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Memoirs of the Queensland Museum | Nature (ISSN 0079-8835) Proceedings of the 13th International Marine Biological Workshop The Marine Fauna and Flora of Moreton Bay, Queensland Volume 54, Part 1 Editors: Peter J.F. Davie & Julie A. Phillips Memoirs of the Queensland Museum | Nature 54(1) © The State of Queensland (Queensland Museum) PO Box 3300, South Brisbane 4101, Australia Phone 06 7 3840 7555 Fax 06 7 3846 1226 Email [email protected] Website www.qm.qld.gov.au National Library of Australia card number ISSN 0079-8835 NOTE Papers published in this volume and in all previous volumes of the Memoirs of the Queensland Museum may be reproduced for scientific research, individual study or other educational purposes. Properly acknowledged quotations may be made but queries regarding the republication of any papers should be addressed to the Editor in Chief. Copies of the journal can be purchased from the Queensland Museum Shop. A Guide to Authors is displayed at the Queensland Museum web site http://www.qm.qld.gov.au/About+Us/Publications/Memoirs+of+the+Queensland+Museum A Queensland Government Project Typeset at the Queensland Museum The use of infrared spectroscopy as a test for species-specific pedal mucus in gastropod molluscs — a comparative study in Moreton Bay and Singapore Shirley S.L. LIM T.L. TAN Natural Sciences & Science Education, National Institute of Education, Nanyang Technological University, 1 Nanyang Walk, Singapore 637616. Email: [email protected] Citation: Lim, S.S.L. & Tan, T.L. 2008 12 01. The use of infrared spectroscopy as a test for species- specific pedal mucus in gastropod molluscs — a comparative study in Moreton Bay and Singapore. In, Davie, P.J.F. & Phillips, J.A. (Eds), Proceedings of the Thirteenth International Marine Biological Workshop, The Marine Fauna and Flora of Moreton Bay, Queensland. Memoirs of the Queensland Museum — Nature 54(1): 349–354. Brisbane. ISSN 0079-8835. ABSTRACT Infrared (IR) spectroscopy was used to study pedal mucous characteristics of eight species of marine gastropods to determine if it was useful for species identification. Three species of Nerita, two morphologically indistinguishable species of Littoraria (L. articulata and L. strigata) and one species each of Nodilittorina, Austrolittorina and Echinolittorina, were compared with a terrestrial snail, Achatina fulica. The IR spectra all showed high absorption in the 900–2100 cm!1 wave number region. No benzene overtones were observed at low wave numbers (1800–2000 cm!1) suggesting that aromatic amino acids could be lacking in core glycoprotein molecules. Strong absorption bands at peaks of 1644 cm!1 and 1545 cm!1 were obtained for all species and attributable to amide-I and amide-II peaks respectively. Amide I (range 1600–1720 cm !1) to Amide II (1500–1600 cm!1) band area ratios ranged significantly: Nerita species: 1: 0.10–1: 0.22; Achatina fulica 1: 0.40; and Littoraria sp. 1: 0.48; Austrolittorina unifastciata: 1: 0.57; Echinolittorina malaccana: 1: 0.65 and Nodilittorina pyramidalis: 1: 0.74. These differences appear useful for distinguishing between the six genera and congeneric species studied. q infrared, intertidal, snail, spectroscopy, Singapore, marine, Moreton Bay, mucus, Queensland. Molluscan mucus is made up of many com- trails, but ignore the trails of other males. Thus, pounds such as lectins, charged muco-polysac - both species-specific and gender specific cues charides, glycoproteins, proteins, uronic acid, are known to occur within gastropod mucus. sialic acid hexosamine and a host of other molec - ules in an aqeous medium (Schlichter 1982; Cottrell et al. (1993) used SDS-PAGE electro- Cottrell et al. 1993, 1994; Furuta et al. 1995; Davies phoresis of mucus to distinguish between seven & Hawkins 1998). It is generally used for the snail species, however, such analysis requires protection of cell surfaces exposed to the external complicated preparation and purification of the environment (Davies & Hawkins 1998). Infor - sample. Recently, infrared (IR) spectroscopy has m ation about the mollusc’s sexual state and been proposed as a simple method to study direction of locomotion is also conveyed to con- molluscan mucus (White et al. 1997; Skingsley specifics and predators by the mucus (Denny et al. 2000), as lengthy sample preparation is 1989). The mud snail, Ilynassa obsoleta, (Say, 1822) unnecessary and results of spectra analyses can follows polarised trails of conspecifics in the be obtained within seconds. Skingsley et al. (2000) pursuit of mates, but ignores non-conspecific successfully used IR spectroscopy of mucus to trails (Bretz & Dimock 1983). Similarly, Erlandsson distinguish between six species of slugs, using & Kostylev (1995) reported that male Littorina two species of terrestrial snails, Helix aspersa littorea track females using cues from conspecific and Cepaea nemoralis for comparison. Memoirs of the Queensland Museum — Nature 2008 54(1) www.qm.qld.gov.au 349 Lim & Tan The identification of many mollusc species is The entire foot of each snail was swiped across difficult, and often requires dissection to study a barium fluoride crystal IR window, thereby subtle differences in internal organ structure. coating it with a thin layer of mucus. This mucus Reid (1986) reported that it was difficult to was analysed ‘raw’ which reduces the chance of distinguish between Littoraria strigata and L. artifact from any derivative procedure. Infrared articulata merely by their shells, and additional spectroscopy in transmission mode was used, examination of anatomical features (e.g. penial i.e., a spectroscopic beam was passed through form) was required. Lee (2003) when conducting the sample and crystal. The mucus was too a field study on the distribution, modes of attach - viscous for injection into a normal transmission ment, and heat tolerance of Singapore littorinids, cell. The presence of liquid water, water vapour had to lump together Littoraria articulata and and barium fluoride bands disrupt the spectral Littoraria strigata under Littoraria sp., as the shells profile, and therefore these were subtracted off-line of these two species are too similar in colour from the raw mucus specimen. The corrected and morphology to be able to separate in the spectra were assigned bands that equate to the field. The objective of the present study was to likely chemical components of the mucus using test IR spectroscopy of pedal mucus for its standard banding protocols developed by efficacy in distinguishing between eight species Williams & Fleming (1987). The IR spectra were in the genera, Austrolittorina, Echinolittorina, recorded at a resolution of 2 cm!1 in the spectral Nodilittorina, Nerita and Littoraria. range of 900–2000 cm!1 using a Perkin-Elmer Fourier transform infrared spectrometer. The MATERIALS AND METHODS sampling procedure was repeated five times Five individuals of Austrolittorina unifasciata for each species, and several individuals were (Gray, 1826) (= Nodilittorina unifasciata, see Reid, used to reduce the likelihood of biological varia - 2007) and Nodilittorina pyramidalis (Quoy & Gai- bility affecting the results. mard, 1833) were collected from the high intertidal RESULTS rocky shore at Hospital Bay, Dunwich, North Stradbroke Island on 23 February 2005, and The IR spectra of all gastropods show high !1 kept in moist containers for transport back to absorption in the 900–2000 cm wave number the laboratory in Singapore. Similarly several region, and this region contains particularly signifi - individuals of Echinolittorina malaccana (Gray, cant information on the organic components of 1839) (= Nodilittorina trochoides, see Reid, 2007), the mucus (see Williams & Fleming 1987). In all Nerita lineata Gmelin, 1791, Nerita planospira Anton, species studied, there were strong absorption !1 !1 1839, Nerita chamaeleon Linnaeus, 1758, and bands at peaks of 1644 cm and 1545 cm . Littoraria sp. were also taken from the Pasir Ris These bands show the presence of amide bonds Nature Area in Singapore. Five individuals of in the core proteins of both glycosaminoglycans the giant African snail, Achatina fulica Bowdich, (GAGS) and proteoglycan molecules, including 1822 were collected from Singapore. The spectrum lectins (Davies & Hawkins 1998), and are obtained from the pedal mucus of this terrestrial attributable to amide-I and amide-II peaks respect- pulmonate was used to compare with the marine ively. The shape of the amide bands, and the gastropods’ spectra. Specimens were used within size of the area under them, provide useful five days of collection. Marine gastropods were information on the presence of b-sheets and wetted with artificial seawater to induce them b-turns (Surewicz et al. 1993). to crawl, while tap water was used for the giant The broad absorption band around the 1400 African snail. Snails may produce a defense cm!1 region indicates the presence of different mucus that could have prop erties different from ionisation states of the carboxylate groups assoc- normal body mucous secre tions, and therefore iated with acid derivatives of sugars and some the snails were handled several times in order amino acid side chains ( COOH « COOG + H+). !1 to habituate them. As long as the stimulus is Generally, CH2 vibration occurs at 1450 cm !1 non-threatening, habituation is rapid (Carew & and CH3 vibration at 1380 cm and bands are due Sahley 1986). to the presence of the core carbon spines of 350 Memoirs of the Queensland Museum — Nature 2008 54(1) Infrared Spectroscopy of Pedal Mucus in Gastropods FIG. 1. Infrared spectra of the pedal
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