Brennan, Gerard P.

Cain, Shaun

Sigwart, Julia D.

Sumner-Rooney, Lauren H.

Several methods have been proposed to ‘clean’ the soft tissues of molluscs of mucus, so that the surface cilia can be examined microscopically. We report the first empirical test of the effectiveness of methods for removing mucus in the pallial cavity surface of chitons. Three methods were compared, at several time intervals: the enzyme hyaluronidase, the mucolytic agent N-acetyl cysteine (NAC), and seawater washing via the natural action of cilia in excised tissue. Treatment in NAC for 10 min produced the best results, and we recommend this protocol as a starting point for further investigation on mucus removal in a broader suite of taxa. We present the first description of the pallial surface cilia in the chiton Lepidochitona cinerea. During the course of this study, we also determined that these chitons were frequently infested with a ciliate protozoan parasite, Trichodina sp., which have been historically reported from chitons but never studied in detail. The parasites were absent where antimucus treatments were effective, but their abundance and large size (about 30-mm diameter) in less successful treatments obscured the view of the pallial cavity surface

Queen's University Belfast Research Portal

A test for mucus removal in the chiton Lepidochitona cinerea(Linnaeus, 1767) (Polyplacophora: Chitonida: Ischnochitonidae)Sumner-Rooney, L. H., Cain, S., Brennan, G. P., & Sigwart, J. D. (2014). A test for mucus removal in the chitonLepidochitona cinerea (Linnaeus, 1767) (Polyplacophora: Chitonida: Ischnochitonidae). Veliger, 51(4), 261-264.Published in:VeligerDocument Version:Publisher's PDF, also known as Version of recordQueen's University Belfast - Research Portal:Link to publication record in Queen's University Belfast Research PortalPublisher rights© The AuthorsThis is an open access article published under a Creative Commons Attribution-NonCommercial-ShareAlike License(https://creativecommons.org/licenses/by-nc-sa/4.0/), which permits use, distribution and reproduction for non-commercial purposes,provided the author and source are cited and new creations are licensed under the identical terms.General rightsCopyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or othercopyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associatedwith these rights.Take down policyThe Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made toensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in theResearch Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk.Download date:15. Feb. 2017A Test for Mucus Removal in the Chiton Lepidochitona cinerea (Linnaeus,1767) (Polyplacophora: Chitonida: Ischnochitonidae)LAUREN H. SUMNER-ROONEYQueen’s University Belfast, Marine Laboratory, 12–13 The Strand, Portaferry, Co. Down, Northern Ireland BT221PF, and Queen’s University Belfast, School of Biological Sciences, Medical Biology Centre, 97 Lisburn Road,Belfast, Northern Ireland BT9 7BLSHAUN CAINEastern Oregon University, La Grande, Oregon 97850, USAGERARD P. BRENNANQueen’s University Belfast, School of Biological Sciences, Medical Biology Centre, 97 Lisburn Road, Belfast,Northern Ireland BT9 7BLANDJULIA D. SIGWARTQueen’s University Belfast, Marine Laboratory, 12–13 The Strand, Portaferry, Co. Down, Northern Ireland BT221PF, and Queen’s University Belfast, School of Biological Sciences, Medical Biology Centre, 97 Lisburn Road,Belfast, Northern Ireland BT9 7BLAbstract. Several methods have been proposed to ‘‘clean’’ the soft tissues of molluscs of mucus, so that thesurface cilia can be examined microscopically. We report the first empirical test of the effectiveness of methods forremoving mucus in the pallial cavity surface of chitons. Three methods were compared, at several time intervals: theenzyme hyaluronidase, the mucolytic agent N-acetyl cysteine (NAC), and seawater washing via the natural action ofcilia in excised tissue. Treatment in NAC for 10 min produced the best results, and we recommend this protocol as astarting point for further investigation on mucus removal in a broader suite of taxa. We present the first descriptionof the pallial surface cilia in the chiton Lepidochitona cinerea. During the course of this study, we also determinedthat these chitons were frequently infested with a ciliate protozoan parasite, Trichodina sp., which have beenhistorically reported from chitons but never studied in detail. The parasites were absent where antimucus treatmentswere effective, but their abundance and large size (about 30-mm diameter) in less successful treatments obscured theview of the pallial cavity surface.INTRODUCTIONTraditionally, molluscan systematics has relied largelyon analyses of shell anatomy. More recently, with theimprovement of visualization techniques such as SEMimaging, shell features have been extended to micro-structural elements in gastropods (e.g., Geiger, 2012),bivalves (e.g., Turner, 2008) and chitons (e.g., Sigwart& Sirenko, 2012). However, another potentially richsource of information that has still not fully exploitedfor systematics is microanatomy of the soft parts of thebody. Previous work on the visualization of soft partsin other molluscan taxa using a scanning electronmicroscope (SEM) has required the removal of mucus(e.g., Nezlin et al., 1994), but there has been nodefinitive protocol reported for use on adult chitons, norany comparison of methods in any taxon. To determinethe best way forward to visualize cilia in the pallialcavity of chitons, we reviewed published techniques fora variety of invertebrates and directly tested the effect onthe chiton Lepidochitona cinerea.The enzyme hyaluronidase has been reported todigest mucus and similar materials in a variety of taxaincluding bivalves (Calabro et al., 2005), proso-branchs (Nezlin et al., 1994), caenogastropods (Ron-kin, 1952), and onychophorans (Mayer & Harzsch,2007). However, the efficacy of proteolytic enzymeshas been refuted (e.g., Stamm & Docter, 1965), andThe Veliger veli-51-04-09.3d 25/7/14 16:43:39 261The Veliger 51(4):261–264 (Month, 2014)THE VELIGER# CMS, Inc., 2014hyaluronidase has not been widely used. N-acetylcysteine (NAC) has also been reported to function asa mucolytic agent in both vertebrate and invertebrateanimals, including a marine bivalve (Moraes & Lopes,2003), a sacoglossan (Pierce et al., 1996), andplanarians (Bocchinfuso et al., 2012), but its primaryuse has been in mammals (e.g., Stamm & Docter,1965). In the absence of a chemical treatment, motorcilia may continue to beat in excised tissue. So byexcising tissue blocks and maintaining them in coldseawater, the mucus could clear ‘‘naturally’’ (SC,personal observation). This is also analogous towashing in seawater or phosphate-buffered saline,which is another treatment suggested previously (e.g.,Buckland-Nicks, 1993). In order to test and optimizea treatment effective for chitons, we experimentallycompared these three approaches.MATERIALS AND METHODSFourteen treatment regimes were tested for theirefficacy in mucus removal. These were all variants onthe three techniques outlined above (hyaluronidase,NAC, and surgical separation) using different time andtemperature intervals. Live specimens of Lepidochitonacinerea collected in Strangford Lough (County Down,Northern Ireland) were used for each treatment. Allanimals were held in aquaria flow-through seawater atthe Queen’s University Marine Laboratory (QML),where one of the authors (JS) has successfully keptlong-term captive specimens. All experiments wereperformed on site in laboratory facilities at QML.Following treated incubation with gentle agitation,specimens were flushed with a pipette. All specimenswere fixed in 4% glutaraldehyde in cacodylate buffer(pH 7.4) and post-fixed in 1% osmium tetroxide incacodylate buffer in preparation for SEM visualization.Tissues were then dehydrated in acetone and preparedfor histology as described by Ruthensteiner (2008).Specimens were dried overnight using hexamethyldisi-lazane (HMDS, Sigma-Aldrich), sputter-coated withgold using a Polaron E5100 SEM Coating System(Quorum Technologies) and visualized in a FEI Quanta200 scanning electron microscope operating at 10 kV.Bovine hyaluronidase (Sigma-Aldrich) was appliedat 4.46 e23 mM in low-calcium artificial seawater(420 mM NaCl, 10 mM KCl, 50 mM MgCl2, 10 mMTrizma, 10 mM MnCl). Specimens were incubated forperiods of 5 min, 10 min, or 1 hr, and at temperatures ofeither 10uC or room temperature (six treatment regimes,two specimens each). Another set of specimenswere treated with 500 mM NAC in an 2-(N-morpholino)ethanesulfonic acid (MES) buffer (50 mM MES, 10 mMsucrose, 90 mM sorbitol, all Sigma-Aldrich, pH titratedto 5.5 using NaOH, adapted from Pierce et al., 1996).These specimens were incubated for intervals of 5 min,10 min, or 30 min, and also at temperatures of either10uC or room temperature (six treatment regimes, twospecimens each). Four L. cinerea were decapitated andkept in seawater at 5uC for either 1 hr or for 24 hr beforefixation (two treatment regimes, two specimens each), amethod to induce ‘‘natural’’ clearing of the tissue by theaction of still-active cilia, which has given good resultsin Tritonia diomedea (SC, personal observation).Finally, one specimen of L. cinerea was fixed withoutmucus removal treatment, as a control.RESULTS AND DISCUSSIONThe development of SEM techniques has been a crucialstep forward in the study of microanatomy across alltaxa, yet recent attempts to characterize the pallialcavity of another chiton, Leptochiton rugatus via SEMwere hampered by excessive mucus obscuring thesurface epithelium (JS, SC, personal observation).The SEM images here (Figure 1) show that the pallialcavity of Lepidochitona cinerea is carpeted withmicrovilli, with large tufts of cilia (6 mm in diameter)of approximately 15–20 mm in length at regularintervals (approximately 25 mm apart, Figure 1A, B).This pattern persisted throughout the pallial cavity andinto the gill row with no visible change in density orfrequency. These are the first images to illustrate thesurface features of the pallial cavity in L. cinerea.The ‘‘success’’ of our treatments was assessed qualita-tively by visualization under SEM, to determine whethercilia were clearly visible, or whether tissue appeared badlydehydrated, or covered in mucus sheets obscuring someor all of the cilia in the pallial cavity. Specimens wereexamined whole; that is, the entire pallial cavity on bothsides was visually examined under SEM although onlysmall portions are illustrated herein (Figure 1). Temper-ature did not have any substantive influence on tissuequality, and we conclude that performing these chemicaltreatments at room temperature does not decrease quality.The different treatments for mucus removal hadvarying rates of success, with NAC generally producingthe best results. Incubation of specimens with hyal-uronidase did not effectively remove mucus from thepallial cavity when incubated for less than an hour(Figure 1C, D), but after longer incubations both ciliaand microvilli were visible and only small spheres ofmucus were present. NAC preserved cilia and microvilliin good condition, and left little mucus when appliedfor 10 min or more (Figure 1A, B). In addition, the useof hyaluronidase appears to have disrupted the tissue,with cracks appearing in the epithelium in hyaluroni-dase-treated samples (not shown), this effect was notpresent under NAC treatment despite identical dehy-dration and preparation for SEM.The difference in mucus coverage observed between thedecapitated specimens (i.e., natural clearing of the tissue)The Veliger veli-51-04-09.3d 25/7/14 16:43:39 262Page 262 The Veliger, Vol. 51, No. 4and the untreated control specimen was minimal. In bothcases, the cilia could not be clearly observed and the discretecilia tufts, visible in other specimens, could not always beclearly identified beneath the mucus ‘‘sheet’’ (Figure 1E, F).An additional novel observation is the presence of aciliate protozoan parasite, Trichodina sp., which alsoobscured the view of the pallial cavity surface. Theseprotozoa, visible at low magnification, had not been notedThe Veliger veli-51-04-09.3d 25/7/14 16:43:40 263Figure 1. SEM images of the anterior pallial cavity of treated L. cinerea. A, B, NAC, 10uC, 10 min; C, D, Hyaluronidase, 10uC,5 min; E, F, Decapitated, 1 hr. Abbreviations: cil, cilia tuft; mic, microvilli; par, Trichodina sp. parasite; muc, mucus. Scale bars:A, C, E 5 100 mm; B, D, F 5 10 mm.L. H. Sumner-Rooney et al., 2014 Page 263during specimen preparation, but on subsequent investi-gation were found to infest the majority of specimensavailable for study. Therefore, we do not believe theinfluence of the parasite confounded our results here byinfluencing either mucus production by the chitons or itsremoval in our experiments. Under treatment conditionswhere the mucus was completely or partially removed,these parasites were also cleared. However, they wereobserved in the untreated, decapitated, and the shorterhyaluronidase-treated specimens and caused a significantobstruction to our view of the pallial cavity due to theirlarge size (around 30 mm across). Their presence here isparticularly notable as they have only been reported twicein the literature for chitons: once in our study species, L.cinerea (Plate, 1898) and one other report in the Pacificchiton Katharina tunicata (Kozloff, 1961).Although the NAC treatment was the most effectivefor Lepidochitona, it may not be a panacea and itseffectiveness requires further investigation across abroad range of molluscan taxa. Two additionalspecimens of Leptochiton asellus (Leptochitonidae)were treated with NAC at room temperature for30 min as described above. Unlike Lepidochitonacinerea, which is a member of the suborder Chitonida,Leptochiton asellus belongs to the other major clade ofliving chitons, Lepidopleurida. However, in this case,mucus deposits continued to obstruct the structures.Ronkin (1952) noted that the susceptibility of mucus todigestion by hyaluronidase varied in different areas inBusycon canaliculatum, and postulated that variation inchemical composition might explain this. It may be thatthe taxonomic differences between chitons extend tothe composition of mucus, with dissimilar proportionsof glycoproteins and proteoglycans being affecteddifferently by the NAC and hyaluronidase chemicaltreatments, and thus giving results of variable quality.Although the applicability of this treatment requiresfurther investigation, our recommended treatment onthe basis of our empirical tests is NAC, effective after10 min as a starting point for other investigators.LITERATURE CITEDBOCCHINFUSO, D. G., P. TAYLOR, E. ROSS, A. IGNATCH-ENKO, V. IGNATCHENKO, T. KISLINGER, B. J. PEARSON &F. MORAN. 2012. Proteomic profiling of the planarianSchmidtea mediterranea and its mucous reveals similaritieswith human secretions and those predicted for parasiticflatworms. Molecular and Cell Proteomics 11:681–691.BUCKLAND-NICKS, J. 1993. Hull cupules of chiton eggs:parachute structures and sperm focusing devices? Biolog-ical Bulletin 184:269–276.CALABRO, C., M. P. ALBANESE, S. MARTELLA, P. LICATA,E. R. LAURIANO, C. BERTUCCIO & A. LICATA. 2005.Glycoconjugate histochemistry and nNOS immunolocal-ization in the mantle and foot epithelia of Tapesphilippinarum (bivalve mollusc). Folia Histochemica etCytobiologica 43:151–156.GEIGER, D. L. 2012. Monograph of the little slit shells. Vol. 1:Introduction, Scissurellidae. Santa Barbara Museum ofNatural History: Santa Barbara, CA. 728 pp.KOZLOFF, E. N. 1961. A new genus and two new species ofancistrocomid ciliates (Holotricha: Thigmotricha) fromsabellid polychaetes and from a chiton. Journal ofEukaryotic Microbiology 8:60–63.MAYER, G. & S. HARZSCH. 2007. Immunolocalization ofserotonin in Onychophora argues against segmentalganglia being an ancestral feature of arthropods. BMCEvolutionary Biology 7:118.MORAES, D. T. D. E. & S. G. B. C. LOPES. 2003. Thefunctional morphology of Neoteredo reynei (Bartsch,1920) (Bivaliva, Teredinidae). Journal of MolluscanStudies 69:311–318.NEZLIN, L. P., R. ELOFSSON & D. A. SKHAROV. 1994.Transmitter-specific subsets of sensory elements in theprosobranch osphradium. Biological Bulletin 187:174–184.PIERCE, S. K., R. W. BIRON & M. E. RUMPHO. 1996.Endosymbiotic chloroplasts in molluscan cells containproteins synthesized after plastid capture. Journal ofExperimental Biology 199:2323–2330.PLATE, L. H. 1898. U¨ber primitive Organisationsverha¨ltnisse,Viviparie und Brutpflege bei Chitonen. Sitzungsberichteder Ko¨niglich Preussischen Akademie der Wissenschaften14:213–217.RONKIN, R. R. 1952. Cytological studies of mucus formationand secretion in Busycon. Biological Bulletin 102:252–260.RUTHENSTEINER, B. 2008. Soft part 3D visualization by serialsectioning and computer reconstruction. Zoosymposia 1:63–100.SIGWART, J. D. & B. I. SIRENKO. 2012. Deep-sea chitons fromsunken wood in the West Pacific. Zootaxa 38:1–38.STAMM, S. J. & J. DOCTER. 1965. Clinical evaluation ofacetylcysteine as a mucolytic agent in cystic fibrosis.Diseases of the Chest 47:414–420.TURNER, J. A. 2008. Digital imaging of micro bivalves.Zoosymposia 1:47–61.The Veliger veli-51-04-09.3d 25/7/14 16:43:43 264Page 264 The Veliger, Vol. 51, No. 4

A test for mucus removal in the chiton Lepidochitona cinerea (Linnaeus, 1767) (Polyplacophora: Chitonida: Ischnochitonidae)

Abstract

Similar works

Full text

Available Versions

Queen's University Belfast Research Portal