Journal of Coastal Life Medicine 2014; 2(5): 376-381 376

Journal of Coastal Life Medicine

Journal homepage: www.jclmm.com

Document heading doi: 10.12980/JCLM.2.201414J3 2014 by the Journal of Coastal Life Medicine. All rights reserved. 襃 Cholinergic neuromuscular junctions in Brachionus calyciflorus and Lecane quadridentata (Rotifera: )

1 2 3 4 Ignacio Alejandro Pérez-Legaspi *, Alma Lilián Guerrero-Barrera , Iván José Galván-Mendoza , José Luis Quintanar , Roberto Rico-Martínez5 1Graduate Studies and Research Division, Boca del Río Institute of Technology, Km. 12 Carr. Veracruz–Córdoba, 94290, Boca del Río, Ver., México 2Morphology Department, Basic Sciences Center, Aguascalientes Autonomous University, Ave. Universidad 940, Aguascalientes, 20100, México 3Confocal Microscopy Unit, Experimental Services Department, CINVESTAV-Zacatenco, National Politechnic Institute, Ave. Politécnico, 2508, Zacatenco, 56000, México, D.F., México 4Pharmacology and Physiology Department, Aguascalientes Autonomous University, Ave. Universidad 940, Aguascalientes, 20100, México 5Chemistry Department, Basic Sciences Center, Aguascalientes Autonomous University, Ave. Universidad 940, Aguascalientes, 20100, México

PEER REVIEW ABSTRACT

Peer reviewer Objective: To iBrachionusdentify the calycifloruspresence of jointLecane muscu quadridentatalar and cholinergic systems in two freshwater Dr. Evangelia Michaloudi, Assistant rMethods:otifer species, and . Professor, School of Biology, Aristotle The muscle actin fibers were stained with phalloidin-linked fluorescent dye, and University of Thessaloniki, 54124 acetylcholine was detected with Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit, and Thessaloniki, Greece. tResults:hen confocal scanning laser mBrachionusicroscopy w calyciflorusas used. +302310998265 Tel: The musculature of Lecane quadridentatashowed a pattern similar to other species E-mail: [email protected] of the same genus, while that of was different from other genera Comments described previously. The cholinergic system was determined by co-localization of both muscles aConclusions:nd acetylcholin e labels in the whole rotifer, suggesting the presence of neuromuscular junctions. The manuscript is dealing with an The distribution pattern of muscular and acetylcholine systems showed aspect for which not much information considerable differences between the two species that might be related to different adaptations is available; the neurotraBrachionusnsmission to particular ecological niches. The confirmation of a cholinergic system in contributes to scalyciflorusystem of rotifers including the development of potential neuro-pharmacological and toxicological studies using rotifers as . This species along with model organism. other rotifers are being studied in order to be used in toxicity tests thus knowledge on the way the neuromuscular system functions is essential. Furthermore increasing the available information further adds to the comparative analysis elucidating ecological and evolutionary KEYWORDS relationships. Acetylcholine, Co-localization, Confocal microscopy, F-actin, Rotifer Details on Page 380

1. Introduction pseudocoelomates invertebrates[1]. The body is typically divided into three regions: the apical end (head) forming a Rotifers are cosmopolitan mainly aquatic or semiaquatic ciliated region known as corona, the trunk and the foot. In microscopic non-segmented, bilaterally symmetric and many species, the corona has two concentric rings of cilia é C I A P L G S * orresponding author: gnaício lejandro rez- egaspi, raduate tudies and Article history: Research Division, Boca del R o Institute of Technology, Km. 12 Carr. Veracruz- R 12 J 2014 ó í é eceived an C rdoba, 94290, Boca del R o, Ver., M xico. Received in revised form 1 Feb, 2nd revised form 8 Feb, 3rd revised form 18 Feb 2014 Tel: +052-229-9860189, 9862818 ext. 129 Accepted 23 Apr 2014 Fax: +052-229-9861894 Available online 28 May 2014 E -mail: [email protected] í Foundation Project: Supported by Consejo Nacional de Ciencia y Tecnolog a (Conacyt) through the scholarship (Grant No. 136453), and Sistema Nacional de Investigadores (Grant No. 49351). Ignacio Alejandro Pérez-Legaspi et al./Journal of Coastal Life Medicine 2014; 2(5): 376-381 377 Lecane that beat in a metachronous pattern creating enough flow of been described although this genus is highly to obtain food and engage in locomotion. Rotifers have a diverse with 163 species recorded so far[27]. Therefore, the muscular pharynx (mastax) with a complex set of chitinous jaws information on Lecanidae would be valuable for comparison (trophi) of high taxonomic importance used to classify rotifer with other rotifer species and would increase knowledge about species. Class Monogononta comprises 70% of rotifer species, the diversity of rotifer muscular systems. The objective of this including 95% of benthonic, sessile, and free swimming study was to reveal the joint muscular and cholinergic system B. calyciflorus L. quadridentata species[1,2]. The ultrastructural organization of rotifers has in two rotifer species: and . been studied by means of scanning and transmission electron microscopy[3]. The rotifer muscular system has been studied Philodina 2. Materials and methods in whole individuals of sp. by specific F-actin fiber recognition with Alexa-488 phalloidin or labeled phalloidin- B. calyciflorus TRITC. Several works have studied the rotifer muscular system The rotifer species studied were (Pallas Euchlanis with confocal laser scanning microscope (CLSM) in 1776) collected from the pond of the water treatment plant at dilatata unisetata Brachionus quadridentatus B. ó ° ’ ° ´ and ( Universidad Aut noma de Aguascalientes (21 55 N, 102 29 quadridentatus Brachionus urceolaris, Floscularia ringens, é L. quadridentata )[4], W), Aguascalientes, M xico, and (Ehrenberg Hexarthra mira Notommata glyphura ° ’ ° ´ , and [5], three species 1832) collected from Lake Chapala (20 12 N, 102 85 W), Jalisco, Proales Filinia novaezealandiae Encentrum é ± ° of the genus [6], [7], M xico. Rotifers were cultured at (25 2) C in plastic Petri mucronatum Dicranophorus forcipatus Hexarthra and [8], dishes with freshwater EPA medium[28], using a bioclimatic Polyarthra Bryceella stylata , Adineta ricciae and [9], [10] and chamber with a 16: 8 Light: Dark period and fed with the Macrotrachela quadricornifera , Brachionus manjavacas Nannochloris oculata [11] green algae (UTEX strain LB2194) grown Epiphanes senta , Squatinella rostrum , Trichotria ’ é and [12] [13] and in Bold s medium[29], following the method of P rez-Legaspi pocillum í [14]. The rotifer nervous system comprising a large and Rico-Mart nez[30]. A hundred amictic females of each cerebral ganglion known as the brain positioned dorsally species were aµdded randomly into microcentrifuge° tube below the corona, paired ventral neurons, which branches containing 500 L of EPA medium and incubated at 4 C for ( E to the mastax, body and foot, and sensory organs mechano, one hour, to precipitate the rotifers. ach° rotifer sample was chemo, and photoreceptors); even a retrocerebral organ centrifuged at 5 000 r/min for 10 min at 4 C to eliminate EPA (paired subcerebral glands, unpaired retrocerebral sac, and medium, and then rested in 1% MgCl2 for one hour to maintain paired ducts leading to the apical surface) is found behind the integrity of individuals. Centrifuged rotifer samples were the brain in many bdelloid and monogonont rotifers[1,2]. fixed in 4% formalin in 0.01 mol/L phosphate-buffered saline This nervous system has been described by showing the (PBS, pH 7.4) for 30 min, centrifuged and rinsed with 0.01 mol/ presence of neurotransmitters (acetylcholine) and enzymes L PBS. Subsequently, rotifers were centrifuged to eliminate the (acetycholinesterase), demonstrating the existence of a medium and permeabilization solution 0.1% Triton X-100 in cholinergic system in rotifers by histochemical methods[1,15,16]. 0.01 mol/L PBS was added for one hour, following the protocol The studies did not include in their analysis individuals of Hochberg and Litvaitis[31]. Brachionus Lecane of the genera or . Other studies have B. 2.1. Acetylcholine labeling described catecholaminergic neurons in rotifers like quadridentatus [17,18]. Other types of neurotransmitters, FMRF (5 ) A A such as amide and serotonin -hydroxytryptamine , cetylcholµine was shown in rotifers using mplex red have been reported in rotifers, using histochemical, reagent (400 mol/L) containing horseradish peroxidase, Alcaligenes immunocytochemical, and CLSM techniques, which indicate choline oxydase from sp., and acetylcholinesterase the presence of a serotonergic nervous system[19-23]. from electric eel (Molecular Probes, Inc. Eugene, OR, USA); Otherwise, acetylcholinesterase receptors were recognized subsequently each rotifer sample was incubated in darkness B. quadridentatus Lecane luna Lecane quadridentata L. in , , ( for 30 min at room temperature. The method of Amplex Red quadridentata Plationus patulus Rotaria neptunia ) A A A K (A12217) , αand using cetylcholine/ cetylcholinesterase ssay it was epifluorescence microscopy and -bungarotoxin fluorescein used to detect acetylcholine following hydrolysis to choline, isothiocyanate (FITC)[24]. Also, the exocytotic membrane and oxidation, by choline oxydase, to betaine and H2O2, which proteins known as SNARE (syntaxin-1, syntaxin-4, SNAP-23, in contact with horseradish peroxidase, reacts with Amplex and SNAP-25) involved in the vesicular release of secretory red reagent in a 1:1 stoichiometry to generate a fluorescent proteins were identified in three rotifer species by mean of product (resorufin). After incubation, rotifers were washed in immunohistochemical and immunoblot techniques[25]. PBS and stained for F-actin fibers. The CSLM studies have been limited to the musculature Brachionus 2.2. F-actin labeling of individuals of some species of the genus : Brachionus plicatilis, B. quadridentatus, Brachionus urceolaris Brachionus manjavacas , and and [4,5,12,26]. FITC-labeled (SIGMA-Aldrich) was added to rotifers to Brachionus calyciflorus B. calyciflorus ( ) has not been studied show mainly the muscular system following the protocol by this method. Neither has the muscle system of the species of Hochberg and Litvaitis with slight modifications[31]. The Ignacio Alejandro Pérez-Legaspi et al./Journal of Coastal Life Medicine 2014; 2(5): 376-381 378 sample was incubated overnight at room temperature in the 3A). The neuromuscular junctions indicate the co-localization dark. Subsequently, rotifer samples were rinsed in 0.01 mol/ of both muscles (green dots) and acetylcholine (red dots). PBS L , mounted on microscopic slides and examined on the A sm B Confocal/two photons Microscope Leica Systems TCS SP5- MO equipped with laser Argon/HeNe 543, using excitation – cm m spectrum in 530 560 nm, and emission detection at ~590 nm. µ rcd The scanning step size was about 0.5 m. The max-projection 1 rfv 1 (flat projections of sections) was obtained by scanning from dv2 dv m series of optical sections in order to make reconstructions of rfv2 rcl L LAS AF the whole by using the eica software. rfv3 rcv rf rcm rfl3 rfl2 rfl1 st 3. Results

3.1. L. quadridentata C D Rotifer CSLM images obtained from the entire specimen n st ( =6) showed various muscles stained by phalloidin (Figure 1A); in the lateral view it is possible to observe a pair of thin circular muscles (cm) in the medial region of the trunk, and two wide pairs of superior and inferior dorso-ventral muscles (dv1-2). Longitudinal musculature showed different strong and wide retractor muscles observed in the dorso-ventral medial tr region as the medial retractor muscles of corona (rcm) inserted from upper ventral-medial region and connected into the muscles of mastax (m); also the wide pyramid shape lateral retractor muscles of the corona (rcl) connected to the upper ( ) sides of the superior mastax sm , while thin ventral retractor Figure 1. L. quadridentata Whole mounts of at dorsoventral examination (20 , muscles of the corona (rcv) were observed in the middle and µ 伊 scale bar: 30 m). M connected into the lateral sides of mastax region. uscles of A: Max projection showed musculature by phalloidin green fluorescent dye ( ) the mastax m were present between the insertions of retractor FITC by means of CSLM; B: Max projection display acetylcholine stained red muscles of corona suggesting that they are connected to the by Amplex red assay using CSLM; C: Max projection revealed both musculature trophi (either ramus or fulcrum). A superior mastax muscle (green) and acetylcholine (rhodamine) by the two pictures using CSLM; D: (sm) was observed above the insertions of retractor muscles Optical light microscopy picture showed. rcm: Medial retractor muscles of of the corona. In the lower ventral-medial region the strong corona; rcv: Ventral retractor muscles of corona; rcl: Lateralretractor muscles of corona; m: Mastax; sm: Superior mastax muscle; dv1-2: Dorso-ventral and broad retractor muscles of the foot (rf), extended from muscles; rfv1-3: Ventral retractor muscles of foot; rfl1-3: Lateral retractor muscles the ventral middle toward the foot, was present in the medial of foot; rcd: Dorsal retractile muscle of corona. st: Stomach; tr: Trophi. region. Several stronger and conspicuous ventral retractor muscles of the foot (rfv1-3) were easily observed, while at 3.2. B. calyciflorus the sides narrow lateral retractor muscles of foot (rfl1-3) were seen; it was clear that the muscles of the foot engrossment, n from the middle to inferior of the trunk, are attached to the The CLSM picture obtained from whole specimens ( = lower foot. A CSLM picture obtained from a whole specimen 42) showed the musculature stained by phalloidin green showed diverse sites marked with acetylcholine (Figure 1B). fluorescence (Figure 2A); in the upper lateral side it was All mastax (m) regions and some dots near the corona region possible to recognize three pairs of dorso-ventral muscles were observed, while the conspicuous stomach (st) region (dv1-3). Longitudinal musculature was represented by long, showed the greatest amount of acetylcholine dye in rotifers. wide retractor muscles of the corona (rc) originating near In Figure 1C a whole mount of rotifer reveals double-staining the mastax and in the middle part of the body extending of both fluorescent markers, one for muscles (phalloidin in anteriorly and connected to the corona region. These green color) and one for acetylcholine (rhodamine in red muscles were present in the contracted state (the animal color) using CSLM. A photograph of a complete rotifer using retracted the corona before dying); and they include pairs of light microscopy showed several structures such as stomach ventral (rcv), lateral (rcl) and dorsal (rcd) retractor muscles (st), and a conspicuous trophi (tr) by optical light microscopy of the corona. The largest mastax muscle corresponded (Figure 1D). Double-labeling revealed co-localization (white to the superior mastax (sm), also under and at both sides dots) of many specific features (neuromuscular junctions) of this muscle, there are medial mastax muscles (mm1-2). L. quadridentata concentrated in the stomach region of (Figure Below them a pair of inferior mastax muscles (im) formed a Ignacio Alejandro Pérez-Legaspi et al./Journal of Coastal Life Medicine 2014; 2(5): 376-381 379 “ ” V shape. In the lower ventral-medial region conspicuous large and strong contracted muscles were evident such as retractor muscles of the foot (rfv1-2). At the sides thin and larger narrow longitudinal muscles (lm) were observed that extended towards the lower body. A pair of thinner and longitudinal muscles corresponded to dorsal tubular

( ) R muscles dtt . Acetylcholine was present in diverse areas O L within the rotifer body (Figure 2B). The corona region 1 (cr) showed a conspicuous presence of acetylcholine. A whole specimen double-marked in the muscular system (by phalloidin in green color) and cholinergic system (acetylcholine stained by rhodamine in red color) showed some of the red dots corresponding to the localization of A B acetylcholine masked by phalloidin (Figure 2C). Double- Figure 3. Max projections showing muscular system (green dots), cholinergic labeling revealed co-localization (white spots) of the specific B. calyciflorus system (red dots), and co-localization of muscular and cholinergic systems ( ) µ points neuromuscular junctions of indicating (white dots) (scale bar: 30 m). L. the proximity or co-localization of both muscles (green dots) A: The area (green circle) corresponds to the region of interest in quadridentata B. calyciflorus and acetylcholine (red dots), mainly in the stomach region . (B) . and the corona with slight points near the mastax region (Figure 3B). A B 4. Discussion rfv1 rfv2 lm ov The co-localization of muscular (phalloidin-stained points) st im ( ) 2 and cholinergic systems rhodamine-stained points in both mm rcd B. calyciflorus L. quadridentata 1 mm rcv and , indicate the existence sm rcl m of cholinergic neuromuscular junctions where acetylcholine dv3 is released from presynaptic vesicles to muscles to cr N dv2 control most movements of the rotifer. erve endings were previously reported using transmission electron microscopy é [3] dtt dv1 by Clem nt and Wurdak . These authors observed synaptic vesicles suggesting that they might contain the acetylcholine C D neurotransmitter. Our CLSM study confirms the presence of acetylcholine as a potential marker of neuromuscular st junctions, and is useful for demonstrating their location within the rotifer body. The Amplex Red Acetylcholine/Acetylcholinesterase Assay m ov Kit makes it possible to detect acetylcholine by rhodamine tr stain using CLSM to demonstrate fluorescence signals in L. quadridentata B. calyciflorus both rotifers studied and . L. quadridentata Clearly shows a remarkable concentration B. calyciflorus c of neuromuscular junctions than . A greater number of neuromuscular junctions results in a finer control L. quadridentata of muscular contractions, probably requires B. calyciflorus Figure 2. B. calyciflorus a greater effort coordinated than to move Entire specimens of with foot retracted viewed at µ P dorsoventral examination (20伊, scale bar: 30 m). into the sediment such as benthic environment. halloidin (A) Max projection illustrated musculature by phalloidin green fluorescent dye stain revealed skeletal muscles in several orientations FITC by mean of CSLM; B: Max projection display acetylcholine showed in red (longitudinal, circular, and visceral) in both rotifer species et al color by Amplex red assay by CSLM; C: Whole specimen double-marked in studied. Kotikova .[4] considered that the visceral their muscular system (by phalloidin in green color) and cholinergic system musculature was represented by several structures: 1) mastax ( ) acetylcholine stained by rhodamine in red color using CSLM; D: Optical muscles; 2) cutaneo-pharyngeal muscles; 3) the network light microscopy image. rcd: Dorsal retractor muscles of corona; rcv: Ventral retractor muscles of corona; rcl: Lateral retractor muscles of corona; sm: of muscles that envelop internal organs of the posterior 4) Superior muscles of mastax; mm1-2: Medial muscles of mastax; im: Inferior part; the muscles near both foot and dorsal groove of the muscles of mastax; dv1-3: Dorso-ventral muscles; rf1-2: Retractor muscles of lorica. The rotifer species studied did not show coronal foot; dtt: Dorsal tubular muscle of corona; lm: Longitudinal muscle; ov: Ovary; muscles (par coronalis or coronal ring muscles). This result st: Stomach; cr: Corona region; st: Stomach; tr: Trophi; m: Mastax; c: Corona. agrees with reports of musculature in rotifers of the genus Ignacio Alejandro Pérez-Legaspi et al./Journal of Coastal Life Medicine 2014; 2(5): 376-381 380 Brachionus L. quadridentata B. calyciflorus [4,5,12,26]. The species where these muscles have pictures of and , respectively. Filinia novaezealandiae Squatinella been reported are [7], Financial support was provided by Consejo Nacional de rostrum Encentrum mucronatum Dicranophorus í [13], and and Ciencia y Tecnolog a (Conacyt) through the scholarship forcipatus [8]. The muscular arrangement pattern reflects (Grant No. 136453), and the support of Sistema Nacional de morpho-ecological adaptations[12]. Therefore, rotifers Investigadores (Grant No. 49351). might vary in the muscle arrangement depending on their adaptations to each ecological niche such as their swimming Comments behavior through the water column for planktonic rotifer or littoral zone for epi-benthic rotifer. Background The two rotifer species examined in this study revealed similar patterns of muscular system with regard to coronal Rotifers are exquisite small metazoan invertebrates highly e.g. retractor muscles and circular incomplete muscles. However, sensitive to pollutants, with life characteristics ( short there are differences in the proportions and arrangement of generation time, reproduce parthenogenetically, produce the muscles around the trophi and foot. These differences resting eggs) which make them good test organisms. On might be due to adaptations for a particular habitat. The that basis among other aspects of their ecology, biology and L. quadridentata epi-benthic rotifer showed stronger systematics their muscular and nervours system is being retractor muscles of the corona (rc) and foot (rf) and this may explored. be an adaptation to move in a more dense benthic habitat. B. calyciflorus Research frontiers The planktonic rotifer showed stronger B. calyciflorus muscles in the retractors of the corona (rc), shorter dorso- has not been studied by CSLM, neither has Lecane ventral muscles (dv1-2) connected to corona, and conspicuous the muscle system of the genus been described, strong muscles of mastax (m) particularly the superior despite the fact that this genus is considered one of the most mastax (sm). This muscular arrangement is similar to that of numerous with 163 species recorded so far. other members of the genus Brachionus[4,5,12,26], suggesting Related reports a conservative feature; therefore, the description of the B. calyciflorus muscular system is useful to increase our The authors applied the methodology that has proven L. knowledge about interspecific variation. In the case of appropriate to reveal the muscular and nervous system of quadridentata a unique pattern among rotifers was observed, the rotifers. different from other rotifers reported. Nevertheless, more Innovations and breakthroughs studies of the muscular system are needed to elucidate phylogenetic relationship in the family Lecanidae, which The research was applied to rotifers that have not been is one of the most species of the phylum Rotifera[27]. It is previously studied accordingly, increasing the relative probable that this type of musculature might be common in knowledge for the phylum rotifera. most Lecanidae, since they live mainly in littoral habitats. Applications In conclusion, this study reveals cholinergic neuromuscular junctions in two rotifer species that have been identified This kind of research is mainly basic research which is previously. The confirmation of a cholinergic system in necessary to understand the function of rotifera in their rotifers contributes to the development of model organisms environment as well as to further exploit their unique to assess the neuro-pharmacological effects related to characteristics and use them in neuropharmacological agonist or antagonist receptors in rotifers, to perform further studies and toxicity tests. toxicity test to assess adverse effects on anticholinesterase Peer review substances (like the ones produced by organophosphate and carbamate pesticides) in laboratory conditions, or to monitor The manuscript is dealing with an aspect for which not aquatic ecosystems susceptible to pesticide pollution. much information is available; the neurotransmission system B. calyciflorus of rotifers including . This species along with Conflict of interest statement other rotifers are being studied in order to be used in toxicity tests thus knowledge on the way the neuromuscular system functions is essential. Furthermore increasing the available We declare that we have no conflict of interest. information further adds to the comparative analysis elucidating ecological and evolutionary relationships. Acknowledgements References

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