGGAAGGTGTTCTC R: ACCACCTCCACGATGAATA 101 FQ896398 Cat F: ATT GGA GAG CGA GGT CCT CT R: TGT TCC CGA CCA AAT CCC AG 316 KC668411.1 MnSod F: TCACCCAATGGTGGAGAA R: TGATCCTTGAACAGCAACAG 117 MF289344.1 Nos F: CCGTCAGCACTAGGTTTATC R: GGTCCACCTACTTGCATTT 102 GQ865598 CypG F: GAGATGGTACTGGAGGAAGA R: CAGCATTAGCCATTGAAAGC 102 EU216759.1 Dscam F: GTCCTTGCGTTCACTTCT R: GTTGGAGCCTCTGGAATATC 86 JX679085 Toll4 F: GAGATCCGAAGTATAGGTTATGC R: AGTCCTCCTGCTGTTGT 101 MF124331 MyD88 F: TGATTCTCTTCGCTGACAAA R: CTCAGACCACCAACCATATC 115 FQ906745.1 Ppae2a F: ACTACCCTAAGCCAGTGAA R: CTCCAAATTGAGTCTGTGTTATG 114 FJ620685.1 A2m F: AAATGACGAATCGGGATCTAC R: CAACCATTCCCTCGTTATGT 115 KJ540280 Atg5 F: AGC TTT GGA CAG GCT TGT GT R: ACG GTG GTT CAA TGC CTT GA 261 KP317125.1 Tabela 1. Podatki o začetnih oligonukleotidih, ki smo jih uporabili v analizi RT-qPCR. Table 1. Data on primers used in RT-qPCR analysis. 11 Acta Biologica Slovenica, 2023, 66 (2) Spremembe v izražanju genov povezanih z imunostjo pri P. scaber s simptomatsko bakterijsko okužbo Okužba P. scaber z bakterijo Rhabdochlamydia porcelli- onis je izzvala spremenjeno izražanje genov, povezanih z imunostjo, v primerjavi z asimptomatskimi, tj. zdravimi organizmi (Sl. 4). V hemocitih okuženih živali smo opazili statistično značilno povečano izražanje genov Cat (p < 0,05), CypG (p < 0,05), Dscam (p < 0,01), Toll4 (p < 0,05) in pa Ppae2a (p < 0,001), medtem ko za gene Nos, A2m in Atg5 nismo zaznali sprememb v izražanju. V primeru genov MnSod (p= 0,082) in MyD88 (p= 0,067) pa smo opazili mejno značilno povečano izražanje. Slika 3. Aktivnost encimov (A) katalaza (CAT) in (B) alfa-2-makroglobulin (A2M) v hemolimfi asimptomatskih P. scaber in osebkih s simptomatsko okužbo z R. porcellionis. Zvezdica nad stolpci na grafu prikazuje statistično značilno razliko v primerjavi z asimptomatskimi (kontrolnimi) živalmi (**; p < 0,01), medtem ko lojtra (#) označuje mejno značilne razlike (0,05 < p < 0,1). Figure 3. Activity of the enzymes (A) catalase (CAT) and (B) alpha-2-macroglobulin (A2M) in the haemolymph of asymptomatic P. scaber and individuals with symptomatic R. porcellionis infection. An asterisk above the graph bars indicates a statistically significant difference compared to asymptomatic (control) animals (**, p < 0.01), while a hash mark (#) indicates a borderline significant difference (0.05 < p < 0.1). Slika 4. Izražanje genov povezanih z imunostjo v hemocitih P. scaber s simptomatsko bakterijsko (R. porcellionis) okužbo. Kontrolo predstavljajo asimptomatske živali, tj. živali brez znakov okužbe. Podatki so predstavljeni kot relativna sprememba v izražanju genov v primerjavi s kontrolo, povprečna vrednost ± standardna napaka je izračunana iz treh neodvisnih vzorcev RNA. Zvezdica nad stolpci na grafu prikazuje statistično značilno razliko v primerjavi z asimptomatskimi (kontrolnimi) živalmi (*, p < 0,05; **, p < 0,01; ***, p < 0,001), medtem ko lojtra (#) označuje mejno značilne razlike (0,05 < p < 0,1). Figure 4. Expression of immune-related genes in haemocytes of P. scaber with symptomatic bacterial (R. porcellionis) infection. Control is represented by asymptomatic animals, i.e., animals without signs of infection. Data are presented as relative expression change compared with control, mean ± standard error calculated from three independent RNA samples. An asterisk above the bars in the graph indicates a statistically significant difference compared to asymptomatic (control) animals (*, p < 0.05; **, p < 0.01; ***, p < 0.001), while a number sign (#) indicates a marginally significant difference (0.05 < p < 0.1). 12 Acta Biologica Slovenica, 2023, 66 (2) Diskusija V raziskavi smo določili spremembe v izražanju genov, povezanih z imunostjo, v hemocitih kopenskih rakov P. scaber z izraženo (tj. simptomatsko) bakterijsko okužbo z R. porcellionis. V primerjavi z asimptomatskimi P. scaber smo v okuženih živalih opazili statistično značilne spre- membe v izražanju imunskih genov Toll4, Dscam, Ppae2a, CypG in Cat, medtem ko je bilo izražanje genov MnSod in MyD88 zgolj mejno značilno spremenjeno (0,05 < p < 0,1). Ti rezultati pomembno prispevajo k razumevanju doga- janja na nivoju transkriptoma v primeru mikrobne okužbe kopenskega enakonožca P. scaber. V okuženih živalih je bilo izražanje genov Toll4 in Dscam pričakovano značilno povečano. Gena namreč kodirata evolucijsko ohranjena receptorska proteina (tj. imunski receptor; PRR), odgovorna za prepoznavo molekularnih vzorcev patogenov oziroma mikroorganizmov (P/MAMP) (Tran in sod., 2020; Sánchez-Salgado in sod., 2021). O podobnem povečanem izražanju Toll in Dscam genov poročajo tudi Li in sod. (2019) v hemocitih rakov Eriocheir sinensis, okuženih z bakterijo Staphylococcus aureus in pa Pan in sod. (2019), v primeru bakterijske Vibrio sp. okužbe raka Macrobrachium nipponense. Znano je, da aktivacija Toll-u podobnih receptorjev sproži prepisovanje genov, ki sodelujejo v protibakterijski obrambi rakov, tj. sintezi anti- mikrobnih peptidov (Pan in sod., 2019), nasprotno pa akti- vacija gena Dscam promovira fagocitozo bakterij, medtem ko novejše raziskave dokazujejo udeležbo produktov gena Dscam tudi v regulaciji signalne poti Toll (Li in sod., 2019). Poleg povečanega izražanja omenjenih genov smo opazili tudi mejno značilno povečanje izražanja MyD88, ki predstavlja pomemben adaptorski protein udeležen v signalni kaskadi Toll (Habib in Zhang, 2020; Gao in sod., 2021), kar sovpada s povečanim izražanjem Toll4. Vezava P/MAMP na plazemske oziroma PRR, vezane v membrano hemocitov, sproži evolucijsko ohranjen proces fagocitoze, tj. požiranje tujih delcev, npr. bakterij, ki so znotraj hemoci- tov uničeni in razgrajeni s strani ROS in RNS (Raman in sod., 2008; Rodríguez-Ramos in sod., 2010). Povečano produk- cijo RNS v hemolimfi enakonožcev v primeru mikrobne okužbe smo dokazali že v raziskavi Dolar in sod. (2020), v kateri poročamo o značilno povečani koncentraciji dušikovega oksida (NO) v hemolimfi P. scaber s simp- tomatsko R. porcellionis okužbo. Nasprotno pa v aktualni raziskavi v okuženih P. scaber nismo zaznali sprememb v izražanju gena Nos, ki kodira encim sintaza dušikovega oksida, odgovornega za produkcijo NO. Koncentracija reaktivnih zvrsti (ROS in RNS) mora biti v celicah in tkivih strogo regulirana, saj lahko povečane koncentracije trajno poškodujejo celice gostitelja. Za uravnavanje nivoja ROS in RNS so odgovorni antioksidativni encimi (npr. CAT in SOD), ki predstavljajo pomembno komponento prirojene imunosti rakov (Gopalakrishnan in sod., 2011; Liu in sod., 2013). V povezavi s tem smo v hemolimfi bakterijsko okuženih P. scaber opazili značilno povečano izražanje gena Cat, medtem ko je bilo izražanje gena MnSod zgolj mejno značilno spremenjeno. Za primerjavo, v raziskavi Dolar in sod. (2020) smo v hemolimfi bakterijsko okuženih P. scaber izmerili značilno povečano aktivnost encima SOD, v primerjavi z asimptomatskimi živalmi, medtem ko smo v aktualni raziskavi dodatno analizirali tudi aktivnost encima katalaza in dokazali mejno značilno povečanje, kar sovpada z rezultati izražanja gena Cat v hemocitih P. scaber. Simptomatska R. porcellionis okužba pri P. scaber izzove aktivacijo celične komponente prirojene imunosti, kar je jasno razvidno iz zmanjšanega deleža semi- granularnih (SGC) in povečanega deleža granularnih hemocitov (GC) v hemolimfi okuženih P. scaber (Dolar in sod., 2020). Spremenjen delež SGC lahko razložimo z migracijo le-teh na mesto okužbe in tvorbe nodulov, o čemer poročata že Kostanjšek in Pirc Marolt (2015), medtem ko je razlog za neznačilno povečanje deleža GC moč iskati v proizvodnji ključnih humoralnih molekul, vključno s komponentami sistema proPO (Herbiniѐre in sod., 2005; Tassanakajon in sod., 2018). To sovpada z opaženim značilno povečanim izražanjem gena CypG, ki kodira regulatorni protein, za katerega se domneva, da v granularnih hemocitih sodeluje pri ohranjanju konformaci- jske celovitosti shranjenih granularnih beljakovin, s čimer je pomembno udeležen v regulacijo sekrecije vsebine citoplazemskih granul, tj. humoralnih molekul (Takaki in sod., 1997; Herbinière in sod., 2008). Domnevamo, da aktivacija signalne poti Toll vodi v prepisovanje protibak- terijskih genov in sintezo humoralnih obrambnih molekul v granularnih in semigranularnih hemocitih. Dodatno smo v simptomatskih P. scaber opazili tudi značilno povečano izražanje gena Ppae2a, odgovornega za aktivacijo proPO sistema oziroma procesa melanizacije (Charoensapsri in sod., 2011). Proces je namreč aktivno udeležen pri tvorbi nodulov in se aktivira ob prisotnosti P/MAMP oziroma molekularnih vzorcev poškodovanih celic in tkiv (t.j. DAMP) (Cerenius in Söderhäl, 2021). Opažena sprememba 13 Acta Biologica Slovenica, 2023, 66 (2) v izražanju gena Ppae2a je lahko neposredno povezana s poškodbo organizma, zaradi vdora bakterij, ali pa gre za posledico aktivacije Toll signalne poti, s katero si proPO sistem deli skupne regulatorne proteine (Cerenius in Söderhäll, 2021). S tem rezultatom sovpada tudi zaznana povečana aktivnost encima alfa-2-makroglobulin, ki pred- stavlja pomembno serinsko proteazo, odgovorno za regu- lacijo proPO sistema in procesa melanizacije (Ponprateep in sod., 2017). Zaključki Na podlagi rezultatov lahko nedvomno zaključimo, da simptomatska bakterijska okužba P. scaber z R. porcel- lionis izzove značilne spremembe v izražanju izbranih kandidatnih genov. Rezultati potrjujejo pomembno vlogo analiziranih genov v imunskem odgovoru v primeru mikrobne okužbe. Hkrati pa preučevani geni, povezani z imunostjo, predstavljajo potencialne biološke označev- alce v okoljskih raziskavah. Zahvala Raziskovalno delo predstavljeno v prispevku je bilo finan- cirano s strani javne agencije za znanstvenoraziskovalno in inovacijsko dejavnost Republike Slovenije (ARIS), v okviru raziskovalnega programa Integrativna zoologija in speleobiologija (P1-0184), projekta (J1-2482) in pogodbe sklenjene z Univerzo v Ljubljani, Biotehniško fakulteto, za usposabljanje mladega raziskovalca (Andraž Dolar). Viri Amparyup, P., Charoensapsri, W., Tassanakajon, A., 2013. Prophenoloxidase system and its role in shrimp immune responses against major pathogens. Fish & Shellfish Immunology, 34 (4), 990-1001. https://doi.org/10.1016/j.fsi.2012.08.019 Becking, T., Giraud, I., Raimond, M., Moumen, B., Chandler, C., Cordaux, R., Gilbert, C., 2017. Diversity and evolution of sex determination systems in terrestrial isopods. Scientific Reports, 7 (1), 1084. https://doi.org/10.1038/s41598-017-01195-4 Cerenius, L., Söderhäll, K., 2021. Immune properties of invertebrate phenoloxidases. Developmental & Comparative Immunology, 122, 104098. https://doi. org/10.1016/j.dci.2021.104098 Charoensapsri, W., Amparyup, P., Hirono, I., Aoki, T., Tassanakajon, A., 2011. PmPPAE2, a new class of crustacean prophenoloxidase (proPO)-activating enzyme and its role in PO activation. Developmental & Comparative Immunology, 35 (1), 115-124. https://doi.org/10.1016/j.dci.2010.09.002 Chevalier, F., Herbiniére-Gaboreau, J., Bertaux, J., Raimond, M., Morel, F., Bouchon, … Braquart-Varnier, C., 2011. The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia. PLoS One, 6 (4), e18531. https://doi.org/10.1371/journal.pone.0018531 Chevalier, F., Herbinière-Gaboreau, J., Charif, D., Mitta, G., Gavory, F., Wincker, P., … Bouchon, D., 2012. Feminizing Wolbachia: a transcriptomics approach with insights on the immune response genes in Armadillidium vulgare. BMC microbiology, 12, 1-18. https://doi.org/10.1186/1471-2180-12-S1-S1 Clark, K.F., Greenwood, S. J., 2016. Next-generation sequencing and the crustacean immune system: the need for alternatives in immune gene annotation. Integrative and comparative biology, 56 (6), 1113-1130. https://doi.org/10.1093/icb/icw023 Cole, A., Morris, J., 1980. A new iridovirus of two species of terrestrial isopods, Armadillidium vulgare and Porcellio scaber. Intervirology, 14 (1), 21-30. https://doi.org/10.1159/000149158 Dolar, A., Mayall, C., Drobne, D., Kokalj, A. J., 2020. Modulations of immune parameters caused by bacterial and viral infections in the terrestrial crustacean Porcellio scaber: Implications for potential markers in environmental research. Developmental & Comparative Immunology, 113, 103789. https://doi. org/10.1016/j.dci.2020.103789 Dolar, A., Selonen, S., van Gestel, C. A., Perc, V., Drobne, D., Jemec Kokalj, A., 2021. Microplastics, chlorpyrifos and their mixtures modulate immune processes in the terrestrial crustacean Porcellio scaber. Science of the Total Environment, 772, 144900. https://doi.org/10.1016/j.scitotenv.2020.144900 Dolar, A., Jemec Kokalj, A., Drobne, D., 2022a. Time-course of the innate immune response of the terrestrial crustacean Porcellio scaber after injection of a single dose of lipopolysaccharide. Frontiers in Immunology, 13, 867077. https://doi.org/10.3389/fimmu.2022.867077 Dolar, A., Drobne, D., Dolenec, M., Marinšek, M., Jemec Kokalj, A., 2022b. Time-dependent immune response in Porcellio scaber following exposure to microplastics and natural particles. Science of The Total Environment, 818, 151816. https://doi.org/10.1016/j.scitotenv.2021.151816 Dolar, A., Drobne, D., Narat, M., Jemec Kokalj, A., 2022c. Tire microplastics exposure in soil induces changes in expression profile of immune-related genes in terrestrial crustacean Porcellio scaber. Environmental Pollution, 314, 120233. https://doi.org/10.1016/j.envpol.2022.120233 Dolar, A., Močivnik, L., Manzano, M. A., Hrga, N., Zakšek, V., Jemec Kokalj, A., … Kostanjšek, R., 2022d. RNA-Seq data of common woodlice Porcellio scaber. Zenodo. https://doi.org/10.5281/zenodo.6673325 14 Acta Biologica Slovenica, 2023, 66 (2) Esteve, M., Herrera, F. C., 2000. Hepatopancreatic alterations in Litopenaeus vannamei (Boone, 1939) (Crustacea: Decapoda: Penaeidae) experimentally infected with a Vibrio alginolyticus strain. Journal of invertebrate pathology, 76 (1), 1-5. https://doi.org/10.1006/jipa.2000.4951 Gao, Q., Tang, Q., Xia, Z., Yi, S., Cai, M., Du, H., … Yang, G., 2021. Molecular identification and functional analysis of MyD88 in giant freshwater prawn (Macrobrachium rosenbergii) and expression changes in response to bacterial challenge. International Journal of Biological Macromolecules, 178, 492- 503. https://doi.org/10.1016/j.ijbiomac.2021.02.177 Gopalakrishnan, S., Chen, F. Y., Thilagam, H., Qiao, K., Xu, W. F., Wang, K. J., 2011. Modulation and interaction of immune-associated parameters with antioxidant in the immunocytes of crab Scylla paramamosain challenged with lipopolysaccharides. Evidence-Based Complementary and Alternative Medicine, 2011, 824962. https://doi.org/10.1155/2011/824962 Habib, Y. J., Zhang, Z., 2020. The involvement of crustaceans toll-like receptors in pathogen recognition. Fish & shellfish immunology, 102, 169-176. https:// doi.org/10.1016/j.fsi.2020.04.035 Halter, T., Köstlbacher, S., Collingro, A., Sixt, B. S., Tönshoff, E. R., Hendrickx, F., … Horn, M., 2022. Ecology and evolution of chlamydial symbionts of arthropods. ISME Communications, 2 (1), 45. https://doi.org/10.1038/s43705-022-00124-5 Herbinière, J., Braquart-Varnier, C., Grève, P., Strub, J. M., Frère, J., Van Dorsselaer, A., Martin, G., 2005. Armadillidin: a novel glycine-rich antibacterial peptide directed against gram-positive bacteria in the woodlouse Armadillidium vulgare (Terrestrial Isopod, Crustacean). Developmental & Comparative Immunology, 29 (6), 489-499. https://doi.org/10.1016/j.dci.2004.11.001 Herbinière, J., Grève, P., Strub, J. M., Thiersé, D., Raimond, M., van Dorsselaer, A., … Braquart-Varnier, C., 2008. Protein profiling of hemocytes from the terrestrial crustacean Armadillidium vulgare. Developmental & Comparative Immunology, 32 (8), 875-882. https://doi.org/10.1016/j.dci.2008.01.007 Hornung, E., Farkas., S., Fischer., E., 1998. Tests on the isopod Porcellio scaber. In: Løkke H., van Gestel C.A.M. (eds.) Handbook of soil invertebrate toxicity tests, John Wiley & Sons Ltd, 207-226. Hornung, E., 2011. Evolutionary adaptation of oniscidean isopods to terrestrial life: structure, physiology and behavior. Terrestrial Arthropod Reviews, 4 (2), 95-130. Jemec Kokalj, A., Dolar, A., Titova, J., Visnapuu, M., Škrlep, L., Drobne, D., … Heinlaan, M., 2021. Long term exposure to virgin and recycled LDPE microplastics induced minor effects in the freshwater and terrestrial Crustaceans Daphnia magna and Porcellio scaber. Polymers, 13 (5), 771. https://doi. org/10.3390/polym13050771 Jemec Kokalj, A., Dolar, A., Drobne, D., Škrlep, L., Škapin, A.S., Marolt, G., … van Gestel C.A., 2022. Effects of microplastics from disposable medical masks on terrestrial invertebrates. Journal of hazardous materials, 438, 129440. https://doi.org/10.1016/j.jhazmat.2022.129440 Jiravanichpaisal, P., Lee, B. L., Söderhäll, K., 2006. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization. Immunobiology, 211 (4), 213-236. https://doi.org/10.1016/j.imbio.2005.10.015 Kostanjšek, R., Štrus, J., Drobne, D., Avguštin, G., 2004. ‘Candidatus Rhabdochlamydia porcellionis’, an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda). International journal of systematic and evolutionary microbiology, 54 (2), 543-549. https:// doi.org/10.1099/ijs.0.02802-0 Kostanjšek, R., Marolt, T. P., 2015. Pathogenesis, tissue distribution and host response to Rhabdochlamydia porcellionis infection in rough woodlouse Porcellio scaber. Journal of invertebrate pathology, 125, 56-67. https://doi.org/10.1016/j.jip.2015.01.001 Li, D., Wan, Z., Li, X., Duan, M., Yang, L., Ruan, Z., … Li, W., 2019. Alternatively spliced down syndrome cell adhesion molecule (Dscam) controls innate immunity in crab. Journal of Biological Chemistry, 294 (44), 16440-16450. https://doi.org/10.1074/jbc.RA119.010247 Liu, Y. T., Chang, C. I., Hseu, J. R., Liu, K. F., Tsai, J. M., 2013. Immune responses of prophenoloxidase and cytosolic manganese superoxide dismutase in the freshwater crayfish Cherax quadricarinatus against a virus and bacterium. Molecular Immunology, 56 (1-2), 72-80. https://doi.org/10.1016/j. molimm.2013.03.023 Liu, Q. N., Tang, Y. Y., Zhang, S. P., Li, Y. T., Wang, G., Zhang, D. Z., Jiang, S-H., Yang, H., Tang, B-P., Dai, L. S., 2022. Characterization and expression analysis of differentially expressed genes in the red swamp crayfish Procambarus clarkii in response to Vibrio cholerae challenge. Aquaculture, 547, 737435. https://doi.org/10.1016/j.aquaculture.2021.737435 Lou, F., Wang, Y., Han, Z., Shui, B., 2022. Comparative transcriptome reveals the molecular regulation mechanism of Charybdis japonica to high-and low- temperature stresses. Frontiers in Marine Science, 9, 849485. https://doi.org/10.3389/fmars.2022.849485 Mengal, K., Kor, G., Kozák, P., Niksirat, H., 2022. Effects of environmental factors on the cellular and molecular parameters of the immune system in decapods. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 276, 111332. https://doi.org/10.1016/j.cbpa.2022.111332 Pan, L., Zhang, X., Yang, L., Pan, S., 2019. Effects of Vibro harveyi and Staphyloccocus aureus infection on hemocyanin synthesis and innate immune responses in white shrimp Litopenaeus vannamei. Fish & Shellfish Immunology, 93, 659-668. https://doi.org/10.1016/j.fsi.2019.08.016 Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic acids research, 29 (9), e45-e45. https://doi. org/10.1093/nar/29.9.e45 Ponprateep, S., Vatanavicharn, T., Lo, C. F., Tassanakajon, A., Rimphanitchayakit, V., 2017. Alpha-2-macroglobulin is a modulator of prophenoloxidase system in pacific white shrimp Litopenaeus vannamai. Fish & shellfish immunology, 62, 68-74. https://doi.org/10.1016/j.fsi.2016.12.028 Raman, T., Arumugam, M., Mullainadhan, P., 2008. Agglutinin-mediated phagocytosis-associated generation of superoxide anion and nitric oxide by the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii. Fish & Shellfish Immunology, 24 (3), 337-345. https://doi.org/10.1016/j.fsi.2007.12.005 Rodríguez-Ramos, T., Carpio, Y., Bolívar, J., Espinosa, G., Hernández-López, J., Gollas-Galván, T., … Estrada, M. P., 2010. An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: cloning, characterization and expression analysis. Fish & shellfish immunology, 29 (3), 469-479. https://doi.org/10.1016/j.fsi.2010.05.013 15 Acta Biologica Slovenica, 2023, 66 (2) Sánchez-Salgado, J. L., Pereyra, M. A., Agundis, C., Calzada-Ruiz, M., Kantun-Briceño, E., Zenteno, E., 2019. In vivo administration of LPS and β-glucan generates the expression of a serum lectin and its cellular receptor in Cherax quadricarinatus. Fish & Shellfish Immunology, 94, 10-16. https://doi. org/10.1016/j.fsi.2019.08.061 Sánchez-Salgado, J. L., Pereyra, M. A., Alpuche-Osorno, J. J., Zenteno, E., 2021. Pattern recognition receptors in the crustacean immune response against bacterial infections. Aquaculture, 532, 735998. https://doi.org/10.1016/j.aquaculture.2020.735998 Söderhäll, I., 2016. Crustacean hematopoiesis. Developmental & Comparative Immunology, 58, 129-141. https://doi.org/10.1016/j.dci.2015.12.009 Sun, M., Li, S., Zhang, X., Xiang, J., Li, F., 2020. Isolation and transcriptome analysis of three subpopulations of shrimp hemocytes reveals the underlying mechanism of their immune functions. Developmental & Comparative Immunology, 108, 103689. https://doi.org/10.1016/j.dci.2020.103689 Sun, Y., Zhang, X., Wang, Y., Zhang, Z., 2022. Long-read RNA sequencing of Pacific abalone Haliotis discus hannai reveals innate immune system responses to environmental stress. Fish & Shellfish Immunology, 122, 131-145. https://doi.org/10.1016/j.fsi.2022.01.042 Takaki, Y., Muta, T., Iwanaga, S., 1997. A peptidyl-prolyl cis/trans-isomerase (cyclophilin G) in regulated secretory granules. Journal of Biological Chemistry, 272 (45), 28615-28621. https://doi.org/10.1074/jbc.272.45.28615 Tassanakajon, A., Somboonwiwat, K., Supungul, P., Tang, S., 2013. Discovery of immune molecules and their crucial functions in shrimp immunity. Fish & shellfish immunology, 34 (4), 954-967. https://doi.org/10.1016/j.fsi.2012.09.021 Tassanakajon, A., Rimphanitchayakit, V., Visetnan, S., Amparyup, P., Somboonwiwat, K., Charoensapsri, W., Tang, S., 2018. Shrimp humoral responses against pathogens: antimicrobial peptides and melanization. Developmental & Comparative Immunology, 80, 81-93. https://doi.org/10.1016/j.dci.2017.05.009 Tran, N. T., Kong, T., Zhang, M., Li, S., 2020. Pattern recognition receptors and their roles on the innate immune system of mud crab (Scylla paramamosain). Developmental & Comparative Immunology, 102, 103469. https://doi.org/10.1016/j.dci.2019.103469 Van Gestel, C.A., Loureiro, S., Zidar, P., 2018. Terrestrial isopods as model organisms in soil ecotoxicology: a review. ZooKeys, 801, 127-162. https://doi. org/10.3897/zookeys.801.21970 Wang, W., 2011. Bacterial diseases of crabs: a review. Journal of invertebrate pathology, 106 (1), 18-26. https://doi.org/10.1016/j.jip.2010.09.018 Wang, X. W., Wang, J. X., 2013. Pattern recognition receptors acting in innate immune system of shrimp against pathogen infections. Fish & shellfish immunology, 34 (4), 981-989. https://doi.org/10.1016/j.fsi.2012.08.008 Xu, Z., Liu, A., Li, S., Wang, G., Ye, H., 2020. Hepatopancreas immune response during molt cycle in the mud crab, Scylla paramamosain. Scientific Reports, 10 (1), 13102. https://doi.org/10.1038/s41598-020-70139-2 16 1 University in Ljubljana, Biotechnical Faculty, Dept. Of Biology, Večna pot 111, 1000 Ljubljana, Slovenia * Corresponding author: E-mail address: matevz.likar@bf.uni-lj.si Citation: Likar, M., Grašič, M., Gaberščik, A. (2023). Contribution of neutral processes to the assembly of microbial communities on Phragmites australis leaf litter. Acta Biologica Slovenica 66 (2) https://doi.org/10.14720/abs.66.2.16495 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY SA) license Research Article Contribution of neutral processes to the assembly of microbial communities on Phragmites australis leaf litter Matevž Likar 1,*, Mateja Grašič 1, Alenka Gaberščik 1 Abstract Phragmites australis is a remarkable aquatic plant known for its adaptability, wide ecological range and extensive presence in natural wetlands. When combined with its microbiome, it holds unique potential to enhance the overall functionality of wetland ecosystems. To fully harness this potential in both natural and constructed wetlands, it becomes crucial to understand the dynamics of decomposition regarding the substantial biomass generated by P. australis. However, our understanding of the selective and neutral processes that shape the microbial communities responsible for decomposing P. australis litter remains somewhat limited. In this context, our research reveals that the majority of microbial taxa inhabiting P. australis leaves and litter follow neutral distribution patterns, indicating they are less likely to be specifically adapted to the host plant or habitat. Their presence in the community primarily results from their prevalence in the broader metacommunity and source pool. Nonetheless, this should not be interpreted as these taxa being functionally unimportant or lacking close interactions with their host. Instead, the host environment does not differentially select them, and as a consequence, their distributions are shaped predominantly by neutral processes of dispersal and drift. Keywords Bacterial communities, fungal communities, decomposition, freshwater ecosystem, ephemeral lake 17 Acta Biologica Slovenica, 2023, 66 (2) lower quality, which consequently slows the decomposi- tion processes (Rejmánková & Houdková, 2006). Although the list of potential factors influencing micro- bial communities is extensive, they can be categorized into two primary groups: selective processes, where microbes thrive within an environment due to differences in their ecological fitness, and neutral processes, which encompass passive dispersal dynamics and the impacts of ecological drift (Chase & Myers, 2011). Although much research is focused on interactions between microbes and their environment, the relative contributions of neutral processes in shaping microbial communities associated with hosts have been mainly overlooked. In contrast, these processes have been subjects of study within the broader field of ecology for many decades, experiencing a renewed surge of interest in recent years (Burns et al., 2016; Cao et al., 2019; Heys et al., 2020). The neutral theory derives its name from its core assumption of species having equivalent per-capita growth, death, and dispersal rates, assuming species are ecologically 'neutral' in their fitness. Without differences in these factors, community assembly is governed by sto- chastic processes involving dispersal and drift. Within this framework, organisms within a community are randomly lost over time and are replaced by individuals randomly Pomen nevtralnih procesov pri oblikovanju mikrobnih skupnosti na listnem opadu vrste Phragmites australis Izvleček Phragmites australis je izjemna vodna rastlina, znana po svoji prilagodljivosti, širokem ekološkem razponu in obsežni prisotnosti v naravnih mokriščih. V kombinaciji s svojim mikrobiomom ima edinstven potencial za izboljšanje splošne funkcionalnosti mokriščnih ekosistemov. Za izkoriščanje njenega potenciala v naravnih in zgrajenih mokriščih pa je ključnega pomena razumevanje dinamike razgradnje velike količine biomase, ki jo ustvari P. australis. Naše razumevanje selektivnih in nevtralnih procesov, ki oblikujejo mikrobne združbe, odgovorne za razgradnjo listnega opada P. australis, je še vedno nekoliko omejeno. V tem kontekstu naša raziskava razkriva, da večina mikrobnih taksonov, ki naseljujejo liste in listni opad P. australis, sledi nevtralnim vzorcem porazdelitve, kar kaže, da je manj verjetno, da so posebej prilagojeni gostiteljski rastlini ali habitatu. Njihova prisotnost v združbi je predvsem posledica njihove razširjenosti v širši metaskupnosti in izvornem naboru vrst. Kljub temu to ne pomeni, da so ti taksoni funkcionalno nepomembni ali da nimajo tesnih interakcij z gostiteljem. Le njihovo okolje ne vrši močnega selektivnega pritiska nanje, zaradi česar njihovo porazdelitev oblikujejo pretežno nevtralni procesi razširjanja in ekološkega zdrsa. Ključne besede Bakterijske združbe, glivne združbe, razgradnja, sladkovodni ekosistem, presihajoče jezero Introduction Phragmites australis is a perennial grass-like aquatic plant known for their remarkable adaptability and wide ecologi- cal range. It has the capacity to establish dense, dominant communities within aquatic ecosystems and is among the most prevalent plant species found in wetlands (Kowalski et al., 2015; Stottmeister et al., 2003). Given their extensive presence in natural wetlands and widespread use in con- structed wetlands, Phragmites and its microbiome possess distinctive potential for enhancing the overall functionality of wetland ecosystems. Nevertheless, in order to harness its potential in natural and constructed wetlands, it is important to understand also the decomposition dynamics of the vast biomass produced by P. australis. The decomposition process within wetlands can be influenced by a range of factors, with its hydrology and temperatures playing pivotal roles (Dolinar et al., 2016; Serna et al., 2013) in the formation of microbial commu- nities decomposing the plant litter. Apart from climatic conditions, the composition of litter and site elements, specifically the nutrient content within plant litter, may also substantially influence decomposition rates (Alfreds- son et al., 2016; Sistla et al., 2012). This is because plants in nutrient-deficient environments often produce litter of 18 Acta Biologica Slovenica, 2023, 66 (2) either from within the same community or through the dispersal of individuals from an outside community. Despite simple assumptions of ecological equiva- lence at the base of neutral models, they have proven remarkably successful in predicting the structures of communities, including microbes (Burns et al., 2016; Cao et al., 2019; Heys et al., 2020). These models find utility in modelling microbial systems where the vast diversity of communities makes it challenging to characterize the specific ecological traits of each taxon. Moreover, they enable researchers to quantify the significance of pro- cesses that are challenging to observe directly, such as dispersal, despite their potentially substantial impacts on microbial communities (Kerr et al., 2002; Shen et al., 2018). This study aimed to build on the previous analysis of fungal communities on decomposing P. australis leaves and to evaluate the relative impact the ecological factors and neutral model processes have on the microbial communities on these leaves. Our previous study Likar et al. (2022) showed that a complex network of fungi forms already in the senescent leaves of P. australis and persists to the decomposition phase. Furthermore, it seems that habitat has a lower impact on the formation of the com- munity during the early decomposition phase than on the interaction between its members. In the present study, we assess the ability of neutral models to explain the distribution of microorganisms among a population of decomposing P. australis leaves and then determine the conditions leading to departures from neutral behavior. If a reduction in the fit of the neutral model reflects heightened selection pressures, we assumed that deviations from the neutral predictions should show distinct compositional changes in the communities, particularly in cases where ecological traits are conserved. By investigat- ing these hypotheses, we aim to establish a framework for identifying communities and taxa that might be of particular interest based on the extent to which they deviate from the expectations set forth by neutral theory. Materials and Methods Study location and experimental conditions The lake Cerknica experiences cyclical inundation, sub- merging its surroundings for approximately nine months each year. Within this dynamic ecosystem, P. australis, a robust plant species, thrives in diverse aquatic environ- ments, encompassing both the inner lake region and the periphery bordering the lake's tributaries (Longhi et al., 2008). The experimental conditions followed the procedure described in (Grašič et al., 2022). In brief, we gathered both the upper and lower leaves of P. australis during the con- clusion of the vegetation period when the plants retained their active. We collected leaves from different parts of the plant to cover the possible microbial diversity. Subsequent to collection, the plant material underwent air-drying at room temperature until a consistent weight was achieved. For the decomposition experiment we used litter bags (1 mm × 1 mm plastic mesh) containing 4 g of the plant material. Water level data were monitored from the nearby hydrological station at Gorenje Jezero-Stržen. In order to minimize direct contact with the substrate, the litter bags were affixed to wooden poles. The decomposition phase of our experiment spanned a duration of 45 days, after which the samples were collected and subjected to air-drying until a consistent weight was reached. Following the drying process, we separated the plant material from non-plant material and processed them for metagenomic analysis. Metagenomics In the present dataset, we analysed the sequences obtained by shotgun sequencing (see Likar et al., 2022 for details), deposited at MG-Rast depository under project mgp97071, libraries mgm4915122.3-mgm4915169.3. In short, whole community DNA from common reed leaves was used for shotgun sequencing (Illumina HiSeqX, 2 x 150 nt pair-end, TruSeq Nano kit). The analysis and annotations were executed on the Metagenomics Rapid Annotation (MG-RAST) online server [22], with the default parameters. Taxonomic identification in this analysis utilized an E-value threshold of 1e-5. All sequencing data are openly available on the MG-Rast server. Plant litter elemental analysis Plant litter was analysed using X-ray fluorescence spec- trometry (XRF). Five samples of each treatment were used for multielemental analysis as described in (Grašič et al., 2022). In short: From 100–500 mg of dried and powdered plant material was pressed into pellets using a pellet die and a hydraulic press. 55Fe (25 mCi; Isotope 19 Acta Biologica Slovenica, 2023, 66 (2) Products Laboratories, Valencia, PA, USA) was used as the primary excitation source for the analysis. The analysis of the X-ray spectra was carried out using an iterative least-squares programme, as included in the quantitative X-ray analysis system software package (Vekemans et al., 1994). The quality assurance for the element analysis was determined using standard reference materials: NIST SRM 1573a (tomato leaves as a homogenised powder), in the form of pressed pellets. Statistics All analyses were performed in R (v4.3.1). To estimate the effect of environment and geo-location on the composition of fungal and bacterial communities, were performed variation partitioning using vegan v2.6-4 library. Geographical distances were transformed to rect- angular data using principal coordinates of neighbourhood matrix (PCNM) before the analysis. Prior to analysis, the environmental factors and PCNM vectors were subjected to forward stepwise redundancy analysis to reduce the number of variables used in the variation partitioning. Null models are an essential tool for assessing the issue of multiple assembly processes by mimicking the consequences of random processes, therefore we calcu- lated a modified stochasticy index as describe in (Liang et al., 2020) using NST v3.1.10 library. The null communities are generated by randomizing the observed community structure 1000 times based on a null model algorithm described previously (Stegen et al., 2013). Adherence to the Sloan neutral model was calculated using the R code published by (Burns et al., 2016). Accord- ingly, the OTUs were grouped into three partitions based on whether they occurred more frequently than ('above' partition), less frequently than ('below' partition), or within ('neutral' partition) the 95% confidence interval of the neutral model predictions. For meaningful comparisons among partitions, we rarefied each partition to an equal number of OTUs, matching the size of the smallest partition. Furthermore, we compared the fit of the neutral model to that of a binomial distribution model to ascer- tain whether incorporating drift and dispersal limitations improved the model fit beyond random sampling from the source metacommunity (Sloan et al., 2007). The binomial distribution model represents the scenario where local communities are random subsets of the metacommu- nity in the absence of drift and dispersal limitations. To compare the fit of the neutral and binomial models, we examined the Akaike information criterion for each model. Computation of the Akaike information criterion was per- formed in R, and 95% confidence intervals for all fitting statistics were generated using bootstrapping with 1000 bootstrap replicates. Results and Discussion Step-wise RDA of explanatory datasets selected P con- centration, PCNM1 and PCNM2 for fungal community dataset, whereas complete models with all environmental parameters and PCNMs were selected for the bacterial community dataset. Variation partitioning for fungal communities explained 56% of the overall variation (Fig. 1). Measured environmental parameters explained only 1.5% alone and additional 10.7% in combination with geographical location. Geographical location alone explained the largest portion of the variability. Measured environmental parameters were a little better predictor for bacterial communities and explained 16% when not controlling for geographical location. In contrast to fungal communities, geographical location alone did not explain any variation in the bacterial communities, which showed a very high percentage of unexplained variation. As measured and unmeasured environmental param- eters seem to explain only around 15-55% of the total variation in the microbial communities, we examined the importance of neutral processes on the formation of these communities. Comparing the number of OTUs shared between fresh and decomposing leaves showed that the largest number of OTUs was specific for all three treatments (Fig. 2). This shows the importance of the initial phyllosphere commu- nities on the formation of microbial communities during the decomposition process. The same was true for the enriched and under-enriched fungal OTUs, suggesting that these represent ubiquitous generalist that start as colonisers of fresh leaves and after leaf-fall proceed to decomposition of leaf litter. The second largest group of OTUs in the present study was characteristic for dryer habitats t.i. fresh leaves and leaves decomposing in the dry habitat. Similarly, identification of indicator species yielded a large portion of fungal taxa that were indicative for both the fresh leaves and leaves 20 Acta Biologica Slovenica, 2023, 66 (2) decomposing in the dry habitat (Likar et al., 2022). Only a few fungal OTUs were characteristic for a single treatment: Corynespora was enriched, whereas Marasmius and Hanseniaspora were under-enriched on fresh leaves. In addition, Lycoperdon was under-enriched on decomposing leaves independently of habitat. Interestingly, none of the taxa that deviated from the neutral model were observed as indicator species for either fresh leaves or leaves decomposing in wet or dry habitat (Likar et al., 2022). Modified Raup-Crick with Bray-Curtis dissimilarity showed that the local fungal communities on plant litter were more dissimilar than expected by random chance (Fig. 3a). In contrast, local communities on fresh leaves were as dissimilar as expected by the null model. All treatment showed values which indicate that the turnover in fungal communities was governed by drift alone (-0.95 < RC < 0.95). To evaluate the stochastic processes in the population, we calculated the modified ST (Fig. 3b). Com- munity assembly was relatively more stochastic on fresh leaves (72% stochasticity ratio (ST) and in litter decompos- ing in the dry habitat (68% ST) than in the wet habitat (38% ST). These results suggest that deterministic processes became increasingly important during the decomposition and especially in the wet habitat, where the MST values are under 42%. In contrast to fungal communities, bacterial communi- ties in plant litter showed RC values above 0.95, which show a significant departures from the degree of turnover expected when drift acts alone (Chase et al., 2011). Values of RC > 0.95 indicate that dispersal limitation governs observed compositional differences. MST values under 0.5 that were calculated for bacterial communities well support this. Further more, it seems that selection was increased during the transition from fresh leaves to plant litter, as bacterial communities on fresh leaves showed dissimilarity that was well expected by random chance. While phyllosphere microbial communities of different species exhibit significant dissimilarities (Bao et al., 2019), they all share a similar underlying structure (Wallace et al., 2018). As plants gradually undergo senescence, the vari- ability in phyllosphere microbes tends to incrementally rise (Ferreira et al., 2016), with changes in microbial com- munities on leaves undergoing decomposition (Kembel et al., 2014; Whipps et al., 2008) increasingly influenced by the leaves' physicochemical properties and competition between the microbes. Out of on average 244 bacterial families that were observed on plant material in our study, only one family (0.4% of all the families) did not fall into the neutral model and showed enrichment (Suppl. Table S1). None of the bacterial families showed under-enrichment against the neutral model. This would suggest greater importance Figure 1. Variation partitioning for fungal and bacterial communities using measured environmental parameters and geo-location selected by step-wise redundancy analysis (RDA), as the explanatory datasets. Values < 0 are not displayed. Slika 1. Pojasnitev variabilnosti za glivne in bakterijske združbe z uporabo izmerjenih okoljskih parametrov in geografske lokacije, izbrane s postopno redundančno analizo (RDA) kot razlagalni nizi podatkov. Vrednosti < 0 niso prikazane. 21 Acta Biologica Slovenica, 2023, 66 (2) of dispersal, as selection would increase or decrease the frequency of bacterial taxa in comparison with neutral model. Nevertheless, unclassified - derived from Rhizo- biales group showed frequencies that were above the neutral model. Alphaproteobacteria, which comprised of several symbionts of plants, such as Rhizobium as the most abundant group in the roots of P. australis He et al. (2022) and among the top generalists in the phyllospheres of nine perennial plants in a Mediterranean ecosystem (Vokou et al., 2019). Their presence could be beneficial for the plant and provide a nitrogen source for the growth of plants (Sawada et al., 2003) as well as other physiological benefits (Jaiswal et al., 2021), which could explain the departure from the neutral model. Figure 2. Venn diagrams for fungal communities (All), enriched fractions (Above) and under-enriched fractions (Below) according to Sloan's neutral model for fresh Phragmites australis leaves and leaf litter decomposing in either dry or wet habitat. Numbers represent the absolute number of OTUs and percentages represent their proportions regarding all OTUs in the individual representation. Slika 2. Vennovi diagrami za glivne združbe (All), obogatene frakcije (Above) in podobogatene frakcije (Below) v skladu s Sloanovim nevtralnim modelom za sveže liste Phragmites australis in listni opad, ki se razkraja v suhem ali mokrem habitatu. Številke predstavljajo absolutno število OTU-jev, odstotki pa njihove deleže glede na vse OTU-je v posamezni predstavitvi. 22 Acta Biologica Slovenica, 2023, 66 (2) In contrast to bacterial communities, fungal commu- nities showed higher taxa numbers that deviated from the neutral model. Overall, the frequency with which fungal taxa occurred in individual communities was well described by the neutral model (Suppl. Table S2, Fig. 4). The migration rate was not very variable and ranged from 0.73-0.89. The fit of the neutral model was compared with the fit of a binomial distribution, which represents the absence of processes of drift and dispersal limitation using Akaike information criterion and Bayes information criterion (Table 1). We observed a much better fit of the model, if we used the neutral model compared to binomial distribution model. This suggest that the processes of passive dis- persal and ecological drift have an impact on the fungal communities. This was observed for fungal communities in various biological systems (Gao et al., 2020; Liu et al., 2023; Zhang et al., 2023). Figure 3. Raup-Crick a) and modified stochasticy b) indices for fungal and bacterial communities on the fresh leaves of Phragmites australis and its leaf litter decomposing in either dry or wet habitat. Dashed line represents the MST = 0.5 t.i. limitation between predominant stochasticity or determinism. Different letter depict statistically significant difference of PERMANOVA test at p < 0.05. Slika 3. Raup-Crickov a) in modificirani stohastični b) indeksi za glivne in bakterijske združbe na svežih listih Phragmites australis in njenem listnem opadu, ki se razkraja v suhem ali mokrem habitatu. Črtkana črta predstavlja MST = 0,5 t.i. omejitev med prevladujočo stohastičnostjo ali determinizmom. Drugačna črka prikazuje statistično značilno razliko testa PERMANOVA pri p < 0,05 Plant material/ decomposition habitat AIC neutral BIC neutral AIC binomial Fresh leaves -157.059 -151.219 -218.616 Dry habitat -191.509 -185.655 -223.202 Wet habitat -219.466 -213.976 7.746 Table 1. Akaike and Bayes information criterions for neutral model and binomial distributions for fungal communities on fresh Phragmites australis leaves or leaf litter in either dry or wet habitat. Tabela 1. Informacijsk kriterij Akaike in Bayes za nevtralni model in binomske porazdelitve za glivne skupnosti na svežih listih Phragmites australis ali listnem odpadku v suhem ali mokrem habitatu. AIC…Akaike information criterion; BIC…Bayes information criterion 23 Acta Biologica Slovenica, 2023, 66 (2) Figure 4. Fit of the neutral model. The predicted occurrence frequencies for a) fresh leaves and leaves decomposing in either b) dry or c) wet habitat. OTUs that occur more frequently than predicted by the model are shown in purple while those that occur less frequently than predicted are shown in green. Dashed lines represent 95% confidence intervals around the model prediction (solid line). The number represent the model fit (generalized R2) and migration rate (m) of the model. Slika 4. Prileganje nevtralnega modela. Predvidene pogostnosti pojavljanja za a) sveže liste in liste, ki se razkrajajo v b) suhem ali c) mokrem habitatu. OTU-ji, ki se pojavljajo pogosteje, kot je napovedal model, so prikazani v vijolični barvi, tisti, ki se pojavljajo manj pogosto, kot je predvideno, pa so prikazani v zeleni barvi. Črtkane črte predstavljajo 95 % intervale zaupanja okoli napovedi modela (polna črta). Število predstavlja prileganje modela (posplošen R2) in stopnjo migracije (m) modela. 24 Acta Biologica Slovenica, 2023, 66 (2) Conclusions Our results suggest that the majority of microbial taxa in the studied ecosystem/plant host system are neutrally distributed and are less likely to be specifically adapted to the host. Therefore their presence in the community is largely the result of their abundance in the surrounding meta-community and source pool. Nevertheless, this does not mean that these taxa are functionally unimportant or even that they are not interacting intimately with their hosts. Rather the host environment is not differentially selecting them, and consequently their distributions are the result of neutral dispersal and drift. Supplementary Materials Suppl. Table S1. Observed frequencies for individual bacterial OTUs and their predicted frequencies under the Sloan neutral model. Upper and lower limits of 95% confidence interval for predicted frequencies are also displayed. Suppl. Table S2. Observed frequencies for individual fungal OTUs and their predicted frequencies under the Sloan References Alfredsson, H., Clymans, W., Stadmark, J., Conley, D., & Rousk, J. (2016). Bacterial and fungal colonization and decomposition of submerged plant litter: Consequences for biogenic silica dissolution. FEMS Microbiology Ecology, 92(3). https://doi.org/10.1093/femsec/fiw011 Burns, A. R., Stephens, W. Z., Stagaman, K., Wong, S., Rawls, J. F., Guillemin, K., & Bohannan, B. J. M. (2016). Contribution of neutral processes to the assembly of gut microbial communities in the zebrafish over host development. ISME Journal, 10(3), 655–664. https://doi.org/10.1038/ismej.2015.142 Cao, M. wen, Jia, T., Mi, J., Jing, J. hui, & Chai, B. feng. (2019). Relative roles of niche and neutral processes on turnover of plant, fungal and bacterial communities in arid and semi-arid areas at the regional scale. Basic and Applied Ecology, 40, 43–54. https://doi.org/10.1016/j.baae.2019.08.005 Chase, J. M., Kraft, N. J. B., Smith, K. G., Vellend, M., & Inouye, B. D. (2011). Using null models to disentangle variation in community dissimilarity from variation in α-diversity. Ecosphere, 2(2). https://doi.org/10.1890/ES10-00117.1 Chase, J. M., & Myers, J. A. (2011). Disentangling the importance of ecological niches from stochastic processes across scales. Philosophical Transactions of the Royal Society B: Biological Sciences, 366(1576), 2351–2363. https://doi.org/10.1098/rstb.2011.0063 Dolinar, N., Regvar, M., Abram, D., & Gaberščik, A. (2016). Water-level fluctuations as a driver of Phragmites australis primary productivity, litter decomposition, and fungal root colonisation in an intermittent wetland. Hydrobiologia, 774(1), 69–80. https://doi.org/10.1007/s10750-015-2492-x Ferreira, V., Raposeiro, P. M., Pereira, A., Cruz, A. M., Costa, A. C., Graça, M. A. S., & Gonçalves, V. (2016). Leaf litter decomposition in remote oceanic island streams is driven by microbes and depends on litter quality and environmental conditions. Freshwater Biology, 61(5), 783–799. https://doi.org/10.1111/ fwb.12749 Gao, C., Montoya, L., Xu, L., Madera, M., Hollingsworth, J., Purdom, E., Singan, V., Vogel, J., Hutmacher, R. B., Dahlberg, J. A., Coleman-Derr, D., Lemaux, P. G., & Taylor, J. W. (2020). Fungal community assembly in drought-stressed sorghum shows stochasticity, selection, and universal ecological dynamics. Nature Communications, 11(1). https://doi.org/10.1038/s41467-019-13913-9 Grašič, M., Likar, M., Vogel-Mikuš, K., Samardžić, T., & Gaberščik, A. (2022). Decomposition rate of common reed leaves depends on litter origin and exposure location characteristics. Aquatic Botany, 179, 103513. https://doi.org/10.1016/j.aquabot.2022.103513 He, C., Zheng, L., Gao, W., Ding, J., Li, C., Xu, X., Han, B., Li, Q., & Wang, S. (2022). Diversity and functions of quorum sensing bacteria in the root environment of the Suaeda glauca and Phragmites australis coastal wetlands. Environmental Science and Pollution Research, 29(36), 54619–54631. https://doi.org/10.1007/s11356-022-19564-6 neutral model. Upper and lower limits of 95% confidence interval for predicted frequencies are also displayed. Author Contributions Conceptualization, M.L. and A.G.; methodology, M.L. and A.G.; formal analysis, M.G. and M.L.; writing—original draft preparation, M.L.; writing—review and editing, M.L., M.G., and A.G.; visualization, M.L.; funding acquisition, A.G. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by the Plant Biology (P1-0212, Slovenian Research Agency) research programme. Data Availability All sequencing data are openly available on the MG- Rast depository under project mgp97071, libraries mgm4915122.3-mgm4915169.3. Conflicts of Interest The authors declare no conflict of interest. 25 Acta Biologica Slovenica, 2023, 66 (2) Heys, C., Cheaib, B., Busetti, A., Kazlauskaite, R., Maier, L., Sloan, W. T., Ijaz, U. Z., Kaufmann, J., McGinnity, P., & Llewellyn, M. S. (2020). Neutral processes dominate microbial community assembly in Atlantic Salmon, Salmo salar. Applied and Environmental Microbiology, 86(8). https://doi.org/10.1128/ AEM.02283-19 Jaiswal, S. K., Mohammed, M., Ibny, F. Y. I., & Dakora, F. D. (2021). Rhizobia as a Source of Plant Growth-Promoting Molecules: Potential Applications and Possible Operational Mechanisms. In Frontiers in Sustainable Food Systems (Vol. 4). Frontiers Media S.A. https://doi.org/10.3389/fsufs.2020.619676 Kembel, S. W., O’Connor, T. K., Arnold, H. K., Hubbell, S. P., Wright, S. J., & Green, J. L. (2014). Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest. Proceedings of the National Academy of Sciences, 111(38), 13715–13720. https://doi.org/10.1073/ pnas.1216057111 Kerr, B., Riley, M. A., Feldman, M. W., & Bohannan, B. J. M. (2002). Local dispersal promotes biodiversity in a real-life game of rock-paper-scissors. Nature, 418(6894), 171–174. https://doi.org/10.1038/nature00823 Kowalski, K. P., Bacon, C., Bickford, W., Braun, H., Clay, K., Leduc-Lapierre, M., Lillard, E., McCormick, M. K., Nelson, E., Torres, M., White, J., & Wilcox, D. A. (2015). Advancing the science of microbial symbiosis to support invasive species management: A case study on Phragmites in the Great Lakes. In Frontiers in Microbiology (Vol. 6, Issue FEB). Frontiers Media S.A. https://doi.org/10.3389/fmicb.2015.00095 Liang, Y., Ning, D., Lu, Z., Zhang, N., Hale, L., Wu, L., Clark, I. M., McGrath, S. P., Storkey, J., Hirsch, P. R., Sun, B., & Zhou, J. (2020). Century long fertilization reduces stochasticity controlling grassland microbial community succession. Soil Biology and Biochemistry, 151. https://doi.org/10.1016/j. soilbio.2020.108023 Likar, M., Grašič, M., Stres, B., Regvar, M., & Gaberščik, A. (2022). Original Leaf Colonisers Shape Fungal Decomposer Communities of Phragmites australis in Intermittent Habitats. Journal of Fungi, 8(3), 284. https://doi.org/10.3390/jof8030284 Liu, Y., Ding, C., Li, X., Su, D., & He, J. (2023). Biotic interactions contribute more than environmental factors and geographic distance to biogeographic patterns of soil prokaryotic and fungal communities. Frontiers in Microbiology, 14. https://doi.org/10.3389/fmicb.2023.1134440 Longhi, D., Bartoli, M., & Viaroli, P. (2008). Decomposition of four macrophytes in wetland sediments: Organic matter and nutrient decay and associated benthic processes. Aquatic Botany, 89(3), 303–310. https://doi.org/10.1016/j.aquabot.2008.03.004 Rejmánková, E., & Houdková, K. (2006). Wetland plant decomposition under different nutrient conditions: What is more important, litter quality or site quality? Biogeochemistry, 80(3), 245–262. https://doi.org/10.1007/s10533-006-9021-y Sawada, H., Kuykendall, L. D., & Young, J. M. (2003). Changing concepts in the systematics of bacterial nitrogen-fixing legume symbionts. The Journal of General and Applied Microbiology, 49(3), 155–179. https://doi.org/10.2323/jgam.49.155 Serna, A., Richards, J. H., & Scinto, L. J. (2013). Plant Decomposition in Wetlands: Effects of Hydrologic Variation in a Re-Created Everglades. Journal of Environmental Quality, 42(2), 562–572. https://doi.org/10.2134/jeq2012.0201 Shen, D., Langenheder, S., & Jürgens, K. (2018). Dispersal modifies the diversity and composition of active bacterial communities in response to a salinity disturbance. Frontiers in Microbiology, 9(SEP). https://doi.org/10.3389/fmicb.2018.02188 Sistla, S. A., Asao, S., & Schimel, J. P. (2012). Detecting microbial N-limitation in tussock tundra soil: Implications for Arctic soil organic carbon cycling. Soil Biology and Biochemistry, 55, 78–84. https://doi.org/10.1016/j.soilbio.2012.06.010 Sloan, W. T., Woodcock, S., Lunn, M., Head, I. M., & Curtis, T. P. (2007). Modeling taxa-abundance distributions in microbial communities using environmental sequence data. Microbial Ecology, 53(3), 443–455. https://doi.org/10.1007/s00248-006-9141-x Stegen, J. C., Lin, X., Fredrickson, J. K., Chen, X., Kennedy, D. W., Murray, C. J., Rockhold, M. L., & Konopka, A. (2013). Quantifying community assembly processes and identifying features that impose them. ISME Journal, 7(11), 2069–2079. https://doi.org/10.1038/ismej.2013.93 Stottmeister, U., Wießner, A., Kuschk, P., Kappelmeyer, U., Kästner, M., Bederski, O., Müller, R. A., & Moormann, H. (2003). Effects of plants and microorganisms in constructed wetlands for wastewater treatment. Biotechnology Advances, 22(1–2), 93–117. https://doi.org/10.1016/j.biotechadv.2003.08.010 Vekemans, B., Janssens, K., Vincze, L., Adams, F., & Van Espen, P. (1994). Analysis of X-ray spectra by iterative least squares (AXIL): New developments. X-Ray Spectrometry, 23(6), 278–285. https://doi.org/https://doi.org/10.1002/xrs.1300230609 Vokou, D., Genitsaris, S., Karamanoli, K., Vareli, K., Zachari, M., Voggoli, D., Monokrousos, N., Halley, J. M., & Sainis, I. (2019). Metagenomic characterization reveals pronounced seasonality in the diversity and structure of the phyllosphere bacterial community in a mediterranean ecosystem. Microorganisms, 7(11). https://doi.org/10.3390/microorganisms7110518 Wallace, J., Laforest-Lapointe, I., & Kembel, S. W. (2018). Variation in the leaf and root microbiome of sugar maple ( Acer saccharum ) at an elevational range limit. PeerJ, 6, e5293. https://doi.org/10.7717/peerj.5293 Whipps, J. M., Hand, P., Pink, D., & Bending, G. D. (2008). Phyllosphere microbiology with special reference to diversity and plant genotype. In Journal of Applied Microbiology (Vol. 105, Issue 6, pp. 1744–1755). Blackwell Publishing Ltd. https://doi.org/10.1111/j.1365-2672.2008.03906.x Zhang, C., Wei, G., & Shu, D. (2023). Temporal loss of fungal taxa driven by drift contributes to community stability during soybean development. Applied Soil Ecology, 186. https://doi.org/10.1016/j.apsoil.2023.104819 26 Spoštovane kolegice in kolegi, obveščamo vas, da je v torek, 18. julija 2023, v 89. letu starosti preminil prof. dr. Dušan Zavodnik, izjemni hrvaški zoolog, sistematik, biogeograf in biocenolog. Dr. Zavodnik se je rodil 30. decembra 1934 v Ljubljani, kjer je leta 1954 končal gimnazijo. Diplomiral je iz biologije na Naravoslovni fakulteti Univerze v Ljubljani leta 1985 pod vodstvom prof. J. Hadžija, leta 1964 pa je doktoriral na Biotehniški fakulteti Univerze v Ljubljani pod mentorstvom prof. M. Zeia. Interes za znanstveno delo in življenje v morju je kazal že kot srednješolec, saj je med poletnimi počitnicami opravl- jal prakso v laboratorijih Inštituta v Rovinju in Splitu. Leta 1960 se je zaposlil v novo ustanovljenem Inštitutu za biologijo morja Jugoslovanske akademije znanosti in umetnosti (JAZU) v Rovinju, leta 1964 pa je postal tudi direktor inštituta. Leta 1968 je ustanovil Laboratorij za ekologijo in sistematičnost, leto kasneje pa se je njegov laboratorij pridružil novo ustanovljenemu oddelku Centra za raziskovanje morja Rovinj - Zagreb (CIM Rovinj - Zagreb) v okviru Inštituta Ruđer Bošković (IRB). Po razdelitvi oddelka CIM Rovinj - Zagreb v samostojne organizacijske enote v okviru IRB je bil dr. Zavodnik v obdobju od 1981 do 1984 direktor rovinjskega dela. Leta 1975 je postal znanstveni svetovalec. Kot redni profesor na Univerzi v Zagrebu je predaval na treh kolegijih podiplomskega študija biologije morja in bil mentor številnih diplomskih, magistrskih in doktorskih del. Leta 1999 se je upokojil, a je nadaljeval z delom vse do konca življenja. Znanstveni opus dr. Zavodnika je obsežen in raznolik. Objavil je več kot 150 izvirnih znanstvenih člankov v domačih in tujih revijah ter več deset strokovnih člankov, predvsem o biologiji morja in zaščiti Jadrana. Organiziral je šest mednarodnih znanstvenih konferenc, objavljal je tudi v strokovnih revijah in dnevnem tisku. Bil je član več bioloških društev in je bil v enem mandatu predsednik Jugoslovanskega društva biosistematikov. Osrednje področje njegovih raziskav so bili kompleksni procesi v obalnih in odprto-morskih ekosistemih vzhod- nega dela Jadranskega morja. Posebno pozornost je namenil bodičarjem, školjkam in ribam Jadrana ter življen- jskim skupnostim litoralnih stopnic. Aktivno se je ukvarjal tudi s zgodovino raziskav Jadrana, predvsem z zgodovino matične ustanove v Rovinju. Po upokojitvi se je posvetil biotski raznovrstnosti in biogeografiji favne Jadrana. Med celotno svojo poklicno kariero je terensko delo štel za temelj svojega raziskovalnega in pedagoškega delo- vanja. Zgodaj se je zanimal za samostojno potapljanje, ki ga je štel za skorajda neizogibno metodo terenskega dela vsakega "morskega" biologa. Zato ga lahko smatramo za pionirja in začetnika znanstvenega potapljanja v Rovinju. Bogato znanje in izkušnje terenskega dela je nesebično delil s svojimi sodelavci in študenti. Dr. Dušan Zavodnik je bil pokopan v četrtek, 18. julija 2023, v Rovinju. dr. Andrej Jaklin In memoriam: prof. Dr. Dušan Zavodnik (1934–2023) Acta Biologica Slovenica 2023 Vol. 66 | Št. 2