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63Russian Journal of Nematology, 2018, 26 (2), 115 - 122
Comparative analysis of nematode defaecation
motor program
Georgy A. Slivko-Koltchik1, Victor P. Kuznetsov1, Kirill V. Mikhailov1' 2,
12 12 Dmitry A. Voronov ' and Yuri V. Panchin '
1Institute for Information Transmission Problems of Russian Academy of Sciences, Moscow, Russia 2A.N. Belozersky Institute of Physico-Chemical Biology MSU, Moscow, Russia e-mail:[email protected]
Accepted for publication 24 November 2018
Summary. The nervous system controls most rhythmic behaviours, with one remarkable exception. The defaecation motor program (DMP) in C. elegans is initiated by intestinal cells' pacemaker without participation of the nervous system. Our study on Heterorhabditis megidis and Enoplus brevis demonstrates that DMP machinery is not a unique feature of C. elegans. In C. elegans the role of several genes important for DMP has been experimentally studied. PBO-5/PBO-6 proton-gated ion channel mediates the signal transduction from gut cells to body muscles. We found homologues for this protein in other nematodes and demonstrated pH sensitivity of E. brevis body muscles. These and other DMP hallmark proteins NLP-40, AEX-2 and EXP-1 are also found in E. brevis and other species but not outside nematodes. Some nematodes lack individual proteins of DMP toolkit. For example, mermithids with degenerated digestive systems and with no through gut lost them all. Different nematode species possess the same non-nervous rhythmical DMP mechanism that evolved in the nematode common ancestor.
Key words: Caenorhabditis elegans, Enoplus brevis, Heterorhabditis megidis, nematode evolution.
Rhythmic activity plays an important role in physiology of various species. The period of rhythmic oscillations in different systems can vary from seconds to years resulting in a great variety of mechanisms for the generation and adjustment of physiological rhythms. Rhythmic model system studies are especially important as they allow in depth understanding of the mechanisms of the system functioning. One of the examples is the defaecation in nematodes, which has been studied in detail in the model species Caenorhabditis elegans. The defaecation motor program (DMP) is represented by the coordinated events occurring in the midgut, some muscle cells, and several neurons (Avery & Thomas, 1997). The defaecation process consists of three consecutive series of muscle contractions that occur every 45 s in adult feeding hermaphrodites in the presence of food: posterior body wall muscle contraction (pBoc), anterior body wall muscle contraction (aBoc) and expulsion step (Exp) (Thomas, 1990; Liu & Thomas, 1994). pBoc is a well-studied process, while mechanisms for aBoc and Exp are incompletely examined. DMP is primarily controlled by the mid-gut cells, which can
autonomously generate rhythmical oscillations of calcium concentration in the cytoplasm (Espelt et al., 2005; Teramoto & Iwasaki, 2006). The concentration of calcium initially increases in the posterior cells, and then a wave of high calcium concentration is propagated from the posterior to anterior midgut. This wave temporally coordinates the consecutive acts of defaecation: pBoc, aBoc and Exp. In this process, one of the main mechanisms of signal transmission relies on the body cavity acidification: cells of the midgut pump out protons that act on the muscle cells as a transmitter (Beg et al., 2008; Pfeiffer et al., 2008). In a species related to C. elegans, Heterorhabditis megidis, DMP is accompanied by periodical fluctuations of the membrane electrical potential in the intestinal cells, which are also synchronised through gap junctions (Kuznetsov et al., 2017). So, in these Rhabditida species, C. elegans and H. megidis, the endoderm cells act like brain central pattern generator interneurons, and similarly to neurons these intestinal cells can be synchronised along the cellular network via electrical synapses (gap junctions) (Kuznetsov et al., 2016).
© Russian Society of Nematologists, 2018; doi:10.24411/0869-6918-2018-10011
Fig. 1. Schematic diagram of the posterior part of C. elegans nematode body with the midgut, surrounding muscles and neurons shows the main players in signalling pathways involved in the defaecation behaviour. IP3 (inositol trisphosphate) (cyan cross) and Ca2+ ions (green rhomb) play the key role in the initiation, regulation and propagation of the oscillatory process (Dal Santo et al., 1999). IP3Rs (inositol trisphosphate receptors) are activated by IP3 that forms by hydrolysis of phospholipid PIP2 (phosphatidylinositol 4, 5-bisphosphate) by phospholipase C (PLC) located in the plasma membrane. Calcium wave is initiated by Ca2+ ion release from the endoplasmic reticulum triggered by IP3Rs (Baylis & Vázquez-Manrique, 2012). Wave propagates from the posterior to anterior via gap junctions (INX-16) (Peters et al., 2007). TRPM channels GTL-1/GON-2 (Kwan et al., 2008) increase the intracellular concentration of Ca2+. To keep the process cycled the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) restores Ca2+ to the baseline levels (Hoon Cho et al., 2000). PBO-1, a calcium binding protein, coordinates Na+ (magenta triangle) and H+ (grey circle) ion exchange by PBO-4/NHX-7 with cyclic calcium oscillations (Beg et al., 2008). PBO-4/NHX-7 anion exchanger transfers protons to pseudocoelome and sodium into the cell through the basolateral membrane. On the apical side, NHX-2 ion exchanger transfers protons to lumen and sodium into the cell through the basolateral membrane (Pfeiffer et al., 2008). It is coupled with a high capacity oligopeptide transporter OPT-2/PEPT-1 that absorbs dipeptides (yellow waves) and protons from the lumen (Wagner et al., 2011). PBO-4/NHX-7 proton afflux from the gut cells activates PBO-5/PBO-6 proton-gated ion channel heterodimer, which depolarises the posterior body wall muscles and causes their contraction (pBoc). aBoc is the most incompletely studied event in this process. It is known that killing AVL neuron causes strong defects of the aBoc step (Wang & Sieburth, 2013). Although AVL is GABAergic neuron, GABA dysfunction mutants have a normal aBoc step. Thus anterior body muscles are controlled by AVL motoneurons but do not require GABA in the pathway from Ca2+ wave initiation to muscle depolarisation. Synaptotagmin (SNT-2) localised on the dense core vesicles (DCV) being bound to calcium promote the release of neuropeptide-like protein NLP-40 from the basolateral surface of intestinal cells (Zhao & Schafer, 2013). Secreted NLP-40 activates AEX-2/GPCR receptor on AVL and DVB neurons. cAMP signalling pathway inside the neuron leads to a calcium influx, neuron excitation and GABA release (green octothorpe) (Wang et al., 2013). Excitatory GABA receptor EXP-1, located on enteric muscles (electrically coupled via gap junctions (Altun et al., 2009) anal sphincter, anal depressor (AD) and stomato-intestinal muscles), binds GABA and causes contraction. This results in the final expulsion DMP step.
Fig. 2. Cladogram (Blaxter et al., 1998) of nematode species. Homo sapiens is an outgroup. Presence or absence of four hallmark proteins involved in the nematode defaecation process in various species is shown on the right.
It remained unclear if the defaecation mechanism is a unique feature of the investigated representatives of Rhabditida or exists in other nematodes. In the previous paper we described membrane potential cycling using intracellular electrophysiological techniques in the midgut cells of a non-rhabditid nematode Enoplus brevis (Enoplida) (Slivko-Koltchik et al., 2018) resembling DMP oscillations in C. elegans and H. megidis. We also found that, similarly to C. elegans and H. megidis, the gut cells in E. brevis, are electrically connected through gap junctions. Here we compare molecular elements of the DMP machinery in phylogenetically distant species, including E. brevis and consider its evolutionary origin in nematodes.
At present, the roles of several dozen genes important for DMP in C. elegans have been experimentally studied (Branicky & Hekimi, 2006) (Fig. 1). In combination with the availability of different nematode species genomic sequences, this makes it possible to study the evolution of the DMP within the whole Nematoda phylum.
We have analysed four genes aex-2, nlp-40, pbo-5/6 and exp-1, specifically important for C. elegans DMP execution. The presence of true orthologues of these genes in other species can indicate the presence of DMP in these animals. We conducted the bidirectional best hits search (BBH) (Overbeek et al., 1999) for the selected genes among 18 currently available complete genomes of the Nematoda phylum and carried out phylogenetic analysis of a wide superfamily of the Cys-loop ligand-gated ion channels including PBO-5/6 and EXP-1 proteins (Jaiteh et al., 2016).
Predicted expression of PBO-5/6 in E. brevis was confirmed in physiological experiments that demonstrate body muscle pH sensitivity in this species.
MATERIAL AND METHODS
Identification of proteins homologous to the C. elegans DMP related genes was performed by sequence similarity search using the BLASTP program and the WormBase ParaSite
(https://parasite.wormbase.org) and NCBI protein databases (https://www.ncbi.nlm.nih.gov/). In the case of nematode E. brevis we assembled the available transcriptomic data (ERR660661 from the NCBI BioProject PRJEB7588) with Trinity software (Grabherr et al., 2013) and extracted translated ORF from transcripts with TransDecoder (Haas et al., 2013). The orthology of proteins found by BLASTP search seeded with C. elegans DMP related genes was tested by bidirectional best hits (BBH) method (Overbeek et al., 1999) that identifies the pairs of genes in two different genomes that are more similar to each other than either is to any other gene in another genome. If the reciprocal best BLAST hit coincided with the initially used C. elegans protein, both proteins were considered orthologous. This procedure was used for four proteins searched in 18 various species (Fig. 2). For receptor's phylogenetic tree, selected nematode sequences and H. sapiens receptors (Beg & Jorgensen, 2003) were used. This protein collection was aligned by MUSCLE program (Edgar, 2004) and the phylogenetic tree was constructed by the neighbour-joining algorithm.
Enoplus brevis were extracted from sandy littoral at the White Sea biological station of the Russian Academy of Sciences "Kartesh" in the summer of 2017 and 2018 and kept in seawater at 4°C. The
adult Romanomermis culicivorax were harvested from larvae of chironomids (lake flies) obtained at the local pet store. All physiological experiments were carried out at room temperature (30°C). The posterior fragments of worms containing the body muscles were cut out using fine forceps and scissors, placed in a Petri dish with a bottom coated by silicone rubber, and fastened by thin metal needles or braces, fixing one point and allowing the loose end to move freely. As a bath media for E. brevis we used Millipore-filtered sterile sea water (buffered with 1mM HEPES to pH 7); for R. culicivorax we used Hanks solution (137 mM NaCl, 5.4 mM KCl, 0.25 mM, N2HPO4, 0.44 mM KH2PO, 1.3 mM CaCl, 1.0 mM MgSO4, 4.2 mM NaHCO3; 1mM HEPES; pH = 7.4). To study the action of acetylcholine (ACh) on the posterior body wall muscles, the ACh was added to Petri dish with the final concentration of 10-6 M. To study the effects of pH changes, bath media in Petri dish was replaced by the same media buffered to pH 6. The muscle contractions were video recorded. To visualise the contractions, 30 s video fragments were split to image sequences and uploaded into FIJI image analysis tool (Dobretsov et al., 2017). Total images were acquired by Z-projection algorithm (z-time) with the maximum intensity parameter.
Fig. 3. Phylogeny tree of Cys-loop receptors in nematodes species with Homo sapiens outgroup. Only nematodes with the presence of either exp-1 or pbo-5 were selected. Star - Homo sapiens. Circle - Caenorhabditis elegans. Triangle - Heterorhabditis bacteriophora, Pristionchus pacificus, Ditylenchus destructor, Bursaphelenchus xylophilius, Syphacia muris, Enterobius vermiculari, Enoplius brevis (have both pbo-5 and exp-1). Square - Meloidogyne incognita, Ascaris suum, Thelazia callipaeda, Brugia malayi, Dracunculus medinensis, Gongylonema pulchurum, Trichuris suis, Trichinella spiralis, Soboliphyme baturini (with exp-1 but without pbo-5 gene).
RESULTS AND DISCUSSION
BLAST search seeded by C. elegans orthologues showed that two of the four genes from the selected DMP hallmark genes, namely, aex-2 and its ligand nlp-40, were found only among nematodes (less than E-value 10-87) and were not found in species outside Nematoda species (more than E-value 1010). The other two genes, pbo-5 and exp-1, have homologues with various functions from both inside and outside the phylum Nematoda. To search for the true orthologues of these genes among the all animals, we used the bidirectional best hits method (BBH) that confirmed the presence of pbo-5 and exp-1 in several nematode species. It is known that the BBH in some cases can lead to misinterpretations and therefore it requires verification using phylogenetic analysis (Dick et al., 2017). The pbo-5 and exp-1 genes belong to the widespread class of Cys-loop ligand-gated ion channels such as nicotinic acetylcholine, GABA, glycine, glutamate and serotonin ionotropic receptors; exp-1 encodes unusual excitatory GABA receptors (Beg & Jorgensen, 2003). In Cys-loop
family tree, exp-1 from C. elegans clusters together with its putative orthologues found by BLAST and BBH search in all studied nematodes with the exception of R. culicivorax. At the same time, this phylogenetic tree confirms the presence of C. elegans pbo-5 homologues in eight nematode species, including E. brevis.
The results of our search for DMP related genes within 18 nematodes with sequenced genomes are presented in the Fig. 2. Eight species have all four genes under consideration aex-2, nlp-40, pbo-5 and exp-1; two species do not have pbo-5 and have nlp-40, seven species do not have pbo-5 and nlp-40, and one lacks all four DMP genes. Since the studied genes are found in phylogenetically distant species, including E. brevis, which is located at the root of the phylogenetic tree of Nematoda (Blaxter et al., 1998) , it can be assumed that the common ancestor of Nematoda had all four hallmark DMP genes. Further, in the course of evolution some species lost some of these genes, while the R. culicivorax, which is a parasitic mermithid nematode with degenerated digestive systems and with no through gut, lost them all.
The presence of C. elegans pbo-5/6 orthologues
Fig. 4. Effects of acetylcholine (ACh) and acidification (ApH) on body muscle contraction in Enoplus brevis (A, B, C) and Romanomermis culicivorax (D, E, F) nematodes. Preparations of posterior body with exposed muscles were fastened in chamber by thin metal needles or braces (white arrowhead), fixing one point and allowing the loose end (white arrow) to move freely. To visualise the preparation movement video recordings were taken during the entire experiment; 30 s fragments were split into frames and image stacks were downloaded to the FIJI image analysation tool and Z-projected (z-time) with the maximum intensity parameter. Motion (shown by double arrows) appear as a sequence of the preparation images during 30 s time period. Plain bath medium applied in control (Con) experiments (A, D) produced no effect. Application of acidified medium (pH 6) activated E. brevis muscle and had no effect in R. culicivorax. In the positive control, ACh (10-6M) application produced muscle contraction in both species (C, F). The sequence of the same experimental procedures: control - ApH - ACh with intermediate washouts was repeated with the same results for four times for both nematode species.
in E. brevis implies that its muscle cells are pH sensitive and they will contract in response to external medium acidification. By contrast, in R. culicivorax, which lacks pbo-5/6 genes, the muscles will not respond to pH changes. At the same time, muscle cells of both species will be able to respond to acetylcholine application in the same way due to nicotinic acetylcholine receptors' conservation in all nematode species (Figs 2 & 3). We have developed a preparation where cuticle and hypoderm were removed from the p art of the nematode body, hence muscle cells were exposed to physiological solution and muscle contractions were recorded by a video camera. In the experiments presented in Fig. 4 acidification of the medium in the vicinity of the posterior body muscles caused contraction in E. brevis (Fig. 4B), while R. culicivorax muscle cells showed no response (Fig. 4E). However, the application of acetylcholine to the medium resulted in contraction of the posterior muscles in both E. brevis and R. culicivorax (Fig. 4C, F). The muscle contraction was not caused by a mechanical effect of the media replacement in the experimental chamber, given that during control replacements the contraction has not occurred (Fig. 4A, D). Thus, our data from physiological experiments confirms the results of bioinformatics research for DMP related genes in nematodes.
Previously in the physiological experiments we have demonstrated the presence of a specific rhythmic activity associated with DMP in the midgut of E. brevis and H. megidis (Kuznetsov et al., 2017; Slivko-Koltchik et al., 2018). New bioinformatics and physiological study reported here confirms that the intricate DMP machinery first discovered in C. elegans is not a unique feature of this species but is a nematode-specific biological creation that evolved in the common ancestor of nematodes.
ACKNOWLEDGEMENTS
The Russian Foundation for Basic Research supported this work (project no. 18-04-00705).
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Г.А. Сливко-Кольчик, В. Кузнецов, К. Михайлов, Д.А. Воронов и Ю.В. Панчин.
Сравнительный анализ моторной программы дефекации нематод.
Резюме. Обычно ритмические формы поведения у животных управляются нервной системой. Но есть одно исключение. Дефекационная моторная программа (ДМП) нематоды C. elegans управляется пейсмекером из кишечных клеток без участия нервной системы. Наши предыдущие исследования на нематодах Heterorhabditis megidis и Enoplus brevis показали, что механизм ДМП не является уникальной особенностью вида C. elegans. Роль нескольких генов, необходимых для организации ДМП, подробно изучена. Протон-зависимые ионные каналы РВО-5/РВО-6 являются важными посредниками при передаче сигнала от кишечника к мышечным клеткам тела. Мы нашли гомологи этого белка в других нематодах и показали чувствительность мышц нематоды E. brevis к изменению уровня рН. РВО-5/РВО-6 и белки ^Р-40, АЕХ-2 и ЕХР-1 были обнаружены нами в геноме E. brevis и у других нематод, но не у видов за пределами типа Nematoda. Некоторые представители типа потеряли определенные белки, необходимые для ДМП. Например, все перечисленные гены потеряны у мермитид - паразитов с дегенерированной пищеварительной системой, не имеющих сквозной кишки. Различные виды нематод имеют сходный, независимый от нервной системы механизм ДМП, возникший у общего предка всех нематод.
