Vol. 23 (1) REVISTA DE LA SOCIEDAD ESPAÑOLA DE MALACOLOGÍA Oviedo, junio 2005 Iberus | Revista de la SOCIEDAD ESPAÑOLA DE MALACOLOGÍA Comité DE REDACCIÓN (BOARD OF EDITORS) EDITOR DE PUBLICACIONES (EDITOR-IN-CHIEF) Serge Gotas Universidad de Málaga, España DIRECTOR DE REDACCIÓN (EXECUTIVE EDITOR) Gonzalo Rodríguez Casero Apdo. 156, Mieres del Camino, Asturias, España EDITORA EJECUTIVA (MANAGING EDITOR) Eugenia M* Martínez Cueto-Felgueroso Apdo. 156, Mieres del Camino, Asturias, España EDITORES ÁDJUNTOS (ÁSSOCIATE EDITORS) Francisco Javier Conde de Saro Benjamín Gómez Moliner Ángel Antonio Luque del Villar Emilio Rolán Mosquera José Templado González Jesús S. Troncoso Embajada de España, Japón Universidad del País Vasco, Vitoria, España Universidad Autónoma de Madrid, Madrid, España Universidad de Vigo, Vigo, España Museo Nacional de Ciencias Naturales, CSIC, Madrid, España Universidad de Vigo, Vigo, España ComiTÉ EDITORIAL (BOARD OF REVIEWERS) Kepa Altonaga Sustacha Eduardo Angulo Pinedo Rafael Araujo Armero Thierry Bockeljau Ridiger Bieler Sigurd v. Boletzky Jose Castillejo Murillo Karl Edlinger Antonio M. de Frias Martins José Carlos García Gómez Gonzalo Giribet de Sebastián Edmund Gittenberger Angel Guerra Sierra Gerhard Haszprunar Yuri |. Kantor María Yolanda Manga González Jordi Martinell Callico Ron K. 0'Dor Tokashi Okutani Marco Oliverio Pablo E. Penchaszadeh Winston F. Ponder Carlos Enrique Prieto Sierra M* de los Ángeles Ramos Sánchez Francisco Javier Rocha Valdés Paul 6. Rodhouse Joandoménec Ros ¡ Aragones María Carmen Salas Casanovas Gerhard Steiner Victoriano Urgorri Carrasco Anders Warén PORTADA DE /berus Universidad del País Vosco, Bilbao, España Universidad del País Vasco, Bilbao, España Museo Nacional de Ciencias Naturales, Madrid, España Institut Royal des Sciences Naturelles de Belgique, Bruselas, Bélgica The Field Museum, Chicago, Estados Unidos Laboratoire Arago, Banyuls-sur-Mer, Francia Universidad de Santiago de Compostela, Santiago de Compostela, España Noturhistorisches Museum Wien, Viena, Austria Universidade dos Acores, Acores, Portugal Universidad de Sevilla, Sevilla, España Harvard University, EE.UU. Notional Notuurhistorisch Museum, Leiden, Holanda Instituto de Investigaciones Marinas, CSIC, Vigo, España Zoologische Staatssammlung Múnchen, Miinchen, Alemania AN. Severtzov Institute of Ecology and Evolution, Moscú, Rusia Estación Agrícola Experimental, CSIC, León, España Universidad de Barcelona, Barcelona, España Dalhousie University, Halifax, Canada Nihon University, Fujisawa City, Japón Universitá di Roma “La Sapienza”, Roma, Italia Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Buenos Aires, Argentina Australian Museum, Sydney, Australia Universidad del País Vasco, Bilbao, España Museo Nacional de Ciencias Naturales, CSIC, Madrid, España Instituto de Investigaciones Marinas, CSIC, Vigo, España British Antarctic Survey, Cambridge, Reino Unido Universidad de Barcelona, Barcelona, España Universidad de Málaga, Málaga, España Institut fir Zoologie der Universitút Wien, Viena, Austria Universidad de Santiago de Compostela, Santiago de Compostela, España Swedish Museum of Natural History, Estocolmo, Suecia Iberus gualterianus (Linnaeus, 1758), una especie emblemática de la península Ibérica, que da nombre a la revista. Dibujo realizado por José Luis González Rebollar “Toza”. ¡4 Í 208 E A SÓN, WA A 21308) Ea BRAR REVISTA DE LA SOCIEDAD ESPAÑOLA DE MALACOLOGÍA Vol. 23 (1) Oviedo, junio 2005 Iberus Revista de la SOCIEDAD ESPAÑOLA DE MALACOLOGÍA Iberus publica trabajos que traten sobre cualquier aspecto relacionado con la Malacología. Se admiten también notas breves. /berus edita un volumen anual que se compone de dos o más números. INSTRUCCIONES PARA LOS AUTORES Los manuscritos deben remitirse a: Serge Gofas, Editor de Publicaciones, Departamento de Bio- logía Animal, Universidad de Málaga, Campus de Teatinos, s/n, 29071, Málaga, España. Los trabajos se entregarán por triplicado (original y dos copias). Se recomienda a los autores leer cuidadosamente las normas de publicación que se incluyen en cada número de la revista. SUBCRIPCIONES Iberus puede recibirse siendo socio de la Sociedad Española de Malacología, en cualquiera de sus formas, o mediante intercambio. Aquellos socios que deseen adquirir números atrasados deberán diri- girse al bibliotecario. Los no socios deberán ponerse en contacto con BACKHUYS PUBLISHERS, P.O. Box 321, 2300 AH Leiden, The Netherlands. Tel.: +31-71-51 70 208, Fax: +31-71-51 71 856, Correo Elec- trónico: backhuysCeuronet.nl Los resumenes de los artículos editados en esta revista se publican en Aquatic Science and Fisheries Abstracts (ASFA) y en el Zoological Records, BIOSIS. Contents list published in Aquatic Science and Fisheries Abstracts and Zoological Records, BIOSIS. Dep. Leg. B-43072-81 ISSN 0212-3010 Diseño y maquetación: Gonzalo Rodríguez Impresión: LOREDO, S. L. - Gijón O Sociedad Española de Malacología —__——T— Iberus, 23 (1): 1-13, 2005 Toxic effects of taraxerol extracted from Codiaeum variega- tum stem-bark on target vector snail Lymnaea acuminata and non-target fish Toxicidad del taraxerol extraído del tallo de Codiaeum variegatum sobre el caracol Lymnaea acuminata y sobre peces Ram P YADAV*, Sudhanshu TIWARÍ* and Ajay SINGH*! Recibido el 26-V-2003. Aceptado el 7-IV-2004 ABSTRACT The active compound taraxerol (triterpene) extracted from the stem-bark of Codiaeum var- iegatum Blume (Euphorbiaceae) was administered for 24h or 96h to the freshwater snail lymnaea (Radix) acuminata Lamarck in order to test its lethality. It was observed that the molluscicidal activity of taraxerol was time as well as dose dependent for the snail at all the exposure periods. There was a significant negative correlation between LC values of taraxerol and exposure period, thus LCso values decrease from 1.69 mg/L (24h) to 0.74 mg/L (96h) against lymnaea (Radix) acuminata Lamarck. Toxicity experiments were also carried out on non-target freshwater fish Channa punctatus (Bloch) (Channidae, Ophi- cephalidae), which shares the habitat with snails, for environmental toxicity, if any. Exposure of sub-lethal (40% and 80% of LC50)] doses of taraxerol over 24h caused signifi- cant alterations in carbohydrates and nitrogenous metabolism in nervous, hepatopancreas and ovotestis tissues of snail Lymnaea acuminata. Fish Channa punctatus also exposed to sub-lethal doses of taraxerol (40% and 80% of LCso 24h of lymnaea acuminata) for 96h periods in order to measure potential effects on non-target organisms , also show significant alteration in carbohydrates and nitrogenous metabolism in muscle, liver and gonadal tis- sues. This study also shows that there ¡is partial recovery in these parameters in both snail and fish after the 7 day of withdrawal of the treatment, which supports the view that it is safe to use plant products as molluscicides for controlling snails in aquatic bodies. RESUMEN Se administró el principio activo taraxerol (un triterpeno) obtenido de los tallos de Codiaeum variegatum Blume (Euphorbiaceae) al caracol Lymnaea (Radix) acuminata Lamarck durante 24 0 96 horas, para probar su letalidad. La actividad molusquicida del compuesto depende tanto del tiempo como de la dosis para ambos periodos de exposición. Hay una correlación negativa significativa entre los valores LC del compuesto y el periodo de exposición, los val- ores LCso descienden de 1,69 mg/L (24h) a 0,74 mg/L (96h) para los caracoles. También se probó el compuesto en el pez Channa punctatus (Bloch) (Channidae, Ophicephalidae), que comporte hábitat con el molusco, para determinar la posible toxicidad ambiental. la exposición durante 24 horas a dosis subletales (40 y 80% de LC50) provocó alteraciones en el metabolismo de carbohidratos y compuestos nitrogenados en tejidos * Natural Product Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur- 273 009 (U.P.) India. 'Corresponding Author: Dr. Ajay Singh, E-mail: ajay_sCsancharnet.in Iberus, 23 (1), 2005 nervioso, del hepatopáncreas y ovotestis del caracol. Exposiciones de 96 horas de los peces a las mismas dosis mostraron los mismos efectos sobre los tejidos muscular, hep- ático y gonadal. El estudio muestra que hay una recuperación parcial de los parámetros en ambos organismos al septimo día tras abandonar el tratamiento, lo que apoya la idea de que es seguro el uso de productos vegetales como molusquicidas en medios acuáticos. KEY WORDS: Lymnaea acuminata, Channa punctatus, molluscicides, taraxerol, metabolism. PALABRAS CLAVE: Lymnaea acuminata, Channa punctatus, molusquicida, taraxerol, metabolismo. INTRODUCTION Once a pesticide is released into the environment, chemical, physical, biolog- ical and other allied factors determine its fate and distribution in the ecosys- tem. Heavy use of pesticides to control pests results in the pollution of the envi- ronment affecting both target species snails, slugs etc. and many non-target species viz. Phytoplankton, Zooplank- ton, fish etc in freshwater ecosystems (GOPAL, KHANNA, ANAND AND GUPTA, 1981). Pesticides interfere with the phys- iological and metabolic function in ani- mals, which sometimes result in death (ARASTA, BAIS AND THAKUR, 1996). With growing awareness of environ- mental pollution caused by synthetic molluscicides (RITCHIE, 1973; SRIVAS- TAVA AND SINGH, 2001), efforts are being made to find molluscicides of plant origin. Being the product of biosynthe- sis, they are highly toxic and easily biodegradable in nature (MARSTON AND HOSTETTMAN 1987; SINGH, SINGH, MISHRA AND AGARWAL, 1996). Two dis- eases - schistosomiasis and fascioliasis - carried by aquatic snails, cause immense harm to man and his domestic animals (BALI, SINGH AND SHARMA, 1986; AGARWAL AND SINGH, 1988). Large pop- ulations of aquatic snails inhabiting freshwater bodies cause serious destruc- tion of freshwater vegetation which ulti- mately affect the growth of organisms feeding on them (REINERT, 1972). Fascioliasis caused by Fasciola hepat- ica, the large liver-fluke, is common in sheep, cattle, goats and other herbivo- rous animals throughout the world. The freshwater snail L. acuminata is the inter- mediate host of Fasciola hepatica and Fas- ciola gigantica (AGARWAL AND SINGH, 1988; SINGH AND AGARWAL 1992; YADAV AND SINGH 2001, 2002), which cause great harm to domestic animals. SINGH AND AGARWAL (1981) reported that 94% of buffaloes slaughtered in Gorakhpur of Uttar Pradesh, India were infected by liver fluke Fasciola gigantica. The aim of the present study is to report on the molluscicidal activity of taraxerol extracted from stem-bark of C. variegatum , against the harmful snail L. acuminata. lts effects on biochemical parameters of target snail L. acuminata and non-target fish Channa punctatus were also studied. Freshwater fish Channa punctatus is a common fish in India and shares the habitat with the snails. MATERIALS AND METHODS The stem-bark of C. variegatum was collected locally from the Botanical garden of D.D.U Gorakhpur University Gorakhpur, where a voucher specimen is deposited and identified by Prof. S.K. Singh (taxonomist), Department of Botany, D.D.U, Gorakhpur University, Gorakhpur (U.P) India. Extraction of active compounds from stem-bark: Pure taraxerol was isolated from the stem-bark of C. variegatum by the method of CHATTERJEE AND BANER- JEE (1977). The stem-bark of C. variega- tum was dried in an incubator at 37"C and dried stem-bark was powdered with the help of a mechanical device. The dried powdered stem-bark (2 kg) of C. variegatum was extracted in Soxhlet YADAV ET AL.: Toxicity of taraxerol from Codiaeum variegatum on Lymnaea acuminata apparatus with petrol, for about 70 hours and a little amount of concen- trated solution was obtained. After evaporation of the solvent by vacuum pump, the isolated compound in dried form was obtained. The organic con- stituents present in stem-bark ¡.e. taraxe- rone-2, taraxerol, taraxeryl acetate-4 and sitosterol were extracted with petrol. Taraxerol is soluble in organic solvents such as CHCl3 and CHCl-MeOH. Iden- tification of the isolated compound was further confirmed with an authentic sample of taraxerol (C32H4809), supplied by Sigma chemical Co. U.S.A. | The extracted compound was stored in an airtight desicator and used for tox- icity experiments. Toxicity experiments were performed by the method of SINGH AND AGARWAL (1988). The freshwater snail L. acuminata were exposed to four different concentrations of taraxerol, 0.7 mg/L, 1.0 mg/L, 1.4 mg/L and 1.7 mg/L respectively for 24h, 48h, 72h and 96h exposure period. Mortality was recorded every 24h up to 96h. Ten snails were kept in 3L de-chlorinated tap water. Control animals were kept in similar conditions without any treat- ment. Each set of experiments was repli- cated six times. Effective doses (LC values), upper and lower confidence limits, slope value, t'ratio, “g' factor and heterogeneity were calculated by Probit log analysis method using POLO com- puter programme of RUSSELL, ROBERT- SON AND SEVIN (1977). For environmental toxicity, if any, the toxic effect of taraxerol was also studied in mixed populations of target organism snails and non-target organ- ism fish. In this experiment, groups of 10 snail L. acuminata and 10 fish C. punc- tatus were put together in 6L de-chlori- nated tap water, which were exposed to 2.92 mg/L (LCo 24h of L. acuminata) of taraxerol for 24h. Treatment protocol for Dose-Response relationship: L. acuminata was kept in glass aquaria containing 6L de-chlori- nated tap water. Each aquarium con- tains 30 experimental animals. Snails and fish were exposed for 24h in the case Of L. acuminata and 96h for fish C. punctatus to sub lethal doses 0.76 mg/L and 1.35 mg/L (40% and 80% of LCso 24h of L. acuminata) of taraxerol. Control animals were held in similar conditions without any treatment. After completion of treatment the test animals were re- moved from aquaria, and washed with freshwater. The nervous, hepatopan- creas and ovotestis tissue of L. acuminata and muscle, liver and gonadal tissue of freshwater fish C. punctatus were quickly dissected out in an ice tray and used for biochemical analysis. In order to see the effect oí with- drawal from treatment, both the experi- mental animals were exposed for 24h in the case of L. acuminata and 96h for fish C. punctatus to sub-lethal doses 1.35 mg/L (80% of 24h LCso of L. acuminata) of taraxerol, following which the test animals were transferred to freshwater. This water was changed every 24h for the next seven days. After this the test animals were removed from aquaria, washed with freshwater and nervous, hepatopancreas and ovotestis tissue of L. acuminata and muscle, liver and gonadal tissues of freshwater fish C. punctatus were quickly dissected out in an ice tray and all the above mentioned biochemical parameters were estimated. Each experiment was replicated at least six times and the values have been expressed as mean +SE of six replicates. Student's “t' test and analysis of vari- ance were applied to locate significant changes (SOKAL AND ROHLF 1973). BIOCHEMICAL ESTIMATIONS Protein: Protein levels were esti- mated according to the method of LOwRkrY, ROSENBROUGH, FARR, RANNDALL (1951) using bovine serum albumin as standard. Homogenates (5 mg/mL, w/v) were prepared in 10% TCA. Total free amino acids: Estimation of total free amino acid was made accord- ing to the method of SPICES (1957). Ho- mogenates (10 mg/mL, w/v) were pre- pared in 95% ethanol, centrifuged at 6000 xg and used for amino acid estima- tion. Iberus, 23 (1), 2005 Nucleic acids: Estimation of nucleic acids (DNA and RNA) was performed, by methods of SCHNEIDER (1957) using diphenylamine and orcinol reagents, respectively. Homogenates (Img/mL, w/v) were prepared in 5% TCA at 900C, centrifuged at 5000 xg for 20 min and supernatant was prepared and used for estimation. Both DNA and RNA have been expressed as 1g/mg tissue. Glycogen: Glycogen was estimated by the Anthrone method of VAN DER VIES (1954) as modified by MAHENDRU AND AGARWAL (1982) for snail L. acumi- nata. In the present experiment 50 mg of tissue were homogenised with 5 mL of cold 5%TCA. The homogenate was fil- tered and 1.0 mL of filtrate was used for assay. Pyruvate: Pyruvate level was measured according to FRIEDEMANN AND HAUGEN (1943). Homogenate (50 mg/mL, w/v) was prepared in 10% TCA. Sodium pyru- vate was taken as standard. Lactate: Lactate was estimated according to BARKER AND SUMMERSON (1941), modified by HUCKABEE (1961). Homogenate (50 mg/mL, w/v) was prepared in 10% cold TCA. Sodium lactate was taken as standard. Protease: Protease activity was esti- mated by the method of MOORE AND STEIN (1954). Homogenate (50 mg/mL, w/v) was prepared in cold distilled water. Optical density was measured at 570 nm. The enzyme activity was expressed in qmol of tyrosine equiva- lent/mg protein/h. Acid and alkaline phosphatase: Activi- ties of acid and alkaline phosphatase were measured by the method of BERGMEYER (1967) and modified by SINGH AND AGARWAL (1983). Tissue homogenate (2% w/v) was prepared in ice cold 0.9% saline and centrifuged at 5000 xg at 0 *C for 15 min. Optical density was measured at 420 nm against a blank, prepared simultaneously. The enzyme activity has been expressed as amount of p-nitrophenol formed /30 min /mg protein in supernatant. Lactic dehydrogenase: Lactic dehydro- genase activity was measured according to the method of ANONYMOUS (1984). Homogenates (50 mg/mL, w/v) were prepared in 1 mL of 0.1 M phosphate buffer, pH 7.5 for 5 min in an ice bath. Enzyme activity has been expressed as nanomol of pyruvate reduced /min/mg protein. Succinic dehydrogenase: Succinic dehydrogenase activity was measured by the method of ARRIGONI AND SINGER (1962). Homogenate (50 mg/mL, w/v) was prepared in 1 mL of 0.5 M potas- sium phosphate buffer, pH 7.6 for 5 min in an ice bath. Optical density was mea- sured at 600 nm. Enzyme activity has been expressed as pumol dye reduced /min/mg protein. Cytochrome oxidase: Cytochrome oxidase activity was measured accord- ing to the method of COOPERSTEIN AND LAZAROW (1951). Homogenates (50 mg/mL, w/v) were prepared in 1 mL of 0.33 M phosphate buffer (pH 7.4) for 5 min in ice bath. Enzyme activity has been expressed in arbitrary units /min/mg of proteins. Acetylcholinesterase: Acetylcholin- esterase was estimated by the method of ELLMAN, (COURTNEY, ANDRES AND FEATHERSTONE (1961) as in 0.1 M phos- phate buffer in ice bath. Optical density was measured at 412 nm at 25 *C. Enzyme activity expressed in mol “SH” hydrolysed /min/mg protein. RESULTS Molluscicidal activity: The toxicity of taraxerol was also time and dose-depen- dent for the freshwater snail L. acumi- nata. There was a significant negative correlation between LC5o values and all the exposure periods for 24h or 96h (Table ID). Thus with an increase in expo- sure periods of taraxerol the LC50 values show a significant decrease from 1.69 mg/L (24h);> 1.30 mg/L (48h);> 0.86 mg/L (72h);> to 0.74 mg/L (96h) respectively, in case of freshwater snail L. acuminata. (Table ID). The active (moiety), of taraxerol which were effective against freshwater snail L. acuminata, would also cause death amongst the fish. Consequently, YADAV ET AL.: Toxicity of taraxerol from Codiaeum variegatum on Lymnaea acuminata Table 1. Toxicity (LC1o, LCso and LCo0) of taraxerol against freshwater snail Lymnaea acuminata at different time intervals. Tabla I. Toxicidad (LCi0 , LCso y LC90) del taraxerol sobre el caracol Lymnaea acuminata a diferentes intervalos de tiempo. Exposure Effective dose Limits (mg/L) Slope value y factor “Prato Hetero-geneity periods LCL UCL 24h 1C10=0.98 0.829 1.089 LC50=1.69 LSD NO 5.397+0.853 0.10 6.33 0.70 1C90=2.92 DIAS, 48h 1C10=0.68 0.494 0.818 1C50=1.30 lo O 4.629+0.649 0.13 7.14 0.50 [C90=2.46 SETS 72h [C10=0.49 0.381 0.586 1C50=0.86 AS US 5.347+0.693 0.07 1.66 0.34 1C90=1.49 1.348 1.759 96h LC10=0.41 0.294 0.514 1C50=0.74 0.640 0.822 5.15240.750 0.08 6.87 0.53 1C90=1.31 1.187 1.542 Botches of ten snails were exposed to four different concentrations of taraxerol. Concentrations given are the final concentrations (w/v) in aquarium water. Regression coefficient showed that there was significant (P<0.05) negative correlation between exposure time and different LC values. LCL = Lower confidence limit; UCL = Upper confidence limit mixed populations of 10 snails (L. acumi- nata) and 10 fishes (C. punctatus) were treated for 24h of the LCoo of taraxerol. Up to the LCoo0 doses for snail L. acumi- nata there was no mortality amongst the freshwater fish C. punctatus. The doses, which can be used for killing the snails, are safe for fish. This is supported by our observations in a mixed population. Effect on freshwater target snail: Data of sub-lethal doses of 40% and 80% of LC5o (0.76 mg/L € 1.35 mg/L) taraxerol exposure to freshwater snail L. acumi- nata are given in Tables II and III. Expo- sure of snails to sub-lethal doses of taraxerol for 24h caused significant alterations in nitrogenous and carbohy- drate metabolism in different body tissues of the freshwater snail L. acumi- nata. Total protein and nucleic acids (DNA and RNA) levels were signifi- cantly reduced, while free amino acid level was significantly enhanced after the exposure to sub-lethal doses in all the body tissues. Acid and alkaline phosphatase activities were significantly reduced, while protease activity was increased after the exposure. Total protein levels were reduced to 31%, 37% and 28% of controls after exposure to sub-lethal doses of 1.35 mg/L of taraxerol respectively in the nervous, hepatopancreas and ovotestis tissue of L. acuminata, respectively. The DNA level was reduced to 41%, 32% and 28% of controls after treatment with 1.35 mg/L of taraxerol in nervous, hepatopancreas and ovotestis tissue Of L. acuminata, respectively. The RNA level was reduced to 36%, 39% and 28% of controls after treatment with sub- lethal doses of 1.35 mg/L of taraxerol respectively in nervous, hepatopancreas and ovotestis of L. acuminata. Total free amino acid levels were induced to 171%, 151% and 174% of controls after treat- ment with sub-lethal doses of 1.35 mg /L of taraxerol respectively in nervous, hepatopancreas and ovotestis of L. acuminata (Table II). Activity of acid phosphatase was inhibited to 75%, 82% and 77% of con- Iberus, 23 (1), 2005 Table IL. Changes in total protein, total free amino acids, nucleic acid (DNA and RNA) (1g/mg) level and activity of protease (umol of tyrosine equivalents/mg protein/h) and acid and alkaline phosphatase (mol substrate hydrolysed/30 min/mg protein) in nervous (NT), hepatopancreas (HP) and ovotestis (OT) tissues of Lymnaea acuminata after exposure to sub-lethal doses of 40% and 80% (0.76 mg/L and 1.35 mg/L) of taraxerol after 24h. Tabla II. Cambios en los niveles de proteínas, aminoácidos libres y ácidos nucléicos (ADN y ARN) (uglmg), actividad de proteasa (umol de equivalentes de tirosinalmg proteínalh) y de fosfatasas ácida y alcalina (umol de sustrato hidrolizado/30 min/mg proteína) en tejido nervioso (NT), hepatopáncreas (AP) y ovotestis (OT) de Lymnaea acuminata tras exposición a dosis subletales de 40% y 80% (0,76 mg/L y 1,35 mg/L) de taraxerol tras 24h. Pe 5 Conta 40% of LC5o (24h) 80% of LC5o (24H) 7 days of the od ll (0.76 mg/L) (1.35 mg/L) withdrawal Protein NT 64.0%0.18(100) 37.1240.24(58) 15.48+0.01(31) 156.80.71* (98) HP. 66.0+2.61(100) 37.6240.91(57) 24.42+0.81 (37) 136.7+1.00* (97) 01 71.0x0.53(100) 38.34+0.87(54) 19.880.84(28) 131.140.45* (96) Amino acid NT. 31.3+1.14(100) 51.64+1.15(165) — 53.52+0.28(171) 29.3+0.23* (103) HP. 26.8+1.51(100) 38.86+0.38(145) 40.40+1.07(151) 23.7+0.38* (105) 0T 34.3+0.68(100) 57.62+0.15(168) 59.68+0.14(174) 21.4+0.10* (104) DNA NT 76.6%0.29(100) 46.72+0.31(61) 31.40+0.21(41) 136.7+0.69* (96) HP. 73.8+0.21(100) 49.44+0.33(67) 23.61+0.29(32) 134.4+0.51* (96) 01 81.5+0.89(100) 44.01+0.21(54) 22.82+0.51(28) 137.740.34* (95) RNA NT 52.31+0.38(100) 26.67+0.27(51) 18.83+0.31(36) 99.9:0.28* (97) HP. 50.21+0.81(100) 30.12+0.77(60) 19.58+0.22(39) 100.0+0.21* (96) OT 55.33+0.41(100) 23.79+2.87(43) 15.49+0.58(28) 98.5+0.17* (93) Protease NT 0.471%0.051(100) 0.664+0.050(141) 0.706*0.051(150) 0.639+0.061* (108) HP 0.492+0.001(100) 0.6834+0.002(139) 0.713+0.001(145) 0.571+0.012* (94) OT 0.474+0.007(100) 0.673+0.003(142) 0.725*0.004(153) 0.739%0.154* (106) Acid phosphatase NT 0.281+0.008(100) 0.258+0.006(92) 0.210+0.004(75) 0.260+0.0123* (92) HP. 0.275+0.007(100) 0.264+0.008(96) 0.225+0.009(82) 0.276+0.013* (93) 0T 0.280+0.008(100) 0.263+0.003(94) 0.215+0.005(77) 0.267+0.012* (93) Alkaline phosphatase NT 0.481+0.021(100) 0.384+0.001(80) 0.307+0.003(64) 0.394+0.002* (91) HP. 0.482+0.004(100) 0.404+0.002(80) 0.307+0.001(68) 0.426+0.003* (92) OT 0.475+0.008(100) 0.384+0.001(81) 0.308%0.002(65) 0.403+0.004* (92) * Significant (P<0.05). Student's *F' test was applied between 80% of LCso (24h) and withdrawal groups. Values are mean +SE of six replicates. Values in parenthesis are percentage changes with control taken as 100% trols after treatment with sub-lethal doses of 1.35 mg/L of taraxerol respectively in nervous, hepatopancreas and ovotestis. The activity of alkaline phosphatase was reduced to 64%, 68% and 65% of controls after treatment with sub-lethal doses of 1.35 mg/L of taraxerol respectively in nervous, hepatopancreas and ovotestis. The protease activity was increased to 150%, 145% and 153% of controls after treatment with sub-lethal doses of 1.35 mg/L of taraxerol respectively in the nervous, hepatopancreas and ovotestis of snail L. acuminata (Table ID. Glycogen and pyruvate levels were significantly reduced, while lactate level was significantly enhanced after the exposure to sub-lethal doses in all the body tissues. Lactic dehydrogenase (LDH), cytochrome oxidase and acetyl- cholinesterase (ACHhE) activities were significantly reduced, while succinic dehydrogenase (SDH) activity was increased after the exposure. YADAV ET AL.: Toxicity of taraxerol from Codiaeum variegatum on Lymnaea acuminata Table III. Changes in glycogen (mg/g), pyruvate (umol/g), lactate (mg/g) level and activity of LDH (umol/mg protein/h), SDH (umol of dye reduced/min/mg protein), cytochrome oxidase (arbitrary unit/min/mg protein) and ACE (pmol “SH” hydrolysed/min/mg protein) after 24h exposure to sub-lethal doses of 40% and 80% (0.76 mg/L and 1.35 mg/L) of taraxerol in nervous (NT), hepatopancreas (HP) and ovotestis (OT) tissues of snail Lymnaea acuminata after 24h. Tabla II. Cambios en los niveles de glucógeno (mglg), piruvato (ymollg), lactato (mg/g) y actividad de LDH (umol/mg proteína/h), SDH (pmol de tinción reducidalmin/mg proteína), citocromo oxidasa (unidad arbitrarialmin/img proteína) y ACHE (umol “SH” hidrolizado/min/mg proteína) tras 24h de exposición a dosis subletales al 40% y 80% (0,76 mg/L y 1,35 mg/L) de taraxerol en los tejidos nervioso (NT), hepatopáncreas (HP) y ovotestis (OT) del caracol Lymnaea acuminata tras 24». a E lissuES Cda 40% of LC5o (24h) 80% of LC5o (24H) 7 doys of the (0.76 mg/L) (1.35 mg/L) withdrawal Glycogen NT 6.8%0.02 (100) 3.12+0.03 (46) 2.31+0.01 (34) 7.340.03* (93) HP. 7.2%0.10 (100) 3.67+0.01 (51) 2.80+0.03 (39) 6.8+0.02* (94) 01 9.1:0.08 (100) 4.09+0.03 (45) 3.00+0.01 (33) 7.3+0.04* (93) Pyruvate NT 0.578%0.03 (100) 0.196+0.04 (34) 0.167+0.23 (29) 0.635+0.27* (91) HP. 0.609+0.03 (100) 0.255*0.27 (42) 0.237+0.11 (39) 0.592+0.08* (90) 0T 0.581+0.04 (100) 0.180+0.18(31) 0.156+0.27 (27) 0.628+0.03* (92) Lactate NT 3.11%0.07 (100) — 5.06+0.15(163) 5.56+0.19 (179) 2.4640.04* (113) HP. 3.79+0.05 (100) 6.59+0.06 (174) 7.20+0.02 (190) 2.83+0.05* (118) OT 3.95+0.07 (100) 6.47+0.08 (164) 7.11%-0.06 (180) 2.4640.03* (113) LDH NT 0.084+0.004 (100) 0.068+0.001 (81) 0.043+0.002 (52 0.065+0.003* (90) HP. 0.096%0.003 (100) 0.081%0.002 (85) 0.056+0.001 (59 0.068+0.004 ( (91) OT 0.087%0.004 (100) 0.072+0.001 (83) 0.043%0.004 (5 0.070+0.003* (90) SDH NT 31.01%0.11 (100) 39.69+0.25 (128) 51.16+0.31* (165) 18.38+0.84* (112) HP. 43.21+0.13 (100) 53.58+0.29* (124) 65.24*0.27* (151) 15.63*0.18* (108) ) 34,42+0.30* (126) 162) — 20.43+0.20* (111) 5 6 ( ( 01 27.32:0.10 (100 (16 (15 44.25+0.34* (16 (55 (02 ) ) 0) ) l 2 ) 16.68+0.04* ) 0) 2 l 3 Cytochrome oxidase NT 18.23+0.12 (100) 11.84+0.16 (65) 10.02+0.18 (92) HP. 716.21+0.14 (100) — 11.34+0.21 (70) 10.05+0.31 13.05+0.12* (90) 01 17.32+0.16 (100) — 11.77%0.15 (68) 10.04+ 0.27 (5 16.18+0.02* (94) ACHE NT 0.068+0.0008 (100) 0.044+0.0003 (65) 0.028+0.0003 (92) 0.064+0.007* (90) HP. 0.088%0.0002 (100) 0.060%0.0007 (69) 0.036%0.0002 (41) 0.085+0.002* (93) OT 0.071:0.0002 (100) 0.046+0.0003 (66) 0.030%0.0004 (43) 0.063*0.007* (90) Details are as given in Table ll Glycogen level was reduced to 34%, 39% and 33% of controls after treatment with sub-lethal doses of 1.35 mg/L taraxerol respectively in nervous, hepatopancreas and ovotestis tissues of L. acuminata. Pyruvate level was reduced to 29%, 39% and 27% of con- trols after treatment with sub-lethal doses of 1.35 mg/L taraxerol respec- tively in nervous, hepatopancreas and ovotestis tissues of L. acuminata. Lactate level was increased to 179%, 190% and 180% of controls after treatment with sub-lethal doses of 1.35 mg/L of taraxe- rol respectively in nervous, hepatopan- creas and ovotestis tissues of snail L. acuminata (Table II. Lactic dehydrogenase activity was reduced up to 52%, 59% and 50% of con- trols after treatment with sub-lethal doses of 1.35 mg/L of taraxerol respec- tively in nervous, hepatopancreas and ovotestis tissue of snail L. acuminata. Activity of cytochrome oxidase was reduced to 55%, 62% and 58% of controls after treatment with sub-lethal doses of Iberus, 23 (1), 2005 Table IV. Changes in total protein, total free amino acids, nucleic acid (DNA 8 RNA) (1g/mg) level and activity of protease (umol of tyrosine equivalents/mg protein/h) and acid and alkaline phosphatase (umol substrate hydrolysed/30 min/mg protein) in muscle, liver and gonadal tissues of freshwater fish Channa punctatus after 96h exposure to sub-lethal doses of 40% and 80% (0.76 mg/L and 1.35 mg/L) of taraxerol and 7 days after withdrawal. Tabla IV. Cambios en los niveles de proteínas, aminoácidos libres, ácidos nucléicos (ADN y ARN) (ug/mg), actividad de la proteasa (umol of equivalentes de tirosinalmg proteínalh) y fosfatasas ácida y alcalina (umol sustrato hidrolizado/30 min/mg proteína) en los tejidos muscular, hepático y gonadal del pez Channa punctatus tras 96h de exposición a dosis subletales al 40% y 80% (0,76 mg/L and 1,35 mg/L) de taraxerol y a los 7 días de retirar el tratamiento. a his a 40% of LC5o (24h) 80% of LCso (24H) 7 days of the Ab (0.76 mg/L) (1.35 mg/L) withdrawal Protein Muscle 171.2+0.75(100) 104.3+0.22(61) 70.1+0.24(41) 162.6+0.68* (95) Liver 151.0+0.65(100) 128.3+0.21(85) 119.2+0.20(79) 146.4+1.00* (97) Gonadal 144.6+1.00(100) 112.7+0.12 (78) 76.4+0.07(53) 134.4+0.43* (93) Amino acid Muscle 38.20+0.23(100) 42.7%0.26(112) 48.5+0.38(127) 42.2+0.20* (110) Liver 22.6+0.40(100) 26.840.44(119) 31.840.40(141) 24.4+0.30* (108) Gonadal 31.0+0.70(100) 42.4+0.02(137) 46.1+0.03(149) 35.6+0.08* (115) DNA Muscle 152.44+0.73(100) 124.8+0.14(82) 94.4+0.14(62) 144.7+0.60* (96) Liver 149.02*0.70(100) 129.8%0.07(87) 105.9+0.13(71) — 137.440.50* (92) Gonadal — 143.00%0.73(100) 111.05+0.44(78) 82.94+0.23(58) 128.7+0.30* (90) RNA Muscle 103.00+0.28(100) 84.41+0.03(82) 69.01%0.04(67) 99.90x0.28* (97) Liver 100.0+0.29(100) 92.00%0.18(92) 72.00+0.18(72) 95.00%0.21* (95) Gonadal 106.60+0.61(100) 83.21+0.40(78) 65.02+0.28(61) 98.07+0.16* (92) Protease Muscle 0.598+0.011(100) 0.825+0.047(138) 0.926+0.015(155) 0.568+0.060* (92) Liver 0.652+0.016(100) 0.796+0.017(122) 0.920%0.014(141) 0.046+0.010* (99) Gonadal 0.601+0.016(100) 0.775+0.013(129) 0.949+0.014(158) 0.552+0.154* (92) Acid phosphatose Muscle 0.302+0.012(100) 0.138+0.010(30) 0.090+0.013(30) 0.277+0.0122*(92) Liver 0.292+0.014(100) 0.110*0.010(38) 0.078+0.009(27) 0.277+0.009* (95) Gonadal 0.281+0.015(100) 0.092+0.007(33) 0.061+0.017(22) 0.275*0.010* (98) Alkaline phosphatase Muscle 0.451%0.010(100) 0.193:<0.006(43) 0.121:0.03(27) 0.410:0.002* (91) Liver 0.400%0.030(100) 0.148+0.006(37) 0.096+0.006(24) 0.368+0.002* (92) Gonadal 0.438+0.012(100) 0.210+0.005 (48) 0.135+0.003(31) 0.420+0.003* (96) *, Significant (P<0.05) when Student's *' test was applied between 80% of LC50 (24h) and withdrawal groups Details are as given in Table || 1.35 mg/L of taraxerol respectively in nervous, hepatopancreas and ovotestis of L. acuminata. Acetylcholinesterase activity was reduced to 92%, 41% and 43% of controls after treatment with sub- lethal doses of 1.35 mg/L of taraxerol respectively in nervous, hepatopancreas and ovotestis of snail L. acuminata. The succinic dehydrogenase (SDH) activity was increased to 165%, 151% and 162% of controls after treatment with sub- lethal doses of 1.35 mg/L of taraxerol respectively in nervous, hepatopancreas and ovotestis tissues of freshwater snail L. acuminata (Table IID. Effect on freshwater non-target fish: Higher doses (LCso of snails) have no apparent toxic effect on non-target fresh- water fish C. punctatus after 24h exposure. But exposure of fish to sub-lethal doses (1.e. 40% and 80% of 24h LCso of snail) 0.76 mg/Land 1.35 mg/L of taraxerol for 96h caused a significant alteration in nitroge- nous and carbohydrates metabolism in YADAV ET AL.: Toxicity of taraxerol from Codiaeum variegatum on Lymnaea acuminata Table V. Changes in glycogen (mg/g), pyruvate (umol/g), lactate (mg/g) level and activity of LDH (umol/mg protein/h), SDH (umol of dye reduced/min/mg protein), cytochrome oxidase (arbitrary unit/min/mg protein) and ACHE (umol “SH” hydrolysed/min/mg protein) in muscle, liver and gonadal tissues of Channa punctatus after 96h exposure to sub-lethal doses of 40% and 80% (0.76 mg/L and 1.35 mg/L) of taraxerol and 7 days after withdrawal. Tabla V. Cambios en los niveles de glucógeno (mgle), piruvato (uymolle), lactato (mg/g) y actividad de LDH (umol/mg proteínalh), SDH (umol de tinción reducidalminímg proteína), citocromo oxidasa (unidad arbitrarialminimg proteína) y ACHE (pmol “SH? hidrolizado/min/mg proteína) en los tejidos muscular, hepático y gonadal del pez Channa punctatus tras 96h de exposición a dosis subletales al 40% y 80% (0,76 mg/L and 1,35 mg/L) de taraxerol y a los 7 días de retirar el tratamiento. E Tissue na 40% of LC5o (24h) 80% of LCso (24H) 7 days of the (0.76 mg/L) (1.35 mg/l) withdrawal Glycogen Muscle 2.20+0.001(100) 1.89+0.004(86) 1.380.04(63) 1.9140.02* (87) Liver 2.980.002(100) 2.17%0.001(73) 2.62+0.03(68) 2.47+0.04* (83) Gonadal 3.00+0.01(100) 2.61+0.02(87) 2.13+0.04(71) 2.73+0.04* (91) Pyruvate Muscle 3.416%0.018(100) 1.702*0.013(50) 1.097%0.024(32) 3.050+0.014* (90) Liver 4.076+0.018(100) 2.446+0.035(60) 1.548+0.007(38) 3.788+0.031* (93) Gonadal 2.993+0.036(100) 1.705+0.014(57) 0.921*0.020(31) 2.721:+0.016* (91) Lactate Muscle 3.816+0.018(100) 4.649+0.090(122) 6.846+0.060(179) 4.221%0.080* (111) Liver 2.323+0.020(100) 3.205+0.020(138) 3.750%0.074 (172) 2.500%0.069* (112) Gonadal 3.816+0.083(100) 4.502+0.088(118) 6.181+0.092(162) 4.159+0.043* (109) LDH Muscle 425.3:0.88(100) 374.2:0.81(88) 259.4:0.83(61) 382.5+0.81* (90) Liver 555.0+1.0(100) 521.7+0.80(94) 394.0+0.81(71) 516.1%0.76* (93) Gonadal 467.1+0.84(100) 420.3*+0.70(90) 350.3+0.81(75) 448.4+0.78* (93) SDH Muscle 59.4+0.21(100) 72.4:0.20(122) 81.3:0.27(137) 64.7+0.20* (109) Liver 62.2+0.20(100) 712.240.20(116) 85.9+0.19(138) 69.30.20* (111) Gonadal 64.4+0.26(100) 85.8+0.16(110) 95.9+0.10(149) 68.2+0.23* (106) Cytochrome oxidase Muscle 28.9140.21(100) 24.86%0.20(86) 17.92*0.24(62) 27.17+0.30* (94) Liver 23.1240.05(100) — 17.10%0.20(74) 15.95:0.24(69) 21.03+0.022* (91) Gonadal 33.20+0.05(100) 32.20+0.10(87) 24.28:+0.13(73) 30.54+0.17* (92) ACHE Muscle 0.091<0.0010(100) 0.045+0.0004(50) 0.034+0.0006 (38) 0.085+0.0002* (94) liver 0.097+0.0009(100) 0.053+0.0004(55) 0.034+0.0003(36) 0.082+0.0001* (92) Gonadal 0.088+0.0020(100) 0.043<0.0006(49) 0.024+0.0005(27) 0.079+0.0004* (90) +, Significant (P<0.05) when Student's “Y test was applied between 80% of LC50 (24h) and withdrawal groups Details are as given in Table || different body tissues of fish C. punctatus (Tables IV and V). Total protein and nucleic acids (DNA and RNA) levels were signifi- cantly reduced, while free amino acid level was significantly enhanced after the exposure to sub-lethal doses in all the studied body tissues. Acid and alka- line phosphatase activities were signifi- cantly reduced, while protease activity was increased after the exposure. Total protein levels were reduced to 41%, 79% and 53%; DNA level was reduced to 62%, 71% and 58% and RNA level was reduced to 67%, 72% and 61% in muscle, liver and gonadal tissue of freshwater fish C. punctatus. Total free amino acid levels were induced to 127%, 141% and 149% of controls after 96h treatment with 1.35 mg/L of LCso of taraxerol in muscle, liver and gonadal tissues, respectively (Table IV). Activity of acid phosphatase was inhibited to 30%, 27% and 22% and Iberus, 23 (1), 2005 activity of alkaline phosphatase was reduced to 27%, 24% and 31% but Pro- tease activity was increased to 155%, 141% and 158% of controls after 96h treatment with 1.35 mg/L of taraxerol in muscle, liver and gonadal tissues of freshwater non-target fish C. punctatus, respectively (Table IV). Glycogen and pyruvate levels were significantly reduced, while lactate level was significantly enhanced after the exposure to sub-lethal doses in the studied body tissues. Lactic dehydroge- nase (LDH), cytochrome oxidase and acetylcholinesterase (ACHE) activities were significantly reduced, while suc- cinic dehydrogenase (SDH) activity was increased after the exposure. Glycogen level was reduced to 63%, 68% and 71% and pyruvate level was reduced to 32%, 38% and 31% in muscle, liver and gonadal tissue of fish. Lactate level was increased to 179%, 172% and 162% of controls after 96h treatment with 1.35 mg/L taraxerol in muscle, liver and gonadal tissues of fish c. punctatus, respectively (Table V). Lactic dehydrogenase (LDH) activity was reduced to 61%, 71% and 75% and activity of cytochrome oxidase was reduced to 62%, 69% and 73% and acetylcholinesterase (ACHE) activity was reduced to 38%, 36% and 27% in muscle, liver and gonadal tissue of fish C. puncta- tus, respectively. Succinic dehydrogenase (SDH) activity was increased to 137%, 138% and 149% of controls after 96h treatment with 1.35 mg/L LC5o taraxerol in muscle, liver and gonadal tissues of fish C. punctatus, respectively (Table V). DISCUSSION Itis clear from the results section that the extracted compound taraxerol from the stem-bark of Codiaeum variegatum has potent molluscicidal activity against the freshwater target snail L. acuminata. More important is the fact that the extracted compound is much more toxic than synthetic pesticides. The present study demonstrated that taraxerol has higher molluscicidal activity than any of 10 the prevalent synthetic molluscicides like carbamate, organophosphate and synthetic pyrethroids. Thus, the 24h LC5so of mexacarbamate (3.5 ppm), aldicarb (30.00 ppm), farmothion (27.00 ppm), cypermethrin (2.5 ppm), perme- thrin (0.82 ppm) and fenvalerate (2.5 ppm) against L. acuminata (SINGH AND AGARWAL, 1981; SINGH AND AGARWAL, 1986; SINGH AND AGARWAL, 1988; SINGH AND AGARWAL, 1991) is higher than that of taraxerol (0.183 ppm), which is about 65 times stronger than the standard mol- luscicide niclosamide (LCso 11.8 ppm) SAHAY, SINGH AND AGARWAL (1991). Statistical analysis of the data on tox- icity brings out several important points. The X? test for goodness of fit (Heterogeneity) demonstrated that the mortality counts were not found to be significantly heterogeneous and other variables (e.g. resistance) do not signifi- cantly affect the LC5o values, as these were found to lie within the 95% confi- dence limits. The slope is thus, an index of the susceptibility of the target animal to the extract used. A steep slope is also indicative of rapid absorption and onset of effects. Since the LC5o of taraxerol is within the 95% confidence limits, it is obvious that in replicate tests of random samples, the concentration response lines would fall in the same range (RAND AND PETROCELLI, 1988). The depletion of protein fraction in different tissues of snail and fish may have been due to their degradation and possible utilization of degraded prod- ucts for metabolic purposes. Mom- MENSEN AND WALSH (1992) reported that proteins are mainly involved in the architecture of the cell, which is the main source of nitrogenous metabolism, and during chronic periods of stress they are also a source of energy. Increment in free amino acids level was the result of breakdown of protein for energy require- ments and impaired incorporation of amino acids in protein synthesis. Inhibi- tions of DNA synthesis might affect both protein as well as amino acid levels by decreasing the level of RNA in protein synthesis machinery (NORDENKJOLD, SODERHALL AND MOLDEUS, 1979). YADAV ET AL.: Toxicity of taraxerol from Codiaeum variegatum on Lymnaea acuminata The increase in the protease activity corroborates the enhancement in the FAA (Free amino acids) level in tissues, the formation of which might be the result of protein hydrolysis in the tissues suggest- ing stimulation during toxic stress. Similar trend of results on protease activ- ity were also reported by several workers in different animals (IT. mossambica (Peters), P. globosa (Swaimson) including mammals (KABEER, SAHIB, SIVA PRASAD AND SAMBASIVA RAO, 1984). SINGH AND AGARWAL (1992) reported that several euphorbious plants significantly reduced the alkaline and acid phosphatase activ- ity in nervous tissue of L. acuminata so the reduction in protein level may be due to the inhibition of alkaline phosphatase activity, as it plays an important role in protein synthesis (PILO, ASNANI AND SHAH, 1972) and other secretory activities (IBRAHIM, HIGAZI AND DEMIAN, 1974). Carbohydrates reserves were depleted to meet energy demand, thus depletion of glycogen may be due to direct utilization for energy generation, a demand caused by active moiety- induced hypoxia. The glycogenolysis seems to be the result of increased secre- tion of catecholamine due to stress. Decrease in pyruvate level is due to higher energy demand during exposure, which suggests the possibility of a shift towards anaerobic dependence due to a remarkable drop in the amount of oxygen. The increase in lactate also sug- gests a shift towards anaerobiosis as a consequence of hypoxia leading to respi- ratory distress (SIvVA PRASADA RAO, 1980). Lactic dehydrogenase (LDH) cat- alyzes the inter-conversions of lactic acid and pyruvic acid during anaerobic conditions. Inhibition of lactic dehydro- BIBLIOGRAPHY AGARWAL, R. A., AND SINGH, D. K., 1988. Harm- ful gastropods and their control. Acta Hy- drochímica et Hydrobiologica 16: 113-138. ANONYMOUS, 1984. Sigma diagnostics TM: Lac- tic dehydrogenase (quantitative, colorimet- ric determination in serum, urine and cere- brospinal fluid) at 400-450 nm. Procedure no. 500. 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P., AND SINGH, A., 2001. Environ- mentally safe molluscicides from two com- mon euphorbiales. Iberus, 19 (1): 65-73. YADAV, R. P., AND SINGH, A., 2002. Toxic effects of latex of Croton tiglium on Lymnaea acumi- nata and Channa punctatus. Iberus 20 (2): 33- 44. O Sociedad Española de Malacología —__——— Iberus, 23 (1): 15-24, 2005 Un avance sobre la composición y microestructura de la concha de lberus gualtierianus morfotipo gualtierianus (Linnaeus, 1758) (Gastropoda: Helicidae) Notes on the composition and microstructure of the shell of /berus gualtierianus morphotype gualtierianus (Linmaeus, 1758) (Gastropoda: Helicidae) Rocío MÁRQUEZ*, José Ramón ARRÉBOLA** y Rafael DELGADO* Recibido el 20-V-2004. Aceptado el 5-VII-2004 RESUMEN Se ha estudiado la concha de Iberus gualtierianus morfotipo gualtierianus (Linnaeus, 1758) desde el punto de vista de su composición mineralógica (difracción de rayos X), composi- ción química (fluorescencia de rayos X, microanálisis de rayos X y calcimetría) y microes- tructura (microscopía electrónica de barrido, MEB). El mineral presente es aragonito (CaCOs) (sobre 99%), con trazas de calcita. Los principales elementos químicos detectados, aparte de Ca, € y O, son Si, Mg, Na y Sr, probablemente como sustituciones en la red cristalina y/o contaminaciones. Los contenidos de CaCO3 están entre 97% y 99%. La concha tiene un espesor de unos 300 pm, con microestructura en capas superpuestas. Dentro de cada una de ellas, el tipo de microestructura se califica como laminar cruzada simple, compuesta de láminas de tercer, segundo y primer orden y con orientaciones distintas de los cristales de aragonito. Rasgos microestructurales destacables son el distinto espesor de las capas, dependiendo de su situación bajo las costillas o estriaciones de la concha, y que el número de capas disminuye en la última media vuelta de espira. La mayoría de estos caracteres son comunes a otros moluscos y en especial a los gasterópodos terrestres, si bien hasta el pre- sente no habían sido establecidos en /. gualtierianus morfotipo gualtierianus. SUMMARY The mineralogical composition of the shell of Iberus gualtierianus morphotype gualtierianus (Linnaeus, 1758) was studied (X-ray diffraction), as was the chemical composition (X-ray fluorescence, X-ray microanalysis and calcimetry) and the microstructure (scanning elec- tron microscopy, SEM). The mineral present is aragonite (CaCO3) (around 99%) with tra- ces of calcite. The main chemical elements detected, apart from Ca, C and O, are Si, Mg, Na and Sr, probably substituting Ca in the crystalline structure and/or as impurities. The CaCOz3 contents are between 97% and 99%. Shell thickness is around 300 pm with supe- rimposed layers. In each of these the microstructure is classified as simple crossed lame- llar, composed of third, second and first order lamellae with differing orientation of the aragonite crystals. Noteworthy microstructural features are the different thicknesses of the layers, depending on their position below the ribs or striations of the shell and the fact that * Departamento de Edafología y Química Agrícola, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja s/n, 18071, Granada, España. ** Departamento de Fisiología y Zoología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes, 6, 41012, Sevilla, España. 15 Iberus, 23 (1), 2005 the number of layers decreases in the last half turn of the whorl. Most of these characteris- tics are common to other molluscs, especially to the terrestrial gastropods, although, until now, these had not been identified in Iberus gualtierianus morphotype gualtierianus. PALABRAS CLAVE: /berus gualtierianus morfotipo gualtierianus, concha, mineralogía, composición química, microestructura, MEB. KEY WORDS: /berus gualtierianus morphotype gualtierianus, shell, mineralogy, chemical composition, micros- tructure, SEM. INTRODUCCIÓN La mayoría de los moluscos se carac- terizan por presentar una concha, verda- dero esqueleto externo que ellos mismos segregan, que posee distintas funciones y que está compuesta en un 95-99% por car- bonato, esencialmente cálcico (CaCO»), y materia orgánica, que representa entre el 5% y el 0,1% restante (HARE Y ABELSON, 1965). El carbonato cálcico de las conchas de los moluscos precipita como calcita o aragonito, siendo la última especie mineral el polimorfo más frecuente (LOWENSTAM Y WEINER, 1989). Aún así, algunos moluscos presentan simultáne- amente los dos polimorfos en sus conchas, bien como constituyentes de una misma capa (MUTVEIL, DAUPHIN Y Curr, 1985), o bien localizados en capas diferenciadas (SABATIER, 1953; DAUPHIN, CUIE, MUTVEI Y DENIS, 1989; LOWENSTAM Y WEINER, 1989). Otras formas de CaCOz3 han sido también reconocidas en la com- posición mineral de las conchas de los moluscos, pero nunca como constitu- yentes principales. Así por ejemplo, la vaterita ha sido identificada en zonas regeneradas de las conchas tras sufrir éstas algún daño estructural (WILBURG, 1964; TAYLOR, KENNEDY Y HALL, 1969; LOWENSTAM Y WEINER, 1989) o en los márgenes de las conchas recientemente mineralizados (LOWENSTAM Y WEINER, 1989; HAsseE, EHRENBERG, MARXEN, BECKER Y EPPLE, 2000), aunque siempre en cantidades inferiores al 1,5%. En la concha de los moluscos apare- cen también ciertos elementos traza, cuyo contenido concreto puede dar información acerca de las condiciones ambientales y paleoambientales del 16 medio en el que se produjo la precipita- ción del mineral. El análisis isotópico (810 y 8C) de las conchas ha aportado, igualmente, información de esa natura- leza (KRANTZ, WILLIAMS Y JONES, 1987). La microestructura de la concha de los moluscos ha sido descrita y analizada por muchos autores (MaAckay, 1952; CARTER, 1980; MUTVEI ET AL., 1985; HE- DEGAARD, 1997; CHATEIGNER, HEDEGA- ARD Y WENK, 2000). BSGGILD (1930) rea- lizó la primera clasificación de la micro- estructura mediante descripciones con microscopía de luz transmitida. Gracias a la introducción de la microscopía elec- trónica en estos estudios (GRÉGOIRE, Du- CHATEAU Y FLORKIN, 1949), y principal- mente desde la utilización del microsco- pio electrónico de barrido (MEB) a finales de la década de los sesenta, la mi- croestructura de las conchas se ha defi- nido más a partir de su micromorfología que a partir de su mineralogía o cristalo- grafía Óptica. Esta situación ha hecho que las clasificaciones iniciales hayan sido redefinidas (KOBAYASHI, 1964, 1971; MACCLINTOCK, 1967; TAYLOR ET AL., 1969, TAYLOR, KENNEDY Y HALL, 1973; GRÉGOIRE, 1972), realizándose importan- tes revisiones de la nomenclatura des- criptiva aplicada a la microestructura y ultraestructura (WILBUR Y SALEUDDIN, 1983; WATABE, 1984; LOWENSTAM Y WEI- _NER, 1989). Cabe destacar, en este sen- tido, los trabajos de CARTER Y CLARK (1985) y CARTER (1990), en los que se in- tegra la nomenclatura de la mineralogía y la microestructura de la mayoría de los grupos de invertebrados y vertebrados, con la intención de poder hacer compa- raciones entre distintos organismos. MÁRQUEZ ET AL.: Composición y microestructura de la concha de /berus gualtierianus El estudio de la microestructura de la concha de los moluscos también se ha empleado para el análisis de los proce- sos y mecanismos que dan lugar a la precipitación del carbonato cálcico y su posterior organización ultraestructural (WATABE Y WILBUR, 1961; WISE Y Hay, 1968). Entre las distintas aplicaciones que tiene el análisis de la microestructura de las conchas se encuentra su utilidad en estudios taxonómicos, filogenéticos y evolutivos (MACKaAY, 1952; WIsE, 1970; TAYLOR, 1973; PoPOv Y BARSKOV, 1978; CARTER Y CLARK, 1985; SCHNEIDER Y CARTER, 2001). En este trabajo se estudia la compo- sición mineralógica y química y la microestructura de la concha de Iberus gualtierianus morfotipo gualtierianus (Linnaeus, 1758), una de las varias formas conquiliológicas conocidas en esta especie. Iberus gualtierianus morfotipo gualtie- rianus es un endemismo andaluz cono- cido popularmente como “chapa”, que vive en tres enclaves muy localizados: Sierra Elvira en la provincia de Granada, Sierra de Jaén en la de Jaén y Sierra de Gádor en la provincia de Almería. Habita en suelos xéricos, sobre calizas y dolomías con modelado de tipo kárstico y una vegetación correspondiente a etapas de regresión del bosque climá- cico. En este ambiente, es capaz de colo- nizar zonas semidesérticas con poco desarrollo de suelo y escasas precipita- ciones anuales. Debido a las presiones de diversa índole que le afectan desde hace varias décadas, y en especial las relacio- nadas con su interés gastronómico (ARRÉBOLA, 2002), ha sido propuesta por la Sociedad Española de Malacología para su incorporación al Catálogo Nacio- nal de Especies Amenazadas. MATERIAL Y MÉTODOS Cuatro conchas (A, B, C y D) de Iberus gualtierianus morfotipo gualtieria- nus fueron recogidas en Sierra Elvira (UTM: VG32), al noroeste de la ciudad de Granada. La composición mineralógica de los cuatro ejemplares ha sido analizada mediante difracción de rayos X (XRD), con un difractómetro Rigaku-Miniflex Ca 2005, equipado con un filtro de Ni, sistema de discriminación de impulsos y contador de gases, según describe GÁMIZ (1987). La composición química de los ejemplares A y B se ha conocido mediante fluorescen- cia de rayos X (XRF) (SORIANO, 1994) y microanálisis de rayos X de energía dis- persiva (EDR) (MÁRQUEZ, 2003). La primera técnica se ha realizado con un espectrómetro secuencial de longitud de onda dispersiva, equipado con un ánodo de Rh y un generador de rayos X de 4 kW de potencia, Philips Magix Pro (PW-2440). Por su parte, el análisis EDR se ha llevado a cabo con un espectrómetro de rayos X de energía dispersiva, Róntec, 288, M-Serie, EDWIN, acoplado a un equipo de MEB (se describirá posteriormente), que permite analizar muestras de superficie rugosa. Para este análisis las muestras fueron meta- lizadas con grafito. Finalmente, los ejem- plares € y D han sido utilizados para ana- lizar el contenido de carbonatos (CaCO%3 equivalente) con el calcímetro de Bernard. Para el análisis de la microestructura de la concha de I. gualtierianus morfo- tipo gualtierianus se han seleccionado, del ejemplar A, muestras de las zonas de crecimiento más reciente de las tres últimas medias vueltas de espira de la concha, ya que según LÓPEZ-ALCÁN- TARA, RIVAS, ALONSO E IBÁÑEZ (1983) cada media vuelta de espira se corres- ponde con un estadio de crecimiento del organismo. Se han observado fracturas frescas de la concha, que facilitan la observación de la microestructura en el MEB (HEDEGAARD, LINDBERG Y BANDEL, 1997), en secciones paralelas y perpendi- culares a las líneas de crecimiento. Los fragmentos de concha fueron pegados directamente sobre el portamuestras de aluminio mediante pegamento de plata coloidal. Posteriormente fueron metali- zados con grafito para facilitar la obser- vación y permitir el análisis EDR. La observación se ha realizado en un microscopio electrónico de barrido Hitachi, modelo S-501 (voltaje de acele- ración de 25 kV; imágenes de electrones 17 Iberus, 23 (1), 2005 Tabla I. Análisis por fluorescencia de rayos X (XRE, ejemplares A y B) y calcimetría (calcímetro de Bernard, ejemplares C y D) de las conchas de /berus gualtierianus morfotipo gualtierianys. Table I. X-ray fluorescence analysis (XRE specimens A and B) and carbonate content (Bernard calcime- ter, specimens C and D) of the shells of lberus gualtierianus morphotype gualtierianus. Resultados XRF Elementos mayores como óxidos (%) Al03 C00 Fez03 K20 MgO Noz20 P205 SiO2 Ei. A 0,05 55,72 0,06 0,07 0,12 0,08 0,02 0,25 Ei. B 0,06 55,44 0,02 0,07 0,08 0,08 0,01 0,30 10,9 624,7 10,5 43,13 99,50 99,40 98,0 21,1 579,2 10,9 43,23 99,29 98/90: 98,20-197 5299772 Colcimetría Elementos menores (ppm) LOI'Y% Suma CaCOs CoCO3 equiva- Sr dr (0%) (4) (0% (40 | lente (%)0 EC ED (a) Pérdida de peso por calcinación (1000 *C), (b) Calculado a partir de los porcentajes de CO; % CaCOz = % CaO x Pm CaCO3/Pm Cad (c) Calculado a partir de los porcentajes de LO; % CaCOz = % LOI x Pm CaCO3/Pm CO»; (Pm, peso molecular); (d) Calcímetro de Bernard secundarios) con el programa de digita- lización de imágenes Scan Vision. RESULTADOS Y DISCUSIÓN El análisis mineralógico pone de manifiesto que las conchas de 1. gualtieria- nus morfotipo gualtierianus están com- puestas, mayoritariamente, por aragonito (ejemplar A, 99,8%; ejemplar B, 99,4%; ejemplar C, 99,5% y ejemplar D, 98,7%), conteniendo cantidades traza de calcita (ejemplar A, 0,2%; ejemplar B, 0,6%; ejemplar C, 0,5%; ejemplar D, 1,3%). El patrón de difracción de los dia- gramas corresponde al del aragonito en la carta JCPDS n* 5-453, con una pequeña reflexión a 0,303 nm correspon- diente a la calcita (carta JCPDS n* 5-586) (JCPDS, 1974). En este resultado, I. gualtierianus mor- fotipo gualtierianus se asemeja a la mayoría de los moluscos, cuya concha es aragoní- tica (LOWENSTAM Y WEINER, 1989). La pre- sencia de pequeñas cantidades de calcita puede interpretarse de varios modos, tal como indican BALMAIN, HANNOYER Y López (1999). Cabe la posibilidad de que esta especie mineral se concentre en deter- minadas partes de la concha, lo que no parece probable, ya que las proporciones en las que aparece son distintas entre los ejemplares, siendo éstos similares macro- morfológicamente. Otra posibilidad es que se trate de una fase secundaria a la for- mación de la concha por un proceso de contaminación natural, dado que estos animales habitan ambientes carbonatados. 18 Por último, cabría el paso de un polimorfo a otro (de aragonito a calcita) generado por el calentamiento durante la molienda; este proceso tampoco parece muy proba- ble dada la escasa velocidad de la reac- ción (CARLSON, 1983). Los resultados del análisis XRF se muestran en la Tabla I. Se observa que entre los elementos mayores destaca por su abundancia el calcio, constituyendo, en óxidos, el 55,72% del ejemplar A y el 55,44% del ejemplar B. Las pérdidas por calcinación (LOL) son elevadas, superiores al 43%. Les siguen en proporción el silicio (en óxidos, 0,25% para el ejemplar A y 0,30% para el ejemplar B), mientras que el resto de los óxidos aparecen con valores inferiores al 0,1% (salvo el de magnesio en el ejemplar A, cuyo valor es 0,12%). De los elementos menores, el estroncio es el que presenta el porcentaje más alto en ambas conchas (624,7 ppm en el ejemplar A y 579,2 ppm en el ejemplar B). Las proporciones de CaO son las espe- radas dada la mineralogía aragonítica, y en menor medida calcítica (ambos poli- morfos del CaCO»), de la concha. Con estas proporciones de CaO, cabe calcular el con- tenido de CaCO> presente, que resulta de 99,4% y 98,9% respectivamente para el ejemplar A y B (Tabla 1). Lo mismo se podría decir de las altas pérdidas por cal- cinación que corresponderían al CO» gene- rado en la descomposición térmica de los carbonatos; en este caso el contenido de CaCO> calculado a partir de ellas es de 98,0% y 98,2% respectivamente para el ejemplar A y B (Tabla ]). El ajuste con las anteriores cifras es aceptable. MÁRQUEZ ET AL.: Composición y microestructura de la concha de /berus gualtierianus x103 Ca Cuentas por segundo 2. .NNO ps 0,0 SS S IO MAAS ON OSO ESAS SEO Energía Cuentas por segundo SS Lore Ss e se 0.0.0.3 1,0 1,53:20.25 510.35) 2401 172/ Energía Figura 1. Espectros de microanálisis de rayos X (EDR) en fracturas frescas de conchas de /berus gualtierianus morfotipo gualtierianus. Izquierda: ejemplar A; derecha: ejemplar B. / Figure 1. X-ray microanalysis spectra (EDR) in fresh fractures of the shells of Iberus gualtierianus morp- hotype gualtierianus. Left: specimen A; right: specimen B. Las proporciones de Si0O»2 (0,25% en el ejemplar A y 0,30% en el ejemplar B) y ADO (0,05% y 0,06% para el ejemplar A y B, respectivamente), son imputables a la contaminación de la concha por silica- tos del suelo, tratándose de componen- tes secundarios. A pesar de que el aragonito es un mineral bastante puro desde el punto de vista químico (DEER, HOWIE Y ZUSSMAN, 1992), el Sr presente en las conchas se incluiría en la red de este mineral susti- tuyendo al Ca, lo cual es un hecho amplia- mente descrito en la bibliografía sobre ara- gonito biogénico (TUREKIAN Y ARMSTRONG, 1960; DoDD, 1967; MASUDA, 1976; MASUDA E HIRANO, 1980). Lo mismo le ocurriría al Mg, pudiendo ser también, en parte, pro- ducto de contaminación de los materiales dolomíticos del suelo. El Na es un catión que también puede estar presente en la red del aragonito, incluso en cantidades elevadas (DAUPHIN Y DENIS, 2000; DAUPHIN, GUZMAN, DENIS, CUIF, Y ORTLIEB, 2003), aunque su propor- ción es mayor en las conchas marinas (WADA Y FUJINUKL, 1974). Otros autores han considerado que la presencia del Na en las conchas de los moluscos, se debe a procesos de contaminación (por ejemplo, EsTES, 1972), o al hecho de ser un catión que forma parte de la composición química de la matriz orgánica de las conchas (LÉCUYER, 1996). El análisis del contenido de carbonato cálcico equivalente de las conchas (Tabla D, registra los siguientes porcentajes: 97,52% para el ejemplar C y 97,72% para el ejemplar D. Estos resultados son de mag- nitudes similares a los obtenidos por aná- lisis XRF ya expuestos (Tabla 1). Se puede, por tanto, afirmar que los ejemplares de [ gualtierianus morfotipo gualtierianus ana- lizados tienen un porcentaje de CaCO3 entre el 97% y el 99% y que se encuentra, de esta forma, dentro del rango de valores conocido para la concha de los moluscos (entre 95% y 99,9%); el resto son compo- nentes orgánicos (HARE Y ABELSON, 1965). El estudio de la composición se ha completado con los resultados de micro- análisis de rayos X (EDR) (Fig. 1) de la superficie de las fracturas frescas de las conchas. Se ha demostrado la composi- ción carbonática al aparecer las líneas correspondientes a los elementos carbono, oxígeno y calcio. Hay, pues, una perfecta coherencia con el resto de los resultados expuestos. Los elementos químicos detec- tados en XRF (Tabla D, distintos al carbono, oxígeno y calcio, no se registran con EDR, al encontrarse sus cantidades por debajo de los límites de detección de esta técnica. El estudio de la concha en fractura fresca bajo el microscopio electrónico de barrido (MEB) ha puesto de manifiesto, en primer lugar, su constitución en capas superpuestas (Figs. 2A, B). Dichas capas presentan estructura laminar cruzada (se describirá posteriormente) y distintas orientaciones de los cristales de aragonito (Fig. 2B). Esta configuración interna de la 1 Iberus, 23 (1), 2005 Tabla IT. Capas reconocidas, y espesores medios de las mismas (um), en una sección perpendicular (A) y en otra paralela (B) a las líneas de crecimiento de la concha estudiada del ejemplar A de Iberus gualtierianus morfotipo gualtierianus. Medidas realizadas en las observaciones con microsco- pio electrónico de barrido (MEB). La capa a es la más interna de la concha y la e la más externa. Table II. Layers observed and their mean thicknesses (um), in both perpendicular (A) and parallel (B) sections to the growth lines of the studied shell of the specimen A of lberus gualtierianus morphotype gualtierianus. The measurements were carried out using scanning electron microscopy (SEM). Layer a is the most internal and layer e the most external. A. Sección perpendicular a las líneas de crecimiento Copos 0 b C d e > (espesor total) Media vuelta de espira esp ds nm. esp dsu nm esp ds. Mo sesp 05 Desp USES ds n Última 132,39 123,14 8 132,60 43,03 8 101,84 14,331 4 366,83 60,16 20 Penúltima 101,14 15,17 8 55,00 16,22 8 61,47 748 8 30/46 9,57 8 55,91 18,93 8 303,98 13,47 40 Antepenúltima 113,24 34,56 8 67,50 36,29 8 46,32 885 8 36,91 911 8 3406 535 8 298,03 18,83 40 B. Sección paralela a las líneas de crecimiento Copos 0 b C d e > (espesor total) Media vuelta de espira esp 05 nm. esp. 05 nm esp. ds n esp “05. .n €5p... 05 Mn esp Último 250,972815 40 53131331054 304,10 21,03 8 Penúltima 77,91 3,91 8 55,57 10,07 8 66,56 7,73 8 5247 919 8 42,335 6,61 8 294,86 7,50 40 Antepenúltima 47,93 13,04 8 60,75 894 8 48,17 1238 8 26,59 5/04 8 24,89 7,52 8 208,33 9,38 40 esp: espesor medio (pm); ds: desviación típica; n: número de medidas realizadas concha ha permitido establecer el número y espesor de las capas en las últimas fases del crecimiento del organismo (Tabla ID. Destaca, como hecho observable, que el espesor de las capas, en general, varía de- pendiendo de la zona que se trate, au- mentando bajo las costillas y decreciendo en las estriaciones (Fig. 2A) presentes en estas conchas (LÓPEZ-ALCÁNTARA, RIVAS, ALONSO E IBÁÑEZ, 1985). Además, cabe destacar que, en la mayoría de los casos, los espesores medidos tienen un cierto grado de espesores aparentes, ya que las secciones observadas son irregulares al ser fracturas frescas y no se disponen total- mente perpendiculares al plano del ob- (Página derecha) Figura 2. Imágenes de microscopía electrónica de barrido de la concha de l/berus gualtierianus morfotipo gualtierianus. A: microestructura en corte fresco de una sección perpendicu- lar a las líneas de crecimiento de la penúltima media vuelta de espira. Presencia de cinco capas con estructura laminar cruzada y orientaciones distintas. La flecha indica una costilla y el incremento de los espesores de las capas debajo de ella. B: detalle de la imagen anterior, demostrando las distintas orientaciones de los cristales en las capas contiguas (por ejemplo, a con b). C: microestructura en corte fresco de una sección paralela a las líneas de crecimiento de la penúltima media vuelta de espira. Se muestran las varillas de aragonito (lt) que componen el tercer orden de la estructura laminar cruzada, y los paquetes de varillas (ls) que constituyen el segundo orden de dicha estructura. D: microestruc- tura en corte fresco de una sección paralela a las líneas de crecimiento de la última media vuelta de espira. Se muestran las unidades estructurales de primer orden de forma tabular (Ip). Las flechas indican las dos distintas orientaciones de los cristales en las láminas de primer orden. E: superficie interna de la concha en la última media vuelta de espira. Se muestran, con flechas discontinuas, las ondulacio- nes (od) y estrías finas (est). La flecha continua señala hacia la abertura de la concha. E: superficie interna de la concha en la última media vuelta de espira. Se destacan con la flecha discontinua las pequeñas protuberancias (pb). La flecha continua señala hacia la abertura de la concha. 20 MARQUEZ ET AL.: Composición y microestructura de la concha de /berus gualtierianus NASA E F Figure 2. Scanning electron micrographs of the shell of Tberus gualtierianus morphotype gualtierianus. A: microstructure in freshly cut section perpendicular to the growth lines of the penultimate half turn of the whorl. Presence of the five layers with crossed. lamellar structure and different orientations. The arrow shows a rib and the increase in thickness of the layers below it. B: detail of A showing different orientations of the crystals in the contiguous layers (e. g. a with b). C: microstructure in freshly cut section parallel to the growth lines of the penultimate half turn of the whorl. The aragonite rods (lt) which constitute the third order of the crossed lamellar structure and the bundles of rods (ls) forming the second order of the structure can be seen. D: microstructure in freshly cut section parallel to the growth lines of the last half turn of the whorl. The first order structural units (lp) can be seen. The arrows show the two different orientations of the crystals in the first order lamellae. E: internal surface of the shell in the last half turn of the whorl. The dotted arrows show the ondulations (od) and fine striations (est). The solid arrow points towards the shell aperture. E: internal surface of the shell in the last half turn of the whorl. The dotted arrows show the small protuberances (pb). The solid arrow points towards the shell aperture. 2] Iberus, 23 (1), 2005 servador, a pesar de los esfuerzos reali- zados en el microscopio para que así sea. Por todo ello se aportan valores medios (Tabla ID. De estos datos se deduce, en primer lugar, que en la sección perpendicular a las líneas de crecimiento de la última media vuelta de espira (Tabla IIA) se reconocen tres capas (a, b y c), mientras que en la sección paralela a dichas líneas sólo se reconocen dos (Tabla IIB). Pensa- mos que esto pueda deberse a la orien- tación relativa de los cristales de arago- nito en las láminas de las capas, de tal forma que, en la sección paralela, dos de ellas, las más internas, tienen una orien- tación similar y se confunden. El número de capas aumenta hasta cinco en la penúltima y antepenúltima medias vueltas de espira en ambas sec- ciones, lo que supone admitir que la concha va cambiando en su microestruc- tura en el transcurso de las fases de desarrollo del animal. | Una cuestión interesante es el espesor de las capas, variable de unas a otras (entre 24,89 um y 250,97 um) (Tabla II). Por otra parte, el espesor total de la concha es de unos 300 um, tendiendo a incrementarse en la última media vuelta de espira res- pecto a las anteriores, lo que apuntaría no sólo a una reconstrucción en el número de capas ya referido, sino también a una pequeña reducción en el volumen de la concha en las medias vueltas de espira más internas. Respecto a la microestructura en detalle de las capas, hay que decir que están constituidas por distintas unida- des estructurales que crecen en comple- jidad y que se denominan, respectiva- mente, láminas de tercer, segundo y primer orden, como corresponde con su calificación de laminar cruzada (BSGGILD, 1930; UOZUMI, IWATA Y TOGO, 1972; CARTER, 1990). Las unidades más pequeñas o de tercer orden, tienen forma de varilla y un espesor medio de 0,49 um (desviación típica, 0,14 um; n, 20) (Fig. 2C). Estas unidades son parale- las entre sí y están unidas generando pequeños paquetes (Fig. 2C) que consti- tuyen las unidades estructurales inter- medias O de segundo orden, con un 22 grosor medio de unos 5 um (4,91 um; desviación típica, 1,16 um; n, 6). A su vez, éstas están unidas entre sí dando lugar a las unidades estructurales mayores o de primer orden que presen- tan una forma plana o tabular (Fig. 2D). De acuerdo con los caracteres descri- tos y según la terminología utilizada por CARTER (1990), esta microestructura podemos clasificarla como laminar cruzada simple (“simple crossed lame- llar”). La microestructura de tipo laminar cruzada es la más extendida en las conchas de los moluscos (B9GGILD, 1930), estando constituida por aragonito salvo en algunas especies (UOZUMI ET AL., 1972). Una carac- terística de esta microestructura (BY9GGILD, 1930; CARTER, 1990) es que la orientación de las unidades aciculares de aragonito (láminas de tercer orden), así como los paquetes que componen (láminas de segundo orden), cambian de orientación entre las láminas de primer orden adya- centes (Fig. 2D). Tanto la constitución en varias capas superpuestas, como la microestructura laminar cruzada, confieren a la concha de 1. gualtierianus morfotipo gualtierianus una gran resistencia mecánica. La superficie interna de la concha es prácticamente lisa (Fig. 2E), si bien en algunas zonas presenta suaves ondula- ciones (Fig. 2E), que pueden incluso llegar a constituir pequeños montículos o pro- tuberancias (Fig. 2F) y que parecen ser un reflejo suavizado de la ornamentación externa. También por zonas, la superficie interna de la concha está microesculpida con estriaciones muy finas (Fig. 2B). 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Scanning Electron Microscopy of molluscan shell ul- trastructures II. Observations of growth sur- faces. Transactions of the American Microsco- pical Society, 87: 419-430. Wise, S. W. JR., 1970. Microarchitecture and mode of formation of nacre (mother-of-pe- arl) in Pelecypods, Gastropods and Cepha- lopods. Eclogae Geologicae Helvetiae, 63: 775- 797. O Sociedad Española de Malacología —__——T— Iberus, 23 (1): 25-31, 2005 Changes in phospholipid and lipid peroxidation levels due to latex of Croton tiglium in freshwater snail Lymnaea acuminata Cambios en los niveles de peroxidación de fosfolípidos y lípidos por efecto del latex de Croton tiglium sobre el molusco dulceacuícola Lymnaea acuminata Digvijay SINGH*!, Ram P. YADAV* and Ajay SINGH* Recibido el 18-VII-2003. Aceptado el 13-1X-2004 ABSTRACT Exposure to 40% and 60% of LCso of 48h of the latex extract of Croton tiglium Linn (Euphorbiaceae) for 24h, 48h, 72h and 96h significantly reduced the endogenous levels of phospholipid and increased the rate of lipid peroxidation in brain and foot tissue of lymnaea (Radix) acuminata Lamarck. Alteration in the levels of phospholipid and rate of lipid peroxidation were time and dose dependent. It is concluded that latex extract of Cro- ton tiglium or its metabolites increase peroxidation of lipids in Lymnaea acuminata, which brings about change in membrane permeability to various ions and may thus be the cause of the toxicity of this plant extract. RESUMEN La exposición a LCso de 48 horas del extracto del latex de Croton tiglium al 40% y 60% durante 24, 48, 72 y 96 horas redujo significativamente los niveles de fosfolípidos e incrementó la tasa de peroxidación de lípidos en los tejidos cerebral y del pie de Lym- naea (Radix) acuminata Lamarck. Ambos cambios dependen tanto del tiempo como de la dosis. Esto confirma que dicho latex o sus metabolitos incrementa la peroxidación de lípi- dos en el molusco, por medio de cambios en la permeabilidad de la menbrana a varios iones. Esta puede ser la causa de la toxicidad del extracto de esta planta. KEY WORDS: Lipid peroxidation, phospholipids level, latex of Croton tiglium. PALABRAS CLAVE: Peroxidación de lípidos, niveles de fosfolípidos, latex de Croton tiglium. INTRODUCTION The snail Lymnaea acuminata is the SINGH AND SINGH, 2002; TRIPATHI AND vector of liver flukes Fasciola hepatica SINGH, 2003; SINGH AND SINGH, 2003). and Fasciola gigantica, which cause Heavy use of synthetic pesticides/ syn- endemic fascialiasis in cattle and liver thetic pyrethroids for the control of stock in northern parts of India (YADAV, aquatic vectors created other serious * Natural Product Laboratory, Department of Zoology, D.D.U. Gorakhpur University, Gorakhpur — 273 009 (U.P.) INDIA 'Corresponding author: Dr. Digvijay Singh. e-mail: digvijaysinOrediffmail.com P4S Iberus, 23 (1), 2005 problems to the aquatic environment (SASTRY AND SHUKLA, 1993; DEVI, 1997). Their hazardous nature has prompted scientists to find out non-disruptive, suitable and newer options for the control of weed and aquatic pests. In recent times, the use of natural pesti- cides has gained popularity all over the world. These plant products are the focus of attention as a suitable alterna- tive to synthetic pesticides due to some ideal properties i.e. low cost, easy avail- ability and biodegradability in nature (MARSTON AND HOSTETTIMAN, 1985; SINGH AND SINGH, 2002). A large number of plant pesticides are lethal to aquatic snails (GODAN, 1983; SINGH, SINGH, MISRA AND AGARWAL, 1996). SINGH AND AGARWAL, (1984 a, b) for the first time reported that the lattices of euphorbiales are highly toxic to snails. Later SINGH AND AGARWAL (1992; 1993 and 1995) reported several euphorbious species (falta algo :p.ej.species) as plant mollusicicides. It is known that the primary target of these plant molluscicides is the nervous system (SINGH AND AGARWAL, 1984a, b). It was observed that these plant molluscicides affect acetyl- choline, dopamine and non-epinephrine levels in nervous tissues of exposed animals (SINGH AND AGARWAL, 1995). Evi- dences for tissues injury induced by certain anti cholinesterase (Anti-ACHE) pesticides has been shown to be associated with increased fragility of various biological membranes and the structural lipids of biomemebranes undergo peroxidation decomposition (CHVAPIL, RYAN AND BRADA, 1972). YADAV AND SINGH (2001, 2002) reported that latex extract of Codiaeum variegatum and Croton tiglium have high molluscicidal and anti-cholinesterase activity against snails Lymnaea acuminata and Indoplanorbis exustus. In the present work the author is interested in knowing the sub-lethal effects of the latex extract of Croton tiglium on the endogenous level of phospholipids and rate of lipid peroxidation level in nervous and foot tissues of the freshwa- ter snail Lymnaea acuminata to see if it can serve as effective molluscicides. 26 MATERIAL AND METHODS Adult Lymnaea acuminata (2.4+0.4 cm in length) were collected from local ponds and acclimatized in the laboratory for 96h in dechlorinated tap water. Batches of 50 snails were kept in 12L glass aquaria con- taining 6L-dechlorinated tap water. Snails were treated according to methods of SINGH AND AGARWAL (1986). Experiments were carried out at 24-26 *C. No food was given during the course of the experiment. There was no mortality in either controls or experiments. Oxygen concentration was normal in control animals as there was no sign of oxygen deficiency stress. 40 snails / 5L water were used which caused no oxygen stress in test snails. The snails were exposed to 40% (0.016 mg/L) and 60% (0.024 mg/L) of 48h LC5o of 0.04 mg/L for 24h, 48h, 72h and 96h. These doses were based on 48h LCso values reported by YADAV AND SINGH, (2001). After 24h, 48h, 72h or 96h the snails were removed from the aquaria and rinsed in water. Control groups were kept in de-chlorinated tap water without any treatment. Endogenous levels of phospholipids were determined in nervous and foot tissue by the method Of FISKE AND ROW, (1925) as modified by DITTMER AND MICHAEL (1965), and mod- ified for snails by SINGH, SINGH AND AGARWAL (1993). The rate of lipid perox- idation was determined in nervous and foot tissues by the methods of (UTELY, BERMHEIM AND HOCHTEIN, 1967). Phospholipids levels estimation: Brain and foot tissue were dissected out and placed on filter paper for removal of adherent water. 300 mg each of brain and foot tissues were used for each replicate. Brain tissue was pooled from eighty five snails. The tissues were then homogenized in chloroform/methanol mixture (2:1v/v) using a Teflon pestle to give a 5% w/v homogenate . Supernatants were filtered and two drops of 0.05M NaCl solutions was added to the supernatant and left for 24h. After 24h the upper layer was dis- carded. 1 mL of lipid layer was placed in a test tube and solvent was removed by heating in a boiling water bath. 1 mL of distilled water and 0.4 mL of 10% trichloroacetic acid was added to this and SINGH ET AL.: Latex of Croton tiglium changes lipid peroxidation in Lymnaea acuminata Table 1. /n vivo effect of exposure to 40% and 60% of 48h LCso of latex of Croton tiglium on the levels of phospholipids in the nervous and foot tissues of snail L. acuminata after exposure to 24h, 48h, 72h and 96h. Values are mean +SE of six replicates. Values in parenthesis indicate % of control. Student test *t” test demonstrated that the treated values were significantly different from control (P<0.05). Two-way analysis of variance demonstrated that the changes were dose and time dependent. 300 mg nervous and foot tissues were taken for all estimations. Table 1. Efecto in vivo de la exposición al 40% y 60% de 48h LC5o del latex de Croton tiglium en los niveles de fosfolípidos en los tejidos nervioso y del pie de L. acuminata tras 24h, 48h, 72h y 96h de exposición. Los valores son la media +SE de 6 réplicas. Valores en paréntesis indican % del control. El test “t” de Student muestra que los valores tratados son significativamente diferentes de los del control (P<0.05). El análisis de varianza de dos vías demuestra que los cambios dependen de la dosis y el tiempo. Se usaron 300 mg de tejido nervioso y del pie en todas las estimaciones. Tissues 40% of LCso (48h) 60% of LCso (48h) Control Broin 23.26+0.15 (100) 23.2640.15 (100) Foot 21.01%0.31 (100) 21.0140.31 (100) 24h Brain 17.45+0.18 (62.12) 13.26+0.22 (57.00) Foot 18.00+0.18 (85.67) 16.30.17 (77.58) 48h Brain 14.19+0.39 (60.99) 13.520.7 (51.52) Foot 14.95+0.26 (71.15) 13.22+0.19 (62.92) 72h Brain 13.02+0.19 (55.96) 11.00+0.25 (47.29) Foot 11.29+0.33 (53.73) 10.64+0.39 (50.64) 96h Brain 9.2+0.23 (39.55) 8.67+0.20 (37.27) Foot 9.63+0.34 (45.83) 8.23+0.28 (39.317) heated. After this, 0.4 mL of 2.5 % Ammo- nium molybdate solutions and 0.2 mL of amino naphthosulphonic acid was added and the mixture heated at 80*C for 15 min, cooled and diluted with 4 ml distilled water. After 5 minutes the absorbance was read at 640 nm with the help of a spec- trophotometer (No. 106 S.No. 3265 Sys- tronics). The blank consisted of 1 mL dis- tilled water, 0.4 mL 10% dichloroacetic acid, 0.4 mL 2.5% ammonium molybdate solution, 0.2 mL of aminonaphthosul- phonic acid and 4.0 mL distilled water but no tissue homogenate. Standards curves were prepared by reading the absorbance of different concentrations of monopotas- sium phosphate solutions containing 0.008 to 0.04 mg of pure salt. Lipid peroxidation estimation: Brain and foot tissues (100 mg each) were homoge- nized in chilled 0.15 M KCI solution using a Teflon pestle to give a 10% w/v homogenate. 1 ml aliquots of the homogenate were incubated at 37*C for 2h, following which 1.0 ml of 10% (w/v) trichoroacetic acid was added. After thor- ough mixing, the mixture was centrifuged at 2200g for 10 min and 1ml of supernatant was then taken with an equal volume of 0.67% (w/v) 2-thiobarbituric acid (Sigma Chemical Company, USA) and kept in a boiling water bath for 10 min, cooled and diluted with one ml of distilled water. The absorbance read at 535 nm against a blank containing only KCl solution and results were expressed as mol of malonal- dialdehyde formed per 30 min /mg tissue. The extinction coefficient was 1.56x105 as described by UTELY ET AL., (1967). The methods of SOKAL AND ROHLF (1973) were applied for analysis of students “t” test and two-way analysis of variance (P<0.05). RESULTS The endogenous levels of phospho- lipids in the brain and foot tissues were 23.26 and 21.01 mg/g, respectively. In vivo 24h, 48h, 72h and 96h exposure to 27 Iberus, 23 (1), 2005 Table IL. Zn vivo effect of exposure to 40% and 60% of 48h LCso of Croton tiglium on the rate of lipid peroxidation in the nervous and foot tissues of snail L. acuminata after exposure to 24h, 48h, 72h and 96h. Values are mean +SE of six replicates. Values in parenthesis indicate % of control. Student test “t” test demonstrated that the treated values were significantly different from control (P<0.05). Two-way analysis of variance demonstrated that the changes were dose and time depen- dent. 600 mg nervous and foot tissues were taken for all estimations. Table 11. Efecto in vivo de la exposición al 40% y 60% de 48h LC5o del latex de Croton tiglium en los niveles de fosfolípidos en los tejidos nervioso y del pie de L. acuminata tras 24h, 48h, 72h y 96h de exposición. Los valores son la media +SE de 6 réplicas. Valores en paréntesis indican % del control. El test “t” de Student muestra que los valores tratados son significativamente diferentes de los del control (P<0.05). El análisis de varianza de dos vías demuestra que los cambios dependen de la dosis y el tiempo. Se usaron 600 mg de tejido nervioso y del pie en todas las estimaciones. Tissues 40% of LCso (48h) 60% of LCso (48h) Control Brain 2.49+0.24 (100) 2.49+0.24 (100) Foot 1.61+0.06 (100) 1.61%0.06 (100) 24h Brain 3.00+0.07 (120.48) 3.33+0.13 (133.73) Foot 1.89+0.02 (117.39) 2.41+0.04 (149.68) 48h Brain 3.28+0.065 (131.72) 3.61+0.053 (144.97) Foot 2.01+0.06 (124.84) 2.64+0.05 (163.97) 12h Brain 3.64+0.060 (146.18) 4.19+0.058 (168.27) Foot 3.16+0.039 (196.27) 3.75+0.11 (232.91) 96h Brain 5.18+0.04 (208.03) 6.42+0.07 (257.83) Foot 4.73+0.17 (293.78) 6.40%0.30 (397.51) 40% and 60% LCso (48h) of C. tiglium caused a significant change in levels of phospholipids (Table I) and activity of lipid peroxidation (Table II) in the nervous and foot tissues of the snails. Treatment with 40% LCso of latex extract of C. tiglium for 24h, 48h, 72h and 96h decreased the levels of phospholipids in the brain tissues up to 62.1, 60.99, 55.96 and 39.55 % of the control, respectively (Table I). Likewise in foot tissue, it decreased to 85.67, 71.15, 53.73, and 45.83 % of the control respectively (Table ID). With exposure to a higher dose i.e. 60% of 48h LCso for 24h, 48h, 72h and 96h, there was a further decrease in the endogenous levels of phospholipids (Table ID. The rate of lipid peroxidation in the brain and foot tissues of control snails was 2.53 and 1.66 mol malonaldialde- hyde formed/ 30min/ mg, respectively. Exposure to the two doses of lattices of C. tiglium increased the rate of lipid per- oxidation in both the nervous and foot tissues. Exposure to 40% of LC5o for 24h, 28 48h, 72h and 96h increased this to 120.48, 131.72, 146.18 and 208.03 % of control in brain tissue and to 117.39, 124.84, 196.27 and 293.78 % of control in foot tissue, respectively. Exposure to 60% of LCso (48h) caused a greater increase in the rate of lipid peroxidation (Table ID). Analysis of variance demon- strated that the increase in the rate of lipid peroxidation was time as well as dose dependent (P<0.05). DISCUSSION The data obtained indicate that exposure of Lymnaea acuminata to latex of C.tiglium, increased the rate of lipid per- oxidation along with decrease in the endogenous levels of phospholipids in the nervous and foot tissue of the snail. That these effects were indeed caused by lattices of C. tiglium is evidenced by the fact that both changes were time as well as dose dependent. SINGH ET AL.: Latex of Croton tiglium changes lipid peroxidation in Lymnaea acuminata Biological membranes are known to be rich in polyunsaturated fatty acids. In fact, lipids may account for 28% to 79% of the mass of cell membranes. While the inner mitochondrial membrane con- tains about 60% proteins and only 20% lipids, the myelin sheath of the brain contains as much as 80% lipids and only 20% protein. Most snail membranes, however, contain nearly equal amounts of protein and fat. Membrane lipids are mainly phospholipids and most of them are polyglycerides. Up to 20% of the phospholipids are acidic which are neg- atively charged and are associated with membrane proteins due to lipid protein interaction (LEHNINGER, 1987). Several workers reported that syn- thetic / plant pesticides cause repetitive discharges due to prolongation of sodium current and increased depolarization (NARAHASHI, 1983; UPRETI ET AL, 1991; SIDDIQUI, SAYEED, ZAFAR AND ISLAM, 2002). The site of action is most likely to be within the lipophilic environment of the membrane in the neighbourhood of sodium proteins (OSBORNE AND SMALL- COMBE, 1983; PATEL, FULLONE AND ANDERS, 1992). Any change in the struc- ture of the concentration of lipids would radically alter the structure of membranes, bringing about a change in their perme- ability characteristic. Indeed, lipid perox- idation has been reported to play an important role in a wide variety of patho- logical and degradative conditions (Bus AND GIBSON, 1979). During lipid peroxidation, oxygen reacts with polyunsaturated lipids to form lipid radicals and semistable hydro- peroxides. During this process hydrogen is abstracted from a carbon-carbon bond of unsaturated lipid. This process requires an initiator radical, which may BIBLIOGRAPHY Bus, J. S. AND GIBSON, J. E., 1979. Lipid perox- idation and its role in toxicity. In: Hodgson, E., Bend, J. R. and Philpot, R. M. (eds) Review in biochemical Toxicology 1: 125-149. come from the metabolic degradation of an external agent (TAPPEL, 1973; ZIA AND IsLam, 2000). According to BUS AND GIBSON (1983) certain classes of xenobi- otics such as parquets and inorganic sub- stances such as carbon tetrachloride lose a single electron but under aerobic condi- tions get re-oxidized immediately to their original compound with the concurrent formation of a super-oxide radical. Toxic- ity of these compounds is due to their ability to initiate the generation or induc- tion of this superoxide anion radical. A similar mechanism may be possible in the case of L. acuminata treated with lat- tices of C. tiglium. Formation of activated oxygen can have extremely detrimental consequences not only for phospholipids but also for proteins, nucleic acids and polysaccharides (BUS AND GIBSON, 1983; SINGH ET AL., 1996). Even through the mechanism of formation of superoxide in the case of C. tiglium is not known yet, it is possible that the metabolic degradation of this extract produces an initiator organic radical which is capable of start- ing the peroxidation reaction. It is clear that the latex extract of Croton tiglium is highly effective at a lower dose and may eventually prove to be a very useful mol- luscicide .. Their effectiveness / attractive- ness as molluscicides in comparison to synthetic pesticides lie in their plant origin, which makes them inexpensive and more easily biodegradable and safe for non-target organismos. ACKNOWLEDGEMENTS One of the authors (Digvijay Singh) is thankful to CSIR, New Delhi (San. Letter No. 9/57 (163)/2K2/ EMR - I dated 4.9.2002) for financial support. Bus, J. S. AND GIBSON, J. E., 1983. Oxygen acti- vation and lipoperoxidative metabolism of toxicity of pesticides and other xenobiotics. In: Miyamoto, J. and Kerney, P. C. (eds) Pes- ticides Chemistry. Human welfare and Envi- ronment. Pergmon Press, London € New York, 3 Pp-457 - 62. 29 Iberus, 23 (1), 2005 CHVAPIL, M., RYAN, J. N. AND BRADA, Z., 1972. Effect of selected chelating agents and met- als on stability of liver lÍysosome. Biochemistry and Pharmacology 21:1097-1105. Devi U., 1997. Heavy metal toxicity to gastro- pod, Morula granulata: tolerance to copper, mercury, zinc and cadmium. Journal of En- vironmental Biology 18 (3): 287-290. DITTMER, J. C. AND MICHAEL, A. W., 1965. Quantitative and qualitative analysis of lipids and lipid components. In: methods of Enzymology. Colowick, S. P. and Kaplam, N. O. (eds). Vol XIV. Academic Press. Pp - 483. FiskE, C. H. AND ROW, Y., 1925. Quantitative and qualitative analysis of lipids and lipid com- ponents. In: methods of Enzymology. Colow- - ick, S. P. and Kaplam, N. O. (eds). Vol. XIV. Academic Press. Pp 483. GODAN, D., 1983. Pest sludge and snail, Biology and control (ed., Dora Godan) translated by Sheila Gruber, Springer verlag, Berlin, Hei- delberg, New York. LEHNINGER, A. L., 1987. Principles of Bio- chemistry. 2nd Indian reprints. CBC Pub- lisher and Distributors Delhi Pp - 1011 MARSTON, A. AND HOSTETTMANN, K., 1985. Plant molluscicide. Phytochemistry 24: 639- 652. NARAHASHI, T., 1985. Nerve membrane ionic channels as the primary target of pyrethroids. Neuro toxicology 6: 3-22. OSBORNE, P. AND SMALLCOMBE, A., 1983. Site of actions of pyrethroid insecticides In: Miyamoto, J. and Kerney, P. C. (eds). Human welfare and Environment. Pergmon Press, London and New York Pesticides Chemistry NON Z: PATEL, N., FULLONE, J. AND ANDERS, M. W., 1992. Brain uptake and metabolism of S-(1,2- dichlorovenyl) glutathione (DCVG) and S- (1,2-Dichlorovinyl)-L cystine (DCVC). Toxi- cology 12: 343-346. SASTRY, K. V. AND SHUKLA, V., 1993. Uptake and distribution of cadmium in tissue of Channa marulius. Journal of Environmental Biology 14 (2): 137 - 142. SIDDIQUI, A., SAYEED, L, ZAFAR, K. S. AND ISLAM, F., 2002. Argemone oil augmented oxidative stress in discrete areas of rat brain. Bulletin of Environmental Contamination and Toxicology 69: 734 - 740. SINGH, A. AND AGARWAL, R. A., 1992. Mollus- cicidal activity of euphorbiales against the snail Indoplanorbis exustus. Acta Hydrochimica et Hydrobiogica 20: 262- 264. SINGH, A., SINGH, D. K. AND AGARWAL, R. A., 1993. Effect of cypermethrin, mexacarbate and phorate on phospholipid and lipi- deroxidation in the snail Lymnaea acuminata. Bulletin of Environmental Contamination and Toxicology 51: 68-71. 30 SINGH, A. SINGH, D. K., MISRA, T. N. AND AGAR- WAL, R. A., 1996. Molluscicides of plant or- gin. Biological Agriculture and Horticulture 13: 205-252. SINGH, D. AND SINGH, A., 2002. Piscicidal effects of some common plants of India used in freshwater bodies against target animals. Chemosphere 49 (1): 45-49. SINGH, D. K. AND AGARWAL, R. A., 1986. Piper- onyl butaoxide synergism with two synthetic pyrethroids against Lymnaea acuminata. Chemosphere 15: 493-498. SINGH, D. K. AND AGARWAL, R. A., 1984a. Al- teration of biogenic level in the snail Lymnaea acuminata by the latex of Euphorbia royleana. Toxicological Letters 21: 309-314. SINGH, D. K. AND AGARWAL R. A., 1984b. Cor- relation of the anti-cholinesterase and mol- luscicidal activity of the latex of Euphorbia royleana Bioss. on Lymnaea acuminata. Journal of Natural Product 47: 702-705. SINGH, A. AND AGARWAL, R. A., 1990. Mollus- cicidal and Anti-Cholinesterase activity of euphorbiales. Biological Agriculture and Hor- ticulture 7: 81-91. SINGH, D. K., SINGH, A. AND AGARWAL, R. A., 1993. Nerium indicum as a potent molluscicide of plant origin. Journal of Medical and Applied Malacology 5: 93-95. SINGH, K. AND SINGH, D. K., 1995. Effect of Azadirachta indica (Neem) on biochemical pa- rameters in ovotestis of Lymnaea acuminata. Malaysian Applied Biology 24: 7-11. SINGH, S. K. AND SINGH, A., 2003. Effect of the plants Thevetía peruviana and Alstonia schol- aris (Family: Apocynaceae) on acetyl- cholinesterase activity of Lymnaea acuminata snails. Egyptian Journal of Schistosomiasis In- fectious Ey Endemic Disease 25: SOKAL, R. R. AND ROHLE, F. J., 1973. Introduc- tion to bio-statistic edited by Freeman, M. N., San Francisco. Pp. 368. TAPPEL, A. L., 1973. Lipid peroxidation damage to cell component. Federation Proceedings 32: 1870-1874. TRIPATHI, P. K. AND SINGH, A., 2003. Toxic ef- fects of dimethoate and carbaryl pesticides on protein metabolism of freshwater snail Lymnaea acuminata. Bulletin of Environmental Contamination Toxicology 70: 146-152. UPRETIL, K. K., DAS, M. AND KHANNA, $. K., 1991. Biochemical toxicology of Argemone oil: effect on hepatic cyt P-450 and Xenobi- otic metabolizing enzymes. Journal of Applied Toxicology 11: 203-209. UTELY, H. C., BERMHEIM, F. AND HOCHTEIN, P., 1967. Effects of sulfhydryl reagent on per- oxidation in microsome. Achieves of Bio- chemistry and Biophysics 188: 29-32. YADAV, R. P. AND SINGH, A., 2001. Environ- mentally safe molluscicide from two common Euphorbiales. Iberus 19 (1): 65-73. SINGH ET 4L.: Latex of Croton tiglium changes lipid peroxidation in Lymnaea acuminata YADAV, R. P. AND SINGH, A., 2002. Toxic effect ZIA, S. AND ISLAM, F., 2000. Selenium altered the of Croton tiglium on L. acuminata and non-tar- levels of lipids, lipid peroxidation and get fish Channa punctatus. Iberus 20 (2): 31-44. sulfhydryl group in straitum and thalamus YADAV, R. P., SINGH, S. K. AND SINGH, A., 2002. of rat Biology of Trace Element Research 77: Molluscicidal activity of Codiaeum variega- 251-259. tum, effects on snail metabolism. Journal of Ecophysiology and Occupational Health 2: 73 - 84. 3] -. => sata AOL dl Ú A ARCO rapri a EN he 4 AE An, Man p A h R y ne CERA a añ , d h p 1 4d Ad 4 : 00 vir A nO , a ¡UH Y V 4 O Sociedad Española de Malacología —__—_——T— Iberus, 23 (1): 33-42, 2005 Cephalopod assemblages caught by trawling along the Southern Tyrrhenian Sea (Central Mediterranean) Asociaciones de cefalópodos capturados por la pesca de arrastre comercial en el Tirreno meridional (Mediterráneo central) Daniela GIORDANO*, Teresa BOTTARI* and Paola RINELLI* Recibido el 26-11-2004. Aceptado el 27-IX-2004 ABSTRACT The teuthofauna assemblages of the Southern Tyrrhenian Sea are identified utilising data from five trawl surveys. A total of 139 hauls were carried out in late spring or the middle of sum- mer between 1995 and 1999, and 25 cephalopod species were found between 18 and 652 m of depth. The assemblages were analysed with the Bray-Curtis similarity index. Depth showed a positive correlation with cephalopod distribution. Four main cephalopod groups were defined: inshore (10-100 m), shelf (101-200 m), slope (201-600 m) and midslope (350- 600 m). Loligo media was the main species for the first group, lllex coindetii for the second, Todaropsis eblanae for the third group and Todarodes sagittatus for the last group. RESUMEN Se identifican las asociaciones de teutofauna capturadas por los arrastreros comerciales en el Tirreno meridional. Se analiza la fauna de cefalópodos recogida en 139 lances realiza- dos en primavera o mediados del verano durante cinco campañas de pesca de arrastre rea- lizadas entre 1995 y 1999. Se identificaron 25 especies de cefalópodos capturadas entre 18 y 652 m de profundidad. El análisis de las asociaciones faunísticas se realizó mediante el índice de similitud de Bray-Curtis. La profundidad tiene una correlación positiva con la distribución de los cefalópodos, pudiéndose definir cuatro grupos principales: costero o lito- ral (10-100 m), de la plataforma (101-200 mj), del talud (201-600 m) y del talud medio (350-600 m). Loligo media fue la principal especie del primer grupo, lllex coindetiidel segundo, Todaropsis eblanae del tercero, y Todarodes sagittatus del último grupo. KEY WORDS: Biogeography, cephalopods, trawl fishery, Tyrrhenian Sea, faunal assemblages, Mediterranean Sea. PALABRAS CLAVE: Biogeografia, cefalópodos, pesquería de arrastre, asociación faunistica, Mar Tirreno, Mediterráneo. INTRODUCTION The geographic and bathymetric dis- QUETGLAS AND SÁNCHEZ, 1998; SORIANO, tributions of demersal cephalopods have SÁNCHEZ LIZASO AND GUERRA, 2003; VIL- been studied in detail in different Mediter- LANUEVA, 1995); the coasts of Libya and ranean areas: Western Mediterranean Sea Tunisia (BONNET, 1973); Marmara Sea (SARTOR, BELCARI, CARBONELL, GONZÁLEZ, (KATAGAN, SALMAN AND BENLI, 1993; * Istituto per Ambiente Marino Costiero IAMC-CNR, Messina Section, Spianata San Raineri 86, 98122 Messina, Italy. Corresponding author: Daniela Giordano. E-mail: daniela.giordanoCiamc.cnr.it 33 Iberus, 23 (1), 2005 UNSAL, UNSAL, ERK AND KABASAKAL, 1999); Eastern Mediterranean Sea (RUBY AND KNUDSEN, 1972; KNUDSEN, 1981; SALMAN, KATAGAN AND BOLETZKY, 1999); Ligurian Sea (RELINI, DE RossI, PIANO AND Z_AAMBONI, 2002; BERTULETTI AND ORSI RELINI, 1986; ORSI RELINI AND BERTULETTI, 1989; WURTZ, 1979; OrSI RELINI, 1995); Sar- dinian Sea (BONNET, 1965; CUCCU, ADDIS, DAMELE AND MANFRIN PICCINETTI, 2003); Tyrrhenian Sea (BERDAR, POTOSCHI, CAV- ALLARO, CAVALIERE AND LI GRECI, 1983; BERDAR AND CAVALLARO, 1975; LUMARE 1968, 1970; BELCARI, BIAGI, BIAGI, DE RANIERI, MORI AND PELLEGRINI, 1986; MANNINI AND VOLPI, 1989; BELCARI AND SARTOR, 1993; BELCARI, SARTOR AND DE RANIERI, 1998; WURTZ, MATRICARDI AND BELCARI, 1992; SPEDICATO, MINETTI, REBORA, MATRICARDI AND WURTZ, 1990; GIORDANO AND PERDICHIZZI, 1998; BELLO AND ARCULEO, 1994; GIORDANO AND CAR- BONARA, 1999); Sicilian Channel (JEREB AND RAGONESE, 1991, 1994; RAGONESE, JEREB AND DI STEFANO, 1992); Adriatic Sea (BELLO AND MOTOLESE, 1983; GUESCINI AND PICCINETTI MANERIN, 1986; GAMULIN- BRIDA AND ILIJANIC, 1972; CASALI, MANFRIN PICCINETTI AND SORO, 1998; BELLO, 1990; PASTORELLI, VACCARELLA AND DE Zio, 1995); lonian Sea (PANETTA, D'ONGHIA, TURSI AND CECERE, 1986; 'TURSI AND D'ONGHIA, 1992; D'ONGHIA, MATAR- RESE, TURSI, MAIORANO AND PANETTA, 1995); Catalan Sea (MANGOLD-WIRZ, 1963; SÁNCHEZ , BELCARI AND SARTOR, 1998); and Aegean Sea (D'ONGHIA ET AL., 1995; SALMAN, KATAGAN AND BENLI, 2002). In particular, assemblages of cephalopod communities have been studied in different areas of this basin (UNGARO, MARANO, MARSAN, MARTINO, MARZANO, STRIPPOLI AND VLORA, 1999; TSERPES, PERISTERAKI, POTAMIAS AND TSIMENIDES, 1999; (GONZÁLEZ AND SÁNCHEZ, 2002; QUETGLAS, CARBONELL AND SÁNCHEZ, 2000). Information about spatio-temporal distributions of these species and their linkage to environ- mental factors is necessary for the study of many commercial species. The studies focused on demersal assem- blages and in particular on their persis- tence, such as the capability to maintain 34 the same specific composition during a lag time are useful even for manage- ment purposes (BIAGI, SARTOR, ARDIZ- ZONE, BELCARI, BELLUSCIO AND SERENA, 2002). In this respect, this work analyses the structure of cephalopod assem- blages, providing information concern- ing their distribution for all four groups defined: inshore, shelf, slope and mids- lope. MATERIAL AND METHODS The study area is situated in the Southern part of the Tyrrhenian Sea (central Mediterranean) between Suvero Cape (Calabria) and San Vito Cape (Sicily). The data here reported come from five trawl surveys carried out from 1995 to 1999, during the MEDITS project (International bottom trawl survey in the Mediterranean Sea). The experimen- tal surveys were always conducted during late spring or the middle of summer. Hauls were carried out from one hour before dawn until one hour after sunset. A fishing vessel equipped with an experimental trawl net with 20 mm stretched mesh size in the cod-end, and 2-2.5 m of vertical opening (FIOREN- TINI, COSIMI, SALA, LEONORI AND PALOMBO, 1999) was used. A total of 139 hauls were carried out, randomly allo- cated into five bathymetrical strata: “A”: 10-50 m; “B”: 51-100 m; “C”: 101-200 m; “D”:> 201-500. m; +*E*%%9 5UESO0B (BERTRAND, GIL DE SOLA, PAPACONSTAN- TINOU, RELINI AND SOUPLET , 2002). The duration of each haul was 30 minutes at depths less than 200 m and one hour at greater depths. All the data were standardised to one hour haul duration. All cephalopods were identi- fied and counted on board. A multivari- ate approach, on the basis of the calcula- tion of a triangular similarity matrix (group-average linkage), by depth, year and abundance (number of speci- mens/hour), according to BRAY AND CURTIS (1957), with relative dendrogram and non metric plan Multi Dimensional Scaling (MDS) was elaborated (CLARKE GIORDANO £7 AL.: Cephalopod assemblages along the Southern Tyrrhenian Sea 0 Bray-Curtis Similarity 20 40 60 80 100 AAA AA 100 m 200 m litoral shelf slope Figure 1. Dendrogram showing similarities between hauls for the 1996 surveys. Mean depth of each haul is presented. Figura 1. Dendrograma que muestra las similitudes entre los arrastres de 1996. Se indica la profundi- dad media de cada arrastre. AND WARWICK, 1994). Because the annual variations of cephalopod distrib- ution were not significant, the five surveys were pooled together to calcu- late the total cluster. Hauls in which only one species was caught and the species with abundance values lower than 3 were not considered for the analysis. To establish which taxa con- tributed most to the separation of one group from another, the SIMPER routine was used (CLARKE, 1993). Abundance values were also analysed by means of univariate indices, in relation to the four assemblages evidenced by cluster analy- sis: total mumber of taxa (S), total number of individuals (N), richness of Margalef (d), Shannon-Wiener diversity (H”) and Pielou's evenness (J) indices. The cumulative abundances were illus- trated in order of dominance as K-domi- nance curves. RESULTS Twenty-five species of cephalopods were collected belonging to three orders and eight families. In Table I the system- atic list of species, drawn up according to BELLO (1986), within the bathymetri- cal range of each species, is reported. In particular, Onychoteuthis banksii, Ancistroteuthis lichtensteini and Abralia veranyi were caught only once during the period studied. The cephalopods collected were dis- tributed between 18 and 652 m of depth. The more coastal species was repre- sented by Sepia officinalis, confined within the depth of 68 m. Fifty percent of the species showed a wide bathymet- rical range that included the shelf and the beginning of the slope. The widest distribution was shown by Eledone cir- rhosa (72-584 m), Scaeurgus unicirrhus (38-549 m), Todaropsis eblanae (61-613 m) and Pteroctopus tetracirrhus (118-633 m). Alloteuthis subulata, Octopus vulgaris and Eledone moschata were found only on the continental shelf. On the contrary, Octopus salutii and Rossia macrosoma were caught at over 200 m of depth. His- tioteuthis bonnellii was found exclusively in the deepest stratum. The dendrogram of all surveys (Fig. 1) showed three principal clusters: l, inshore (<100 m); IL, shelf (80 — 200 m) and III, slope (200 — 600 m). The coastal group was characterized by Loligo media 39 Iberus, 23 (1), 2005 Table I. Cephalopod species captured from 1995 to 1999 off the Southern Tyrrhenian Sea, showing their depth range and mean depth of occurrence with the associated standard deviation (SD). Tabla 1. Especies de cefalópodos capturadas desde 1995 a 1999 en el mar Tirreno meridional, indicando su rango de distribución batimétrica, profundidad media y la desviación estándar asociada (SD). Min Max Media SD Class CEPHALOPODA Cuvier, 1798 Subclass COLEOIDEA Bather, 1888 Order SEPIOIDEA Naef, 1916 Family Sepiidae Leach, 1817 Genus Sepia Linnaeus, 1758 Sepia officinalis Linnaeus, 1758 40 68 495 12.66 Sepia orbignyana Férussac in Orbigny, 1826 61 432 185.5 96.87 Sepia elegans Blainville, 1827 40 345 137,3 14.02 Family Sepiolidae Leach, 1817 Subfamily Sepiolinae Leach, 1817 Genus Sepiola Leach, 1817 Sepiola rondeletii Leach, 1817 110 395 276 115 Genus Rondeletiola Naef, 1921 Rondeletiola minor (Naef, 1912) 117 297 207 127.28 Subfamily Rossiinae Appellof, 1898 Genus Rossia Owen in Ross, 1835 Rossia macrosoma (Delle Chiaie, 1830) 2d 596 484.5 115.56 Genus Neorossia Boletzky, 1971 Neorossia caroli (Joubin, 1902) 123 608 309 195:9 Order TEUTHOIDEA Naef, 1916 Suborder MYOPSIDA Orbygny in Férussac 8. Orbigny, 1840 Family Loliginidae Lesueur, 1821 Genus Loligo Lamarck, 1798 Loligo vulgaris Lamarck, 1798 18 29 75.72 12 Loligo forbesi Steenstrup, 1856 12. 505 134.2 127.3 Loligo media (Linnaeus, 1758) 18 E al 15.43 Genus Alloteuthis Wulker, 1920 Alloteuthis subulata (Lamarck, 1798) 16 143 109,5 47.4 Suborder OEGOPSIDA Orbigny, 1845 Family Enoploteuthidae Pfeffer, 1900 Subfamily Enoploteuthinae Pfeffer, 1900 Genus Abralia Gray, 1849 Abralia veranyi (Ruppell, 1844) 310 ES6 Family Onychoteuthidae Gray, 1847 Genus Onychoteuthis Lichtenstein, 1818 Onychoteuthis banksii (Leach, 1817) 358 358 Genus Ancistroteuthis Gray, 1849 Ancistroteuthis lichtensteinii (Férussac, 1839) 562 562 Family Histioteuthidae Verrill, 1881 Genus Histioteuthis Orbigny, 1840 Histioteuthis bonnellii (Férussac, 1834) 53] 645 565.5 53.33 Histioteuthis reversa (Verrill, 1880) 383 652 565.3 89.4 Family Ommastrephidae Steenstrup, 1857 Subfamily Illicinae Posselt, 1890 Genus /llex Steenstrup, 1880 Illex coindetii (Verany, 1839) 60 NES 128.7 36 GIORDANO ET AL.: Cephalopod assemblages along the Southern Tyrrhenian Sea Table I. Continuación. Tabla I. Continuation. Genus Jodaropsis Girard, 1890 Min Max Media SD Todaropsis eblonae (Ball, 1841) 61 0135.3235] 152.2 Sufamily Todarodinae Adam, 1960 Genus Jodarodes Steenstrup, 1880 lodarodes sagittatus (Lamarck, 1798) 114 593 438.5 Order OCTOPODA Leach, 1818 Suborder INCIRRATA Grimpe, 1916 154.69 Family Octopodidae Orbigny in Férussac 8. Orbigny, 1840 Subfamily Octopodinae Orbigny in Férussac 8. Orbigny, 1840 Genus Octopus Cuvier, 1798 Octopus vulgaris Cuvier, 1798 Octopus salutii Vérany, 1836 Genus Scaeurgus Troschel, 1857 27 114 62.86 25.85 MIL ESTA 10.87 Scaeurgus unicirrhus (Delle Chiaje in Férussac 8. Orbigny, 1840) 00 549 1747 110.8 Genus Pteroctopus P. Fischer, 1882 Pteroctopus tetracirrhus (Delle Chiaje, 1830) UA A 147.83 Subfamily Eledoninae Grimpe, 1921 Genus Eledone Leach, 1817 Eledone moschata (Lamarck, 1798) 18 143 115.4 92.82 Eledone cirrhosa (Lamarck, 1798) IDEA ZOO OY and Loligo vulgaris. In the shelf group (Table lla) Illex coindetíí and Loligo media prevailed, followed by Sepia orbignyana, Sepia elegans and Scaeurgus unicirrhus. The slope group (Table Ilb) was charac- terized by Todaropsis eblanae. The Multi- dimensional Scaling analysis (30%: Stress 0.15) also showed four groups, well discriminated by depth (Fig. 2). There is also a midslope group (Fig. 3), limited to a few hauls, in which Todaro- des sagittatus was the prevalent species. Analysing the univariate indices (Table IMM), elaborated for the four groups, the highest biodiversity was observed in the shelf. In this macrostra- tum, the highest values of Margalef and Shannon-Wiener indices were recorded as well as the highest number of species and individuals. The highest values of H' found at this bathymetrical level can be explained by the relative homogene- ity in abundance of the most frequent species. However, a trend was observed according to the depth as evidenced also by the cumulative abundance curve, in which the shelf group showed the highest biodiversity and the midslope the lowest. In this last group in fact both d and H' values recorded the lowest values (0.973 and 1.063, respectively). Finally the Evenness (J') values ranged from 0.616 to 0.661. The higher value was recorded for the last cluster. Also in the second cluster this value is quite high. DISCUSSION AND CONCLUSIONS The 25 cephalopod species distrib- uted in 8 families recorded in this study account for 42.4% of species reported from the Mediterranean (MANGOLD AND BOLETZKY, 1988; GUERRA, 1992). Absent were certain species of the Sepiolidae family and pelagic species. In compari- 37 Iberus, 23 (1), 2005 Table Ia. Indicator species and related data from the SIMPER analysis. Abbreviations: AA, average abundance, contribution of each species in each group; AT, average term, average Bray-Curtis con- tribution of each species to distinguish between groups. The ratio (AT/SD), the percentage contri- bution to the separation (%), and the cumulative percentage (Cum %) are shown for each group comparison. Tabla lla. Especies indicadoras y datos relacionados, según el análisis SIMPER. Abreviaturas: AA, abundancia promedio, contribución de cada especie en cada uno de los grupos; Average Term (AT), con- tribución promedio de Bray-Curtis de cada especie en la diferenciación de los grupos. Cada grupo viene definido, además, por la relación AT/SD (ratio), la contribución porcentual (%), y el porcentaje acumu- lativo de cada especie (Cum %). Average dissimilarity: 84.1 inshore AA shelf AA l. vulgaris 106.88 0.90 [. coindetii 0.58 26.62 L. vulgaris 18.88 0.02 S. elegans 0.05 0.41 S. orbignyana 0.00 8.86 S. unicirrhus 0.00 0.29 E. cirrhosa 0.05 0.13 Average dissimilarity: 99 inshore AA shelf AA L. media 106.88 + 0.00 L. vulgaris | 18.88 0.00 T. eblanae 0.00 0.46 P tetracirrhus 0.00 2.74 0. vulgaris 0.09 0.00 S. orbignyana 0.00 2.89 l. coindetii 0.04 1.67 E. moschato 2.00 0.19 Average dissimilarity: 92.7 inshore AA shelf AA L. media 21.43 0.00 l. coindetii 26.62 1.67 S. elegans 757 0.00 S. orbignyana 8.86 2.89 T. eblanae 1.14 10.56 S. unicirrhus 6.54 0.85 E. cirrhosa 3.30 0.67 P tetracirhus 0.22 2.14 son, TURSI AND D'ONGHIA (1992) caught 24 species of cephalopods in the lonian Sea, D'ONGHIA ET AL. (1995) 29 species in the North Aegean Sea, QUETGLAS ET AL. (2000) found 30 species in the Balearic Sea, SÁNCHEZ ET AL. (1998) found 36 species in the northern Tyrrhenian Sea and 47 in the Catalan Sea and SORIANO ET AL. (2003) found 18 species in the upper continental slope of Alicante (western Mediterranean). 38 AT Ratio % Cum (%) 34.64 1.39 41.18 41.18 0.50 0.67 13.74 54.92 0.47 1.03 13.22 68.13 0.28 0.71 7.70 15.83 5.69 0.53 6.17 82.6 0.21 0.6 5,45 88.05 0.13 0.6 3.57 91.62 AT Ratio % Cum (9) 47.82 1.48 48.3 48.3 0.80 1.07 19.25 67.56 9.70 0.64 98 17.35 3.8] 0.67 3.05 81.2 0.13 0.63 3.17 84.37 2.68 0.32 1 87.08 2.43 0.56 2.46 89.54 2.37 0.76 2.39 91.93 AT Ratio % Cum (9%) 19.36 1.03 20.89 20.89 17.55 0.82 18.94 39.83 10.85 0.82 11.71 MD 10.51 0.69 11.34 62.88 9.67 0.70 10.43 13.31 117 0.69 8.38 81.69 4.95 0.63 5.34 87.04 3.47 0.83 35 90.79 In previous surveys of the study area before 1995 (GIORDANO AND PERDICHIZZI, - 1998), 32. speciesimolk cephalopods were identified. In general, a similar cephalopod distribution pattern was observed from 1994-1995 trawl surveys, but the sampling was carried out at a different period (in autumn) and with a different net (with 36 mm of stretched mesh). The species caught in the previous faunistic list in the same area were: Sepiola affinis, GIORDANO ET AL.: Cephalopod assemblages along the Southern Tyrrhenian Sea Table ITb. Tabla I1b. Average dissimilarity: 100 inshore AA midslope AA L. media 106.88 0 L. vulgaris 18.88 0 Í. sagittatus 0 / 0. vulgaris DS 0 S. orbignyana 0 3.6 E. moschata 2 0 Average dissimilarity: 97.18 inshore AA midslope AA L. media 21.43 0 Í. coindefii 26.62 0 S. elegans 9.57 0 S. orbignyana 0.86 3.6 Í. sagittatus 0.16 / S. Unicirrhus 6.54 0.2 E. cirrhosa 3 0 E. moschato 0.22 0 Average dissimilarity: 92.38 inshore AA midslope AA T. eblanae 10.56 0 /. sagittatus 0.3 / P tetracirrhus 2.81 0.4 S. orbignyana 2.22 3.6 R. macrosoma 0.19 l l. coindefii 1.67 0 S. unicirrhus 0.85 0.2 E. cirrhosa 0.67 0 Sepiola ligulata, Sepiola robusta, Sepietta oweniana, Sepietta neglecta (Sepiolidae), Chiroteuthis veranii (Chiroteutidae) and Bathypolypus sponsalis (Octopodidae). On-board observation of catches indi- cated that the demersal fauna sampled is strongly influenced by depth. The AT Ratio % Cum (%) 50.79 1.52 50.79 50.79 ES 1.08 21.3 72.09 9.23 0.85 9.23 81.32 3.63 0.62 3.63 84.94 3.5] 0.42 3.5] 88.46 2.61 0.74 2.61 91.07 AT Ratio % Cum (%) 20.96 1.05 21.57 ES 18.87 0.81 19.42 40.98 11.87 0.84 12.21 53.19 11.6 0.72 11.93 65.13 8.66 0.99 8.9] 74.04 8.4] 0.68 8.66 82.7 5.64 0.61 5.8 88.5 2.65 0.64 Di 91.22 AT Ratio % Cum (0) 3805 0.95 DS IS NDS 18 23.33 48.5 11.48 0.96 12.42 60.92 10.36 0.57 11.21 712.14 6.78 0.6 7.34 79.48 5.67 0.6 6.14 85.61 3.49 0.57 3.78 89.39 2.93 0.46 35 92.56 depth range of each species with mean depth and standard deviation are reported in Table I. Eledone cirrhosa (32- 584 m), Scaeurgus unicirrhus (38-549 m), Todaropsis eblanae (61-613 m), Sepia elegans (40-432 m), Loligo media (18-383 m) and Illex coindeti (60-593 m) showed Table III. Univariate diversity indexes for each cluster. Abbreviations: S, number of species; N, number of specimens; d, richness of Margalef; J, Pielow's evenness; H”, Shannon-Wiener diversity. Tabla UI Índices univariados de diversidad de cada grupo. Abreviaturas: S, número de especies; N, número de ejemplares; d, riqueza según Margalef; J, uniformidad según Pielou; H”, diversidad de Shannon- Wiener. Cluster S N inshore 8 709 shelf 15 2267 slope 12 529 midslope 5 61 d J H 1.066 0.616 1.28 1.812 0.652 1.765 1.754 0.019 1.539 0.973 0.661 1.063 9 Iberus, 23 (1), 2005 Stress: 0.15 MX 18-80 m V 196-283 m O 204-600 m <> 366-585 m Figure 2. Multidimensional scaling ordination analysis for the five surveys. Figura 2. Análisis de ordenación de escalamiento multidimensional para las cinco campañas. a wide distribution range, extending up to the continental slope. During the present study four main cephalopod groups were defined: inshore (10-100 m), shelf (101-200 m), slope (201-600 m) and midslope (350- 600 m). 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O Sociedad Española de Malacología Iberus, 23 (1): 43-48, 2005 Onchidoris neapolitana (Delle Chiaje, 1844) (Gastropoda: Nudibranchia: Onchidorididae): una nueva especie de molusco para la fauna andaluza Onchidoris neapolitana (Delle Chiaje, 1844) (Gastropoda: Nudibranchia: Onchidorididae): a new molluscan species for the Andalousian fauna Alma SÁNCHEZ-SANTOS* Recibido el 6-VI1-2004. Aceptado el 24-1-2005 RESUMEN En este trabajo se redescribe el doridoideo Onchidoris neapolitana (Delle Chiaje, 1844) a partir de varios ejemplares recolectados en la zona del Estrecho de Gibraltar. Este hallazgo constituye la primera cita de esta especie en el litoral andaluz y amplía su área de distribución hacia el oeste. ABSTRACT In this paper, the doridoidean nudibranch Onchidoris neapolitana (Delle Chiaje, 1844) is redescribed on the basis of several specimens collected from the Strait of Gibraltar. This record constitutes the first one of this species from the Andalousian littoral and extends the known range of this species to the western. PALABRAS CLAVE: Nudibranchia, Onchidoris neapolitana, Estrecho de Gibraltar, Sur de España. KEY WORDS: Nudibranchia, Onchidoris neapolitana, Strait of Gibraltar, Southern Spain. INTRODUCCIÓN El género Onchidoris está represen- tado en el litoral peninsular ibérico por nueve especies, cuya distribución cono- cida en dicho ámbito geográfico es reco- gida por CERVERA, TEMPLADO, GARCÍA- GÓMEZ, BALLESTEROS, ORTEA, GARCÍA, Ros Y LUQUE (1988). Una de estas especies es Onchidoris neapolitana, descrita por vez primera en las costas de Italia (localidad tipo Nápoles) y con una distribución medite- rránea. En el mar Mediterráneo, además de la costa italiana (golfo de Nápoles, SCHMEKEL, 1968; SCHMEKEL Y PORT- MANN, 1982), las otras citas conocidas proceden de las costas de Francia (Banyuls, PRUVOT-FoL, 1951) y España (Ros, 1975, 1978, 1985; Ros Y ÁLTIMIRA, 1977; PEREIRA Y BALLESTEROS, 1979; ALTI- MIRA, HUELIN Y ROs, 1981; ORTEA Y BALLESTEROS, 1982; HUELIN Y Ros, 1984; BALLESTEROS, 1985), donde todas las referencias de esta especie restringen su área de distribución al litoral catalán. Según CERVERA ET AL. (1988), el litoral catalán constituye el límite más occiden- * Urb. San García, C/ Trucha, 85. 11207 Algeciras (Cádiz), Spain; e-mail: almasanchez830 hotmail.com Á3 Iberus, 23 (1), 2005 conducto uterino vagina conducto deferente ampolla hermafrodita ampolla na B hermafrodita próstata Y glándula femenina receptáculo seminal bolsa copulatriz 3 Figura 1. Anatomía externa de un ejemplar de 8 mm de Onchidoris neapolitana. A: vista dorsal; B: vista ventral; C: detalle de un rinóforo; D: disposición de las hojas branquiales y tubérculos intra- branquiales. Figura 2. Fotografía al microscopio electrónico de barrido de la rádula de un ejemplar de 7 mm (fijado). Escala: 30 pm. Figura 3. Sistema reproductor de un ejemplar de 8 mm (fijado). Figure 1. External anatomy of one specimen of 8 mm of Onchidoris neapolitana. A: dorsal view; B: ventral view; C: detail of a rinophore; D: detail of the arrangement of the branchial leaves and intra- branchial tubercles. Figure 2. Scanning electron micrographs of the radula of one specimen of 7 mm long (preserved). Scale bar: 30 ym. Figure 3. Reproductive system of' one specimen of. 8 mm (preservea). 1 mm tal conocido de su área de distribución. Así, Ros (1975) cita por primera vez para el litoral peninsular ibérico a O. neapolitana como Lamellidoris (Atalodoris) neapolitana (Delle Chiaje, 1844) en base a dos ejemplares recolectados en Cada- qués e Islas Medas (Gerona), asociados al briozoo incrustante Schizobrachiella sanguinea (Norman, 1868). Posterior- mente, PEREIRA Y BALLESTEROS (1979) citan nuevamente esta especie como L. (A.) neapolitana en base a un ejemplar recolectado en Tossa de Mar (Gerona) sobre el alga rodofícea Sphaerococcus coronopifolius Stackhouse, 1797, no apor- tando ninguna información anatómica y cromática. Años más tarde, ORTEA Y 44 BALLESTEROS (1982), en su estudio monográfico del género Onchidoris, incluyen una descripción de la anatomía externa y coloración de O. neapolitana a partir de un ejemplar de 9 mm de longi- tud, recolectado también en Tossa de Mar y sobre el alga rodofícea Sphaerococ- cus coronopifolius. En este mismo trabajo, los autores consideran dudosa la cita de Ros (1975), debido a que los caracteres anatómicos externos en base a los cuales se llevó ha cabo la diagnosis diferencial específica, son del todo genéricos. En el océano Atlántico, esta especie ha sido citada en Gran Bretaña por PruvoT-FoL (1954) y España (Asturias) (ORTEA, 1976). Sin embargo, las diagno- SÁNCHEZ-SANTOS: Redescripción de Onchidoris neapolitana sis específicas del material en los que ambos registros atlánticos fueron basados han sido materia de controver- sia. Así, mientras que ORTEA Y BALLES- TEROS (1982) consideraron la cita de PrRUuvoT-FoL (1954) como muy dudosa, CATTANEO-VIETTI Y THOMPSON (1989), basándose en ella, incluyeron a O. neapo- litana entre las especies con distribución atlanto-mediterránea. En cuanto a la presencia de O. neapolitana en la costa atlántica de la Península Ibérica, ORTEA Y URGORRI (1979) y BALLESTEROS (1985), tomando como referencia la cita de ORTEA (1976), consideraron también para esta especie una distribución atlanto-mediterránea. Sin embargo, ORTEA Y BALLESTEROS (1982), en su revi- sión del género Onchidoris en el litoral ibérico, establecen como únicas referen- cias válidas las procedentes de la costa mediterránea española. En este trabajo se redescribe esta especie a partir de ejemplares proceden- tes del Sur de España (Estrecho de Gibraltar), así como algunos datos bioló- gicos inherentes a su hábitat y la puesta. MATERIAL Y MÉTODOS En junio de 2003 se recogieron varios ejemplares en la zona del Estrecho de SISTEMÁTICA Gibraltar que atribuimos a Onchidoris neapolitana y que constituyen el objeto del presente trabajo. Se redescribe la especie a partir de dichos ejemplares y se aportan algunos datos biológicos referidos a su hábitat y a la puesta. Los animales, tras 24 horas de con- gelación en agua de mar, fueron preser- vados en etanol al 96%. Para el estudio anatómico interno, dos ejemplares fijados, de 7 y 8 mm respectivamente, fueron disecados por incisión dorsal prestando especial atención a la morfo- logía del sistema reproductor y de la rádula. La genitalia fue examinada y dibujada bajo un microscopio óptico con cámara clara. Para estudiar la mor- : fología radular, el aparato bucal fue extraído y mantenido en una solución de KOH al 10% con el objeto de disol- ver el tejido orgánico circundante. La rádula, así aislada, fue sumergida en agua, secada y finalmente montada para su examen con microscopía elec- trónica de barrido. El material examinado se encuentra depositado en el Natural History Museum of Los Angeles County (LACM), en el Departamento de Biolo- gía de la Universidad de Cádiz (UCA) y en el Departamento de Biología Animal y Ecología de la Universidad de Granada (UGR). DORIDOIDEA Pelseneer, 1894 Familia ONCHIDORIDIDAE Alder y Hancock, 1845 Género Onchidoris Blainville, 1816 Onchidoris neapolitana (Delle Chiaje, 1844) (Figs. 1-3) Idalia neapolitana Delle Chiaje, 1844, Napoli 8: appendice prima tomo 2: 5-12 [Localidad tipo: Nápoles] Anatomía externa: Cuerpo muy apla- nado. El noto es espiculoso, ovalado y sobresale ampliamente al pie (Fig. 14). De los tres tipos de disposición espicu- lar característicos del género, sólo se pudo apreciar la disposición radial, en el margen del noto, y la transversal en la zona central del mismo. La oblicua no pudo ser observada. La superficie dorsal del noto presenta papilas digitiformes (Fig. 14). Las de la zona central son más cortas que las laterales y marginales, proyectándose estas últimas más allá del margen notal claramente, mientras que del área intrabranquial son más cortas que las laterales y marginales. El 45 Iberus, 23 (1), 2005 número de papilas presentes en el área intrabranquial oscila entre 5 y 8. El número de laminillas rinofóricas oscila entre 6 y 7 (Fig. 1C). Orificios rinofóricos lisos y sin vaina. La branquia está cons- tituida por hojas branquiales unipinna- das alineadas alrededor del ano for- mando un óvalo abierto posteriormente (Fig. 1D). El tamaño de las hojas decrece desde la central hasta las posteriores. El número de hojas branquiales oscila entre 12, en ejemplares de 5, 6 y 7 mm de longitud, y 14 en un ejemplar de 9 mm de longitud. Coloración: El noto es de color rojo- anaranjado con pequeñas manchas pardas de geometría irregular, más den- samente concentradas en la zona central del noto. En el borde del noto se apre- cian bandas de color pardo grisáceo en disposición radial con manchas naranja intercaladas entre ellas. Las papilas son de color pardo claro translúcido. Las áreas que rodean los orificios rinofóricos y branquial están despigmentadas. Los rinóforos son gris oscuro, mientras que las branquias son de color naranja. La superficie del área intrabranquial está despigmentada con papilas ligeramente más claras que en el resto del dorso. El pie y el velo bucal son naranja claro. Anatomía interna: La rádula es bise- riada, con ausencia de diente raquídeo. El diente lateral más interno (Fig. 2) es de gran tamaño, tiene una placa basal cuadrangular grande con el borde inte- rior curvado y orientado hacia fuera, el borde exterior presenta una cúspide prominente y alargada en cuyo borde interno presenta 7-8 dentículos bien desarrollados. Asimismo, destaca la pre- sencia de una prolongación posterior por debajo de la cúspide. El diente más externo está formado por una placa basal pequeña y cóncava en cuyo borde inferior posee una pequeña cúspide rudimentaria (Fig. 2). La fórmula radular de un ejemplar de 7 mm es 22 x 1.1.0.1.1. La armadura labial está com- puesta por uncinos simples. El sistema reproductor (Fig. 3) pre- senta un conducto hermafrodita que se continua en una ampolla hermafrodita pequeña y curvada. El conducto defe- 46 rente es alargado y no presenta región prostática morfológicamente diferen- ciada, desembocando en el atrio genital. El pene es inerme. El conducto vaginal, más corto y delgado que el conducto deferente, conecta con una bolsa copula- triz esférica. El receptáculo seminal tiene forma de saco curvado y se conecta a la vagina cerca de la bolsa copulatriz. La bolsa copulatriz, el receptáculo seminal y la ampolla hermafrodita son de tamaño similar. Un corto y muy delgado conducto uterino conecta directamente con la bolsa copulatriz en un punto adyacente a la conexión de esta con la vagina. Discusión: La coloración y la anato- mía externa de los ejemplares del Estre- cho de Gibraltar son muy similares a las que muestran SCHMEKEL Y PORTMANN (1982) en los ejemplares recolectados en el golfo de Nápoles. Uno de los ejempla- res estudiados por dichos autores pre- senta una rádula con un diente lateral interno con 4-6 dentículos. Las diferen- cias respecto al número de dentículos de nuestro material probablemente se deban a una cierta variabilidad en este carácter. Asimismo, a nivel del sistema reproductor hemos observado diferen- cias entre ambos materiales respecto al tamaño y la forma del receptáculo seminal y la bolsa copulatriz, así como la ausencia de un conducto uterino cla- ramente definido en el esquema que presentan SCHMEKEL Y PORTMANN (1982, pág. 118, fig. 7.28a). Sin embargo, el esquema de estos autores es una repre- sentación idealizada del sistema repro- ductor, y no un estudio morfológico detallado. Por ello, tales diferencias con nuestras observaciones no deben consi- derarse significativas. Respecto al ejemplar del litoral catalán descrito por ORTEA Y BALLESTE- ROS (1982), las semejanzas entre ambos materiales son grandes. La variación en el número de laminillas rinofóricas y hojas branquiales observada en nuestros ejemplares se debe probablemente a la variabilidad intraespecífica de la especie. En cuanto a la coloración, la única diferencia observada es la presen- cia en todos los ejemplares del Estrecho SÁNCHEZ-SANTOS: Redescripción de Onchidoris neapolitana de Gibraltar de una zona despigmen- tada alrededor de los orificios rinofóri- cos y branquial. ORTEA Y BALLESTEROS (1982) no describen la anatomía interna de su material, por lo que no hemos podido compararla con la del nuestro. De las especies de Onchidoris citadas en las costas de la Península Ibérica, únicamente Onchidoris depressa (Alder y Hancock, 1842) y Onchidoris tridactila Ortea y Ballesteros, 1982, ambas con dis- tribución atlántica, comparten con O. neapolitana la presencia de papilas en su noto. Sin embargo, desde el punto de vista del aspecto externo, O. depressa difiere de esta última por su modelo cro- mático y disposición en forma de herra- dura de las hojas branquiales (ORTEA Y BALLESTEROS, 1982; THOMPSON Y BROWN, 1984; PICTON Y MORROW, 1994) y O. tridactila por su diseño cromático y la presencia de una vaina rinofórica pro- vista de tubérculos y por diferencias en la puesta (ORTEA Y BALLESTEROS, 1982). Desde el punto de vista de la anato- mía interna, la morfología radular de O. depressa, O. tridactila y O. neapolitana es muy similar, si bien, los datos existentes de estas tres especies, revelan pequeñas diferencias en el número de dentículos en el diente lateral más interno. Así, en O. neapolitana este sería de 4-8 (SCHME- KEL Y PORTMANN, 1982; presente trabajo), en O. depressa, sería de 4-5 (THOMPSON Y BROWN, 1984) y en O. tri- dactila es de 3-4 en el holotipo de la especie (ORTEA Y BALLESTEROS, 1982). El segundo diente lateral es idéntico en las tres especies. Dada la similitud en la morfología radular, las diferencias observadas en el número de dentículos pueden ser debidas a una variación intra-específica u ontogenética de esta estructura. Esto último, unido al hecho de que la captura de O. neapolitana en aguas del Estrecho de Gibraltar indica que las tres especies muestran áreas de distribución solapadas en el Atlántico, sugieren la posibilidad de que sean sinónimas. Para aclarar este aspecto habría que examinar el material tipo de las tres especies. En cuanto al sistema reproductor, estas tres especies no pueden ser com- paradas ya que el único conocido es el de O. neapolitana. Existe otra especie mediterránea no citada en las costas ibéricas, también con papilas en su noto, Onchidoris bouvieri (Vayssiere, 1919). Sin embargo difiere de O. neapolitana por la presencia de una espícula en el interior de las proyeccio- nes papilares y la disposición en círculo de las hojas branquiales (ORTEA Y BALLESTEROS, 1982). En cuanto a la biología de O. neapo- litana, según la literatura, esta especie se encuentra asociada a varias especies de briozoos incrustantes de diversos géneros (Chorizopora Hincks, 1879; Cribrilaria Silén, 1941; Escharina Milne- Edwards, 1836; Microporella Hincks, 1877; Parasmittina Osburn, 1952; Schis- mopora MacGillivray, 1888; Schizobra- chiella Canu y Bassler, 1920; Schizoma- vella Canu y Bassler, 1917; Schizoporella Hincks, 1877; Smittoidea Osburn, 1952; Tubulipora Lamarck, 1816) de las que se alimentaría (Ros, 1975, 1978). Todos nuestros ejemplares fueron siempre encontrados asociados a formaciones del briozoo incrustante Schizobrachiella sanguinea, sobre el que mostraban una acusada homocromía. La puesta de algunos ejemplares pudo ser observada en el medio siempre depositadas sobre S. sanguinea, y consistía en una cinta transparente enrollada en espiral de dos vueltas con los huevos de color naranja. El hallazgo de O. neapolitana en aguas del estrecho de Gibraltar consti- tuye la primera cita de esta especie para el litoral andaluz. Asimismo, en las colecciones malacológicas del Museo Nacional de Ciencias Naturales de Madrid existe un ejemplar de esta especie (con el n* de catálogo 15.05/804), capturado en el Peñón del Cuervo (Málaga), el 18 de julio de 1985 (colectado e identificado por José Tem- plado). Esta nueva adición a la fauna de gasterópodos opistobranquios del litoral andaluz supone una considerable ampliación de su área de distribución, cuyo límite más occidental en el Medite- rráneo estaba situado en el litoral catalán. 47 Iberus, 23 (1), 2005 AGRADECIMIENTOS Muy especialmente deseo expresar mi más profunda gratitud al Dr. Angel Valdés (Natural History Museum of Los Angeles County, LACM) por su decisiva ayuda en la realización del estudio ana- tómico interno y por la revisión crítica del manuscrito. Asimismo, mi agradeci- miento a los Drs. Luis Sánchez-Tocino (Universidad de Granada) y Juan Lucas BIBLIOGRAFÍA ALTIMIRA, C., HUELIN, M. F. Y Ros, J. D., 1981. Molluscs bentónics de les illes Medes (Gi- rona). I. Sistemática. Butlleti de la Institucio Ca- talana d'Historia Natural, 47 (Seccio de Zoo- logia 4): 69-75. BALLESTEROS, M., 1985. Contribución al conoci- miento de los Sacoglossos y Nudibranquios (Mo- llusca: Opisthobranchia). Estudio anatómico, sis- temático y faunístico de las especies del Medite- rráneo español. Resumen de la Tesis Presentada para aspirar al grado de Doctor en Ciencias Biológicas, Universitat de Bar- celona. Centre de Publicaciones Intercanvi Cientific I Extensio Universitaria, 46 pp. CATTANEO-VIETTI, R. Y THOMPSON, T. E., 1989. Mediterranean opisthobranch molluscs: a zoogeographic approach. Bollettino Malaco- logico 25(5-8): 183-204. CERVERA, J. L., TEMPLADO, J., GARCÍA GÓMEZ, J. C., BALLESTEROS, M., ORTEA, J. A., GARCÍA, F. J., ROS, J. Y LUQUE, A. A., 1988. Catálogo actualizado y comentado de los opistobran- quios (Mollusca, Gastropoda) de la Península Ibérica, Baleares y Canarias, con algunas re- ferencias a Ceuta y la Isla de Alborán. Iberus, suplemento 1.: 1-84. DELLE CHIAJE, S., 1844. Descrizione e notomia degli animali invertebrati della Sicilia citeriore osservati vivi negli anni 1822-1830, Napoli 8: appendice prima tomo 2, pp. 5-12. HUELIN, M. F. Y Ros, J., 1984. Els molluscs ma- rins de les Illes Medes, pp. 457-504. In: J. Ros, I. Olivella, € J. M. Gili (eds.) Els sistemes na- turals de les Illes Medes, Institut d'Estudis Ca- talans. ORTEA, J. A., 1976. Catálogo brevemente comen- tado de la fauna de moluscos marinos gaste- rópodos y bivalvos existentes en el estuario de Villaviciosa. Asturnatura, 3, pp. 109-120. ORTEA, J. A. Y URGORRI, V., 1979. Una nueva es- pecie de Onchidoris (Moluscos, opistobran- quios, Doridáceos) del Norte de Noroeste de España, Onchidoris cerviñoi n. sp. Cahiers de Biologie Marine, 20: 507-513 48 Cervera Currado (Universidad de Cádiz) por sus valiosos comentarios sobre el manuscrito. Por último, a los alumnos de doctorado Manuel Caballer Gutiérrez (Universidad de Cantabria) y Leopoldo Moro Abad (Museo de Cien- cias Naturales, Tenerife) por facilitarme gran parte de la información bibliográ- fica. ORTEA, J. A. Y BALLESTEROS, M., 1982. Sobre al- gunos Onchidoris Blainville, 1816 (Mollusca, . Opisthobranchia, Doridácea) del Litoral Ibé- rico. Investigación Pesquera, 46 (2): 239-254. PEREIRA, F. Y BALLESTEROS, M., 1979. Gasteró- podos del litoral mediterráneo español. II. Tossa de Mar. Gerona. Primer Simposio de Bentos Marino, 223-235 pp. PIcTON, B. E. Y MORROW, C. C., 1994. A Field Guide to the Nudibranchs of the British Isles, Immel Publishing, 143 pp. PRUVOT FoL, A., 1951. Etude des nudibranches de la Méditerranée (2e partie). Archives de Zoologie Experimentale et Generale. Paris. 88 (1): 1-80, pls. 1-4. PRUVOT FoL, A., 1954. Mollusques Opisthobran- ches. Faune de France, Paris (Lechevalier) 58: 1-460. Ros, J. D., 1975. Opistobranquios (Gastropoda: Euthyneura) del litoral ibérico. Investigación Pesquera., 39 (2): 269-372. Ros, J. D., 1978. La alimentación y el sustrato en los opistobranquios ibéricos. Oecologia Aquatica, 3: 153- 166. Ros, J., 1985. Els poblaments d'Opistobranquis de coves submarines mediterranies: noves da- des i comentaris sobre llur afinitat faunis- tica. Butlleti de la Institucio Catalana d'Histo- ria Natural, 52 (Seccio de Zoologia, 6): 87-94. Ros, J. D. Y ALTIMIRA, C., 1977. Comunidades bentónicas de sustrato duro del litoral NE es- pañol. V. Sistemática de moluscos. Miscelá- nea Zoológica, 4 (1): 43-45. SCHMEKEL, R. L., 1968. Ascoglossa, Notaspidea und Nudibranchia im Litoral des Golfes von Neapel. Revue Suisse de Zoologie, 75(6): 103- 15: SCHMEKEL, R. L. Y PORTMANN, A., 1982. Opist- hobranchia des Mittelmeeres. Nudibranchia und Sacoglossa. Springer-Velarg. Berlin, 410 pp. THOMPSON, T. E. Y BROwN, G. H., 1984. Biology of opisthobranch molluscs, Vol. 2. The Ray So- ciety, London, 229 pp. O Sociedad Española de Malacología Iberus, 23 (1): 49-65, 2005 Contribution to the knowledge of the family Caecidae: 16. Revision of the Caecidae of HEaster Island (Chile) (Caenogastropoda: Rissooidea Gray J. E., 1847) Contribución al conocimiento de la familia Caecidae: 16. Revisión de los Caecidae de la Isla de Pascua (Chile) (Caenogastropoda: Rissooidea J. E. Gray, 1847) Bret RAINES* and Mauro PIZZINI** Recibido el 26-X-2004. Aceptado el 11-11-2005 ABSTRACT With the exception of two species, members of the family Caecidae from Easter Island have been previously neglected. Based on type and additional material, a revision of the species known to date from Easter ls. is herein proposed, with the description of 5 new species: Caecum rehderi spec. nov., C. heterochromum spec. nov., C. pascuanum spec. nov., C. rapanviense spec. nov., C. campanulatum spec. nov. RESUMEN Con la excepción de dos especies, los miembros de la familia Caecidae de la Isla de Pascua han sido previamente desatendido. Basado en el tipo y en el material adicional, se propone aquí dentro, una revisión de las especies conocidas hasta la fecha de la Isla de Pascua, y incluye la descripción de 5 nuevas especies: C. rehderi esp. n., C. heterochro- mum esp. n., C. pascuanum esp. n., C. rapanuiense esp. n., C. campanulatum esp. n. KEY WORDS: Mollusca, Caenogastropoda, Rissooidea, Caecidae, taxonomy, new species, Easter Island, Western Pacific. PALABRAS CLAVE: Mollusca, Caenogastropoda, Rissooidea, Caecidae, taxonomia, nuevas especies, Isla de Pascua, Pacífico Occidental. INTRODUCTION Easter Island (Fig. 1) is found in a to- tally isolated position approximately 3,500 Km from the coast of Chile. From a zoogeographical point of view, Easter ls. is in a very peculiar area within the east- ern Indo-Pacific region, and to the point that, in 1965, Schilder proposed the Ra- panuian province as a separate biogeo- graphical province from the Polynesian province. The submarine seascape fea- tures widely scattered corals affixed to the rugged volcanic substrate. The de- pauperate benthic community employs a variety of adaptive strategies for survival in an environment stressed by waves, currents and the absence of mineral nu- * Research Associate, Natural History Museum of Los Angeles County , P.O. Box 612 Victorville, CA 92393 USA. e-mail: rainesbkPyahoo.com ** Largo della Caffarelletta, 6, 00179 ROME (Italy). e-mail: ma.pizziniClibero.it AS Iberus, 23 (1), 2005 trients. Most of the corals and other bot- tom invertebrates are typical of the Indo- Pacific reefs, but reefs have not formed. The circulation pattern, and espe- cially the marked upwelling between the South Pacific and Mentor currents, contributes to the isolation of the area. In terms of marine ecosystems, this in- sularity has also produced a high degree of radiation in many groups, and partic- ularly in the caenogastropods. Thus the fauna can essentially be described as typical Pacific fauna with a relatively high number of endemic species. The is- land's marine benthic fauna is generally characterised by a high degree of species diversity and a low abundance, and this is true for both hard and soft bottoms. The meiobenthic family Caeci- dae also follows this pattern. The Caecidae of the Easter Island have been scarcely studied in the past, with the exception of the work of REHDER (1980). Past surveys have reported endemicity rates within the molluscan fauna ranging from 37% to 42% (REHDER, 1980, DISALVO, RANDALL AND CEA, 1988, RAINES, 2002). Rehder also indicates that some species appear to have a dual relationship with certain species from Hawaii, as well as species from the Kermadec Islands. In prepara- tion for his 1980 publication, Rehder reviewed all previous studies and expe- ditions. In addition to examining all the Easter Is. material in US museums, he also examined the full store of material housed in the Museo National de Histo- ria Natural in Santiago, Chile. In all, Rehder examined over 7,000 specimens, of which 3480 were collected during his trip to the island in 1974. In the mid 1980's, DiSalvo and his team conducted a comprehensive survey of Easter Island's sublittoral marine environment. The authors of the present work had the good fortune of having access to examples of all the molluscan material collected during DiSalvo's investigation. Other than the few publi- cations that we have been working on (OLIVER, 1915, SCHILDER, 1965, REHDER, 1980, DISALVO ET AL., 1988, RAINES, 2002) no other serious work on molluscs has 50 been completed during the last ten years. With regard to Caecidae, during the three trips to Easter Island, the authors of the present work collected more than 350 specimens of this family. Rehder mentions only two species: Caecum cf. solitarium Oliver, 1915, and C. amydroglyptum Rehder, 1980. In all other publications, which we have reviewed, Caecidae are listed simply as “Caecum species”. No other descriptions or illus- trations were provided. So we present herein a revision of the species known to date from Easter Is., based on type and additional mater- ial, with the description of five new species. Abbreviations used: AMS: Australian Museum Sydney, Sydney (Australia) CM: Canterbury Museum, Christchurch (New Zealand) LACM: Natural History Museum of Los Angeles County, Los Angeles (U.S.A.) MNHN: Museum National d'Histoire Naturelle, Paris (France) MPR: Mauro Pizzini collection. NHML: Natural History Museum, London (U.K.) NMNZ: Museum of New Zealand Te Papa Tongarewa, Wellington (New Zealand) NSMT: National Science Museum, Tokyo (Japan) USNM: National Museum of Natural History, Washington D.C. (U.S.A.) WAM: Western Australian Museum, Perth (Australia) Terminology: Abapical: towards the apices (of the septum) Adapical: opposite to the apices direc- tion (of the septum) Aperture, apertural end: the round ante- rior opening of the shell. Apex, apical end: the smaller, narrower, closed posterior end of the tube. Cutting plane: the plane individuated by the edge of the shell at the apex (excluding septum and mucro). RAINES AND PIZZINI: Revision of the Caecidae of Easter Island (Chile) UNITED STA SMAN $ ¡ ¿ SEA j 3 2 NEW NORTH Y - E ¿ ZEALAND AS p [ANÍS t : DOG EAN | Figure 1. Biogeographical provinces within the tropical eastern Indo-Pacific as proposed by SCHILDER (1965), and as illustrated by REHDER (1980): Micronesian: M; Hawaiian: H; Fijian: E; Polynesian: P; Rapanuian: R; Kermadec Islands: K (added by the authors). Figura 1. Las provincias biogeographicas dentro del Indo-Pacífico oriental tropical propuestas por SCHILDER (1965) e ¿ilustradas por REHDER (1980): Micronesian: M; Hawaiano: H; Fijian: E: Poline- sio: P; Rapanuzan: R; Kermadec Islands: K (añadida por los autores). Interspace: area between rings, with / without microsculpture. Meiobenthic: referred to all interstitial molluscs living in sediment of varying granule size. Microsculpture: usually visible at very high magnification or under SEM can be transverse, longitudinal or both. Mucro: small to large prong projecting from the septum. RESULTS Rings, annular sculpture: transverse, raised sculpture (equivalent to the axial sculpture of the normally coiled gastropods). Septum: closure of the shell at the apex, as it sheds earlier stages. Shell(s): the shell, beached without gas- tropod. Spm(s): live collected specimen(s), with soft parts and /or operculum(a). Superfamily RIsso0IDEA Gray J. E., 1847 Family CAECIDAE Gray J. E., 1850 Genus Caecum Fleming, 1813 Diagnosis (BANDEL, 1996): “The shell of the teleoconch is a small, slightly curved tube orna- mented only with growth lines, numerous ring-like collabral lirae ad /or axial ribs. The poste- rior end of the tube is closed by a conical septum. The protoconch is trochospirally or planispi- rally coiled. Uncoiling of the shell begins after metamorphosis”. Type species (BANDEL, 1996): Dentalium imperforatum Kanmaker, 1798 (= trachea Montagu, 1803) from Europe, Mediterranean Sea and Atlantic to southern England. dl Iberus, 23 (1), 2005 100 yum Figure 2. Caecum rehderi spec. nov. A: holotype LACM 3019, gold coated, length 2.08 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 2. Caecum rehderi spec. nov. A: holotipo LACM 3019, metalizado en oro, longitud 2,08 mm, B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Geiger. Caecum rehderi spec. nov. (Fig. 2) Caecum cf. solitarium; Rehder, 1980: 31-32, pl. 5, fig. 11. Type material: Holotype, LACM 3019; 1 paratype, LACM 3020; 1 paratype, USNM 756269; 1 paratype, MNHN. Material examined: 1 specimen, Onetea, Hotuiti (length: 2.42 mm, USNM 756269) (Oct. 1974, leg. H. Rehder); 4 specimens, Punta Rosalia, east of Anakena (Apr. 1998, leg. B. Raines), Type locality: In sand collected along the base of cliffs at 10-20m, off Punta Rosalia, east of Anakena, Easter 1s., Chile. 27? 04' 18” S, 109% 19 45” W. Description: Shell small (holotype measures, length: 2.08 mm; width: 0.42 mm), tube-like, slender, gently arched, semi-translucent to opaque white. Tube seemingly smooth almost glassy, sub- cylindrical, with posterior end only slightly smaller than anterior end. Microsculpture nearly obsolete, with only fine annular growth lines sometimes present under magnification. Anterior end somewhat flared just above aperture 92 with incised annular rings. Aperture cir- cular, but slightly constricted. Posterior end with tapered rim. Septum not retracted, subquadrate lateral outline inclined with elevated edge slightly right of center when viewed frontally. Opercu- lum and soft parts unknown. Original description of C. cf. solitar- ium Rehder, 1980: “Diagnosis. Shell small, 2.4 to 2.7 mm in length, glassy, grayish-white to whitish, slender, gently RAINES AND PIZZINI: Revision of the Caecidae of Easter Island (Chile) Figure 3. Caecum cf. solitarium Rehder (1980). A: USNM 756269, uncoated, length 2.42 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 3. Caecum cf. solitarium Rehder (1980). A: USNM 756269, no metalizado, longitud 2,42 mm; B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Geiger. curved, diameter at posterior end only slightly smaller than at anterior end, where the aperture is slightly con- stricted and somewhat opaque above the aperture; the sculpture consists of fine, rather crowded, subobscure (worn?) annular riblets that gradually and slightly increase in strength toward the aperture; septum exserted, sub- quadrate with a slightly convex surface inclined from an elevated edge at the right dorsal sector to the edge of the posterior rim of the shell at the left ventral sector. Range. Kermadec Islands (and Easter Island ?). Material. 1 specimen from sta E-27A, USNM 756269. Measurements (mm). USNM 756269: length, 2.42; diameter at anterior end, 0.4”. Discussion: Rehder reported a caecid from Easter Island, which he tentatively identified as Caecum cf. solitarium Oliver, 1915. However, it seems that Rehder un- fortunately overlooked several key char- acteristics within Oliver”s description. The first being the septum of C. solitar- tum, which OLIVER (1915) described as “....hemispherical, making an abrupt shoulder at the junction of the shell”; even the septum of Rehder”s specimen (Fig. 3) could be associated to a tale ty- pology. Oliver also mentions, that the sculpture of C. solitarium consists of sim- ple growth lines, while Rehder refers to the sculpture as consisting of suboscure (worn?) annular riblets that gradually and slightly increase in strength toward the aperture. Other main difference be- tween C. solitarium Oliver, 1915 and C. solitarium sensu Rehder is that the first has a nearly uniform diameter, while the second shows the diameter at posterior end only slightly smaller than at ante- 93 Iberus, 23 (1), 2005 Figure 4. Caecum solitarium Oliver, 1915. A: Holotype CM M2867, uncoated, length 1.68 mm; B: detail of aperture; C: microsculpture; D: detail of posterior. SEM imaging by N. Andrews. Figura 4. Caecum solitarium Oliver, 1915. A: Holotipo CM M2867, no metalizado, longitud 1,68 mm; B: detalle de la abertura; C: microescultura; D: detalle trasero. Imágenes al MEB por N. Andrews. rior end. Furthermore, it appears Re- hder did not examine Oliver”s type ma- terial, because if he would have done, he would have noted that the two speci- mens have significantly different ante- rior ends (Figs. 3D, 4B), and that the holotype of C. solitarium is badly broken and lacks the entire posterior end (Fig. 4). The damage to the holotype is old and worn, and possibly occurred in situ suggesting that Oliver may have chosen an imperfect specimen as the type and actually described another in his de- scription. (Scofield, 2002, pers. commu- nication). Rehder”s specimen is, how- ever, consistent with C. rehderi, and therefore, has been designated as the paratype. Although Rehder broke the anterior end of his specimen while mea- suring it, all the pieces were available for examination. The specimens which we found, actually showed a series of 54 small rings along the entire tube; and apart from the relativity of the term's significance, we hold that the difference between our specimens and those de- scribed by the two authors falls within the species” range of variability, in light, above all else, of the high degree of adaptation of the local molluscs to the island's distinguishing geo-climatic con- ditions. It is known that a number of species of Caecidae (i.e. C. lightfootae Pizzini, Nofroni and Oliverio, 1994), though they have the same general shape (septum, tube and aperture), could show a very wide range of vari- ability in terms of the type of sculpture. Remarks: Caecum rehderi seems to be an unusually fragile species. Of the five known specimens, Rehder chipped the aperture of his specimen (USNM 756269) while measurinyg it, the holotype has a small longitudinal crack toward RAINES AND PIZZINI: Revision of the Caecidae of Easter Island (Chile) Figure 5. Caecum amydroglyptum Rehder, 1980. A: holotype USNM 757977, uncoated, length 1.67 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 5. Caecum amydroglyptum Rehder, 1980. A: holotipo USNM 757977, no metalizado, longitud 1,67 mm; B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Geiger. the aperture end, the other paratype has the Kermadec Islands, while Rehder a Chip in the aperture, and the junior author completely crushed another specimen while examining it. Geographical distribution: Oliver described Caecum solitarium only from tentatively indicated the species as being from Easter Is. In our opinion, according to the present knowledge, C. solitarium is restricted to the Kermadec Is. Caecum amydroglyptum Rehder, 1980 (Figs. 5, 6) Caecum amydroglyptum; Rehder, 1980: 32, pl. 5, fig. 12. Type material: Holotype, USNM 757977; 1 paratype, USNM 757978 Material examined: Original types. Holotype, USNM 757977; paratype, USNM 757978, (Oct. 1974, leg. H. Rehder). 132 specimens in sand collected along the base of cliffs at 10-20m, off Punta Rosalia, east of Anakena, 27” 04 18” S, 109* 19 45” W (Apr. 1998, leg. B. Raines). Voucher material: 4 specimens were deposited in each of the following institutions: LACM; USNM 1018792; MNHN; NHML; NMNZ M.273207; NSMT Mo 73562; AMS C.205278; WAM 513783, and 6 specimens (3 adults /3 juveniles), MPR. 13 shells, beach of Anakena Bay, on the northern coast of Easter Is., picked up among the rocky bottom on the west side of the bay at low tide, among com- munities of Dictyotales, with Galaxaura obtusata. (12-1-1995, leg. E. Rolán), MPR. Type locality: Station E-27A, Onetea, Hotuiti: in patch of sand above high tide level. II Iberus, 23 (1), 2005 B 100 um 100 um Figure 6. Caecum amydroglyptum Rehder, 1980. A: Voucher specimen LACM, gold coated length 1.53 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 6. Caecum amydroglyptum Rehder, 1980. A: El espécimen del vale LACM, metalizado en oro, longitud 1,53 mm, B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Getger. Original description: “Shell small, from 1.3 to 1.7 mm in length, curved, rather evenly cylindrical with the ante- rior end in fully grown specimens slightly swollen above the aperture; glassy grayish white to light orange yellow in color; sculpture consists of rather strong, somewhat distantly spaced annular ribs that become more or less obscure in the middle part of the shell, with microscopic, longitudinal wavy striae that are obscure at the ante- rior and posterior ends; septum, low, dome-shaped. ” Additional description: Shell small (mean length: 1.7 mm; width: min. 0.3 mm, max 0.4 mm), curved, colour grayish white. The tube is perfectly cylindrical, except near the aperture, and its sculpture consists of about 36-40 rings, with some in the middle part of 56 the shell being less raised and changing their shape, until they resemble very fine growth lines. Microsculpture formed by longitudinal worm-like striae visible at enlargement of at least 180x. Septum dome-shaped, slightly raised over the cutting plane. Aperture consist- ing of a large protuberance crossed by slightly raised rings. Operculum and soft parts unknown. Remarks: We agree totally with Rehder's conclusions; because we have ”...been unable to identify this species with any published taxon” from either the Indo-Pacific Provinces, the Panamic Prov. or the Chilean Prov. We have found only one species that resembles C. amydroglyptum, which is C. vertebrale Hedley, 1899, from Funafuti Is. It is quite similar to amydroglyptum in terms of the sculpture of the tube, longitudinal RAINES AND PIZZINI: Revision of the Caecidae of Easter Island (Chile) 100 um 100 um Figure 7. Caecum heterochromum spec. nov. A: holotype LACM 3021, gold coated, length 1.42 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 7. Caecum heterochromum spec. nov. A: holotipo LACM 3021, metalizado en oro, longitud 1,42 mm); B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Getger. microsculpture and septum, but the tube, a very strong microsculpture Rehder's species shows a much greater swelling of the tube above the apertural end, which is crossed by small sculp- tured rings, while the adapical part of vertebrale has almost the same diameter of the tube. In addition, C. amydroglyp- tum presents, along the entire length of consisting of worm-like, longitudinal striae, covering also the top of the rings, while that of vertebrale is an indistinct microsculpture not surely comparable to a real striation (pers. observ.). Geographical distribution: This species would appear to be limited to Easter Is. Caecum heterochromum spec. nov. (Figs. 7, 8) Type material: Holotype, LACM 3021; 6 paratypes, LACM 3022; 6 Paratypes, USNM 1018789; 6 Paratypes, MNHN; 6 Paratypes, NHML,; 6 Paratypes, NMNZ M.273205; 6 Paratypes, NS5MT Mo 73560; 6 Paratypes, AMS C.205275,; 6 Paratypes, WAM S13780; 9 Paratypes, MPR. Material examined: 168 specimens: off Hanga Nui; and 8 specimens off the western coastline near Tahai (Dec. 2000, leg. B. Raines). 39 shells, beach of Anakena Bay, on the northern coast of Easter Is., picked up among the rocky bottom on the west side of the bay at low tide, among communities of Dictyotales, with Galaxaura obtusata. (12-1-1995, leg. E. Rolán) MPR. Voucher material: 39 shells, beach of Anakena Bay, on the northern coast of Easter Is., picked up among the rocky bottom on the west side of the bay at low tide, among communities of Dicty- otales, with Galaxaura obtusata. (12-1-1995, leg. E. Rolán) MPR. 7) Iberus, 23 (1), 2005 100 um 100 um Figure 8. Caecum heterochromum spec. nov. A: paratype from lot LACM 3022, gold coated, length 1.53 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 8. Caecum heterochromum spec. nov. A: paratipo de la porción LACM 3022, metalizado en oro, longitud 1,53 mm; B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Getger. Type locality: In sand collected along the base of cliffs at 20m off Hanga Nui, Easter Is., Chile. 27” 07' 46” S, 109* 16' 35” W. Derivation of the name: From the greek etepoc: other and xpopo: colour. Description: Shell small (mean length: 1.6 mm; mean width: 0.4 mm) with the tube subcylindrical in shape in the abapical part and cylindrical up to the vicinity of the aperture (Fig. 8), where there is a slight swelling, fol- lowed by a narrowing of the tube; the aperture is perfectly circular, simple and rimmed by a very slight flaring towards the outside. The septum is dome-shaped and slightly raised over the cutting plane. Its sculpture is extremely vari- able, ranging from specimens with approximately 50 small raised rings that are separated by interstices of corre- 58 sponding breadth and depth, particu- larly in the upper portion of the tube and near the aperture, to others that lack any type of sculpture; these two extreme represent the limits of the species” range of variability, given that they were found in intermediate specimens whose rings are barely visible. The microsculp- ture also presents a wide range of vari- ability, with some specimens not showing any trace of microsculpture, while the surface of other specimens, at an enlargement of 30x, presents a microsculpture consisting of a large number of very fine, worm-like striae RAINES AND PIZZINI: Revision of the Caecidae of Easter Island (Chile) 100 um Figure 9. Caecum pascuanum spec. nov. A: holotype LACM 3023, gold coated, length 1.64 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 9. Caecum pascuanum spec. nov. A: holotipo LACM 3023, metalizado en oro, longitud 1,64 mm; B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Geiger: that follow its axial direction, while others have a rough surface. C. hete- rochromum has an extremely variable colouring and pattern; white, cream- coloured shell with a pattern consisting of brown, zigzagging lines running almost parallel in a horizontal direction (axial). There are also some specimens showing a brown irregular stripe in the middle portion of the shell. The colour- ing is again a creamy white, with an irregular vertical design consisting of unequal spots. Operculum corneous, light brown; its external side consists of a smooth central nucleus and 5-6 con- centric rings that run from this nucleus up to the external border. Soft parts unknown. Remarks: Caecum heterochromum, despite the limited nature of the name, it is, in absolute terms, the most variable of the species to be found on Easter ls. In fact, its range of variability involves not only its colouring and patterns, but also major morphological characteris- tics, such as microsculpture and sculp- ture. Nevertheless, the silhouette, the form of the septum and that of the tube, as well as the apertural end, which, when taken as a whole, constitute the general form, are a constant that we consider to be a distinguishing charac- teristics of this species. Geographical distribution: The species is currently noted only in relation to Easter ls. 59 Iberus, 23 (1), 2005 Figure 10. Caecum rapanuiense spec. nov. A: holotype LACM 3025, gold coated, length 1.58 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 10. Caecum rapanuiense spec. nov. A: holotipo LACM 3025, metalizado en oro, longitud 1,58 mm, B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Geiger. Caecum pascuanum spec. nov. (Fig. 9) Type material: Holotype, LACM 3023; 2 paratypes, LACM 3024; 1 paratype, USNM 1018790; 1 paratype, MNHN,; 1 paratype, NHML; 1 paratype, AMS C.205276; 1 paratype, WAM 513781; 1 paratype, MPR. Material examined: 9 specimens, Hanga-Teo on the northern coastline (Dec. 2000, leg. B. Raines). Type locality: In silty mud collected at 15m in cave off Hanga-Teo on the northern coast, Easter Is., Chile, 27” 03” 37” S, 109* 21' 58” W. Derivation of the name: The name of this new species comes from a latinized adjective formed from the Spanish name of the island, “Isla de Pascua”. Description: Shell small (holotype measures, length: 1.96 mm; width: 0.36 mm), gently curved. The tube, evenly cylindrical for almost its entire length, presents a slightly smaller diameter only in the abapical part. Towards the adapical part, the tube widens visibly in a large varix crossed by 5-6 rings that are sizable but raised to various degrees, being sepa- rated by interspaces that also vary in terms of their depth and width. The 60 septum protrudes to a significant extent over the cutting plane with an unguiform mucro, visible to a greater or lesser extent and oriented towards the dorsal side of the tube. Frequently visible on the cutting plane are residues of what may be a tem- porary septum (PIZZINL, NOFRONI AND OLIVERIO, 1998). Even under intensive enlargement, no microsculptures are visible. Circular aperture. Operculum and soft parts unknown. RAINES AND PIZZINI: Revision of the Caecidae of Easter Island (Chile) Figure 11. Caecum crystallinum Folin, 1879. A: holotype NHML 1887.2.9.2363; B: detail of aper- ture. SEM imaging by K. Way. Figura 11. Caecum crystallinum Folin, 1879. A: holotipo NHML 1887.2.9.2363; B: detalle de la abertura. Imágenes al MEB por K. Way. Remarks: Caecum pascuanum presents numerous morphological analogies with C. rehderi, including the shape of the tube and the septum, though it is set apart by a large varix crossed by rings, which is completely absent in the other. In fact, this varix, though it can be accentuated to a greater or lesser extent - for that matter, consistently within the range of variability - is always present and would appear to represent the dis- tinguishing morphological characteris- tics of this species. Geographical distribution: The species is currently known only in its typical location. Caecum rapanuiense spec. nov. (Fig. 10) Type material: Holotype, LACM 3025; 2 paratypes, LACM 3026; 1 paratype, USNM 1018791; 1 paratype, MNHN; 1 paratype, NHML; 1 paratype, NMNZ M.273206; 1 paratype, NSMT Mo 73561; 1 paratype, AMS C.205277; 1 paratype, WAM 513782; 1 paratype, MPR. Material examined: 12 specimens, off the western coastline near Tahai (Dec. 2000, leg. B. Raines). Type locality: Dredged at 30m in fine sand off the western coastline near Tahai, Easter Is., Chile, ZIAOIF207 5, 109926305 WN: Derivation of the name: This species take its title from the ancient name for Easter Is., which was Rapa Nui. Description: Shell small (dimensions of the spm no. 3 from Easter ls., length: 1.5 mm; width: 0.35 mm), slightly curved. Tube completely smooth showing only a microsculpture consist- ing of very weakly defined growth lines. Septum dome-shaped, with the mucro reduced to a small pedunculum, resem- bling a squashed ball, found on the exte- rior and oriented to the right. Aperture simple, with no varix and only slight swelling: further on the swelling tends to contract, with a slightly reflected lip. Growth stage and soft parts unknown. Remarks: Following an initial exam- ination, we tentatively classified this species as C. crystallinum Folin, 1879, despite the absence of the mucro in the original type (Fig. 11), but it is straighter, and the texture of the shell is different, exhibiting under the microscope fine longitudinal striations (Fig. 11B), while C. rapanuiense shows only very fine growth lines (Fig. 10C). It also resembles C. glabriforme Carpenter, 1857, but this species has fairly strong microsculpture and a large well developed septum. In terms 61 Iberus, 23 (1), 2005 Figure 12. Caecum campanulatum spec. nov. A: holotype LACM 3027, gold coated, length 1.96 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 12. Caecum campanulatum spec. nov. A: holotipo LACM 3027, metalizado en oro, longitud 1,96 mm, B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Geiger. of the general shape of the tube and while our species is completely septum, it resembles C. neocaledonicum smooth. Folin, 1868, but the latter shows some Geographical distribution: Caecum raised rings in the abapical part of the tube near the aperture (PIZZINI, 1998), rapanutense is actually known only from Easter Is., the type locality. Caecum campanulatum spec. nov. (Fig. 12) Type material: Holotype, LACM 3027; 1 paratype, LACM 3028; 1 paratype, USNM 1019067. Material examined: 6 specimens, off Hanga Nui (Dec. 2000, leg. B. Raines); 2 specimens, Punta Rosalia, east of Anakena (Apr. 1998, leg. B. Raines). Type locality: In sand collected along the base of cliffs at 20m, off Hanga Nui, Easter Is., Chile, 27” 07” 46” S, 109” 16' 35” W. Derivation of the name: The name of the new species comes from the latinized adjective campan- ulatus, which refers to the bell-shaped form of the apertural end. Description: Shell small (holotype's slightly near the apertural end, with a dimensions, length: 1.64 mm; width: silhouette that closely resembles that of 0.31 mm). Tube slightly arched, slender, with it's abapical part only slightly smaller then adapical. The tube widens 62 a bell, quickly narrowing itself once again and ending in a sharp edge. The microsculpture is quite obsolete and RAINES AND PIZZINI: Revision of the Caecidae of Easter Island (Chile) A 333 pm Figure 13. Strebloceras subannulatum Folin, 1879. A: syntype NHML 1887.2.9.2308-2310, length 3.0 mm; B: detail of septum. SEM imaging by K. Way. Figura 13. Strebloceras subannulatum Folin, 1879. A: sintiyo NHML 1887.2.9.2308-2310, longi- tud 3,0 mm; B: detalle del septo. Imágenes al MEB por K. Way. scarcely visible, even under intensive optical enlargement, while the sculpture would appear to consist of rings, which are also barely observable, though they are more visible near the aperture. The septum is dome-shaped and scarcely raised over the cutting plane. The aper- ture is circular. Colour translucent, with faint axial brown wavy lines. Opercu- lum and soft parts unknown. Remarks: Caecum campanulatum shows some similarities with Caecum amydroglyptum about the general shape of the tube and the mucro, but the latter has a longitudinal microsculpture cover- ing all the tube, while the first one pre- sents only a microsculpture consisting of a scarcely visible growth striation. Besides the species closely resembles another new sp. currently being studied (PIZZINI AND NOFRONI, submitted). Endemic to the Fiji Is., it is also present in other Indian-Pacific zones. Geographical distribution: Caecum cam- panulatum is currently known only on Easter Is., its typical location. Superfamily RISSOOIDEA Gray J. E., 1847 Family CAECIDAE Gray J. E., 1850 Genus Strebloceras Carpenter, 1859 [1858] Diagnosis (BANDEL, 1996): “The protoconch is trochospirally coiled, and the teleoconch is uncoiled forming a slightly curving tube with slowly increasing diameter. Protoconch and teleoconch remain together during the whole life-time”. Type species (BANDEL, 1996): According to COSSMANN (1896) Caecum edwarsil Deshayes, 1864 [Oligocene of France]; according to GOUGEROT AND LE RENARD (1981), Strebloceras lituus Deshayes, 1861. Strebloceras subannulatum Folin, 1879 [not Caecum (Brochina) subannulatum Folin, 1870 (Mediterranean Sea)] (Figs. 13, 14) Type material: 2 syntypes, NHML 1887.2.9.2308-2310. Material examined: Original types, 2 syntypes NHML 1887.2.9.2308-2310; 23 specimens, Punta Rosalia, east of Anakena (Apr. 1998, leg. B. Raines). Voucher material: 2 specimens were deposited in each of the following institutions: LACM; USNM 1018793; MNHN; NHML; NMNZ M.273208; NSMT Mo 73563; AMS C.205279; WAM 63 Iberus, 23 (1), 2005 Bo 100 um Figure 14. Strebloceras subannulatum Folin, 1879. A: Voucher specimen LACM, gold coated, length [») 200 pm 3.3 mm; B: detail of septum; C: microsculpture; D: detail of aperture. SEM imaging by D. Geiger. Figura 14. Strebloceras subannulatum Folin, 1879. A: voucher espécimen LACM, metalizado en oro, lon- gitud 3,3 mm; B: detalle del septo; C: microescultura; D: detalle de la abertura. Imágenes al MEB por D. Geiger. 513784; and MPR. 4 shells, beach of Anakena Bay, N. of Easter Is.: almost exposed coast, picked up among rocky bottom on the west side of the bay, among communities of Dictyotales, with Galaxaura obtusata. (12-1-1995, leg. E. Rolán) MPR. Type locality: Reefs of Honolulu, 40 fms (73m). Original description: “Minute, double- curved head, vitreous, diaphanous and clear; oblique nucleus of spirals; an- fractibus duobus: postea testa tubularia, latitudine acrescens, curvam duplicem sequens, transversim subannulata, an- nulis latis, minutissime expressis, suba- cutis, late separatis: oblique aperture. Long.: 3 mm, lat.: 0 mm 5” (FoLin, 1879). Additional description: Shell small (average length: 3.5 mm, min. diam. 0.18 mm, max diam. 0.7 mm), whitish; larval shell (diam. of the last whorl about 0.23 mm) is slightly trochospiral, consisting of roughly 2 and a half whorls. The tube is separated from the protoconch by an incision which, when seen in side-view, 64 is horseshoe shaped (Fig. 14B); the tube has a double curve that forms itself on two different levels and is crossed by a microsculpture whose abapical portion consists of fine, sinuous growth stria- tions that gradually transform them- selves, as they grow, into fairly clear-cut, visible rings on the adapical portion of the tube. The aperture is perfectly circu- lar, with an almost sharp, oblique edge. The operculum and the soft parts are unknown. Geographical distribution: Described from Honolulu, its distribution has now extended to the Easter Is. as well. Remarks: The specimens found on Easter Is. are wholly identical to the specimens of RAINES AND PIZZINI: Revision of the Caecidae of Easter Island (Chile) the typical series (Figs. 13, 14) and to the specimens from Hawaii (MPR), despite the fact that the tube of the former is slightly CONCLUSIONS As a result of the present comprehen- sive review and revision of Easter Is. Cae- cidae, we discovered an unusual anomaly regarding the family's endemic- ity rate. Unlike other regions, where the rate is fairly low among this family, over 71% of the known caecid species are endemic to the island. This is the highest rate ever observed within any region. ACKNOWLEDGEMENTS We would like to thank Luis DiSalvo (Coquimbo, Chile) for providing material collected during his survey of the island's marine environment; Jerry Harasewych (National Museum of Natural History) for his support and loan of Rehder”s type and voucher material; Paul Scofield and Neil Andrews (Canterbury Museum) for BIBLIOGRAPHY BANDEL, K., 1996. Philogeny of the Caecidae. Mitteilungen adem Geologischen und Paláon- tologischen Institut der Untversitát Hamburg. 79: 539-115. COSSMANN, A. M., 1896. Appendix no.2 au ca- talogue illustre des coquilles fossiles de lÉocéne des environs de Paris. Annales de la Societe Malacologique de Belgique, 31: 1-94. DIsALVvO, L. H., RANDALL, J. E. AND CEA, A,, 1988. Ecological Reconnaissance of Easter Island Sublittoral Marine Environment. National Geographic Research. 4: 451-473. FOLIN, A. G. L. DE, (1879) 1880. On the Mo- llusca of the H.M.S. Challenger Expedition. The Caecidae, comprising the Genera Paras- trophia, Watsonia, and Caecum.. Proceedings of the Zoological Society of London (16 dec. 1879): 52; 806-812 [no pls]. GOUGEROT, L. AND LE RENAROD, J., 1981. Clefs de determination de petites especes de gas- teropodes de l'Éocéne du Bassin Parisien. 18. Le genre Tenuiscala. Cahiers des Naturalistes, 37 (3): 61-68. OLIVER, W. R. B., 1915. The Mollusca of Ker- madec Islands. Transactions and Proceedings of the New Zealand Institute, 47: 509-568. wider than that of the latter; the length is also greater, but this is due to the fact that the specimens in question are adult. their comments and support in providing SEM images of Oliver's type material; Kathie Way (British Museum of Natural History) for providing SEM images of the Folin type material; Daniel Geiger (Santa Barbara Museum of Natural History) for his time and effort on the remaining SEM work; Lindsey Groves (Natural History Museum of Los Angeles County) and Michel Garcia (Sociedad de Explotación y Exploración Marítima Orca, Ltda.) for their continued support; Emilio Rolán (Spain) for sending some specimens. We also wish to thank both Ms. Caterina Ciu- ferri and Ms. Mary Taylor, who helped us with captions in Spanish, and Ms. Viviana Meyohas for her linguistic assis- tance in English, which proved necessary, on occasion, in the exchange of corre- spondence between the first and the sec- ond author. PIZZINL, M., NOFRONI, Í. AND OLIVERIO, M., 1998. Contribution to the knowledge of the family Caecidae. 4. The temporary septum forma- tion of some caecid species (Caenogas- tropoda: Rissooidea), Iberus, 16 (1): 133-140. PIZZINI, M., 1998. Contribution to the knowledge of the family Caecidae. 7. C. fulvum Kisch, 1959 a junior synonym of C. neocaledonicum Folin, 1868 (Caenogastropoda: Rissooidea Gray J. E., 1847). Argonauta, 11 (2): 33-38. PIZZINL, M. AND NOFRONI, l. Revision of The family Caecidae in the South-West Pacific ocean with description of 23 new species (Mollusca: Gastropoda) (submitted). RAINES, B. K., 2002. Contributions to the knowl- edge of Easter Island Mollusca. La Conchiglia. 304: 11-40. REHDER, H. A., 1980. The marine mollusks of Easter Island (Isla de Pascua) and Sala y Gómez. Smithsonian Contribution to Zoology, 289: 1-167. SCHILDER, F. A., 1965. The Geographical Dis- tribution of Cowries (Mollusca: Gastropoda). The Veliger, 7 (3): 171-183. 65 O Sociedad Española de Malacología —__——T— Iberus, 23 (1): 67-76, 2005 The molluscs of the intertidal algal turf in the Azores Los moluscos del cesped algal intermareale en Azores Sérgio P. ÁVILA* **, Ana C. SANTOS*, Ana M. PENTEADO*, Ana M. RODRIGUES*, Inés QUINTINO* and Maria Inés MACHADO* Recibido el 25-VII1-2004. Aceptado el 1-IV-2005 ABSTRACT The molluscan fauna of the high intertidal algal turf on the island of Pico (Azores) was studied in order to complement existing descriptions of the intertidal biota on these islands. A total of 15,275 specimens belonging to 19 species were found. Five species, the gas- tropods Alvania mediolittoralis Gofas, 1989, Omalogyra atomus (Philippi, 1841), Pisinna glabrata (Megerle von Muhlfeld, 1824) and Skeneopsis planorbis (Fabricius O., 1780), and the bivalve Lasaea adansoni (Gmelin, 1791), account for 98% of the total number of specimens, P. glabrata itself being responsible for 46% of all specimens. In places where patellid limpets are absent, the algal turf extends higher in the intertidal zone. In such places, three very abundant molluscan species characterize molluscan assemblages in the algal turf: Lasaea adansoni, Skeneopsis planorbis and Pisinna glabrata. Lasaea adansoni and Skeneopsis planorbis virtually disappear in the transition from algal turf to algal fronds, the only (very) abundant species being P. glabrata and, with less importance, Omalogyra atomus and Sinezona cingulata (Costa O. G., 1861). RESUMEN Se estudia la fauna malacológica del cesped algal del intermareal superior en la isla de Pico (Azores), con el fin de complementar los trabajos ya existentes sobre la biota inter- mareal de estas islas. Se encontraron un total de 15275 ejemplares de 19 especies distin- tas. Cinco, los gasterópodos Alvania mediolittoralis Gofas, 1989, Omalogyra atomus (Philippi, 1841), Pisinna glabrata (Megerle von Muhlfeld, 1824) y Skeneopsis planorbis (Fabricius O., 1780), y el bivalvo Lasaea adansoni (Gmelin, 1791), suman el 98% del total de ejemplares, de ellas P. glabrata representa al 46%. En aquellos lugares donde están ausentes los patélidos, las algas se extienden hasta nive- les superiores en el intermareal. En estas zonas hay tres especies de moluscos que carac- terizan las asociaciones malacológicas algales: Lasaea adansoni, Skeneopsis planorbis y Pisinna glabrata. lasaea adansoni y Skeneopsis planorbis desaparecen en la transición entre el cesped algal y la zona de algas frondosas. La única especie abundante aquí es P. glabrata, y en menor medida Omalogyra atomus y Sinezona cingulata (Costa O. G., 1861). KEYWORDS: micromolluscs, intertidal, algal turf, Azores. PALABRAS CLAVE: micromoluscos, intermareal, cesped algal, Azores. * Departamento de Biologia, Universidade dos Agores, Rua da Máe de Deus, PT-9500 Ponta Delgada, Acores, Portugal. ** CIRN (Centro de Investigacio de Recursos Naturais), Departamento de Biologia, Universidade dos Acores, PT 9500 Ponta Delgada — Acores. E-mail: avilanotes.uac.pt 67 Iberus, 23 (1), 2005 INTRODUCTION Located in the middle of the northern Atlantic and therefore relatively isolated, the oceanic islands that form the Azores archipelago (36” 55' to 39% 45” N, 24? 45' to 31? 17 W) are a “live-laboratory” where patterns and processes of disper- sion, colonization and speciation can be studied and ecological, evolutionary and biogeographical theories be tested. Lajes do Pico, a small town located in the south coast of Pico island, Azores (Fig. 1), is one of the most interesting places in the littoral of the Azores, because of its very diverse marine fauna and flora. As a consequence of a number of biological studies (AZEVEDO, 1990, SANTOS, 1992; AZEVEDO, RODRIGUES, MENDIZABAL AND ARRUDA, 1995; MORTON, BRITTON AND MARTINS, 1996, 1998: ÁviLA, 1998), this area was chosen as worthy of protection to conserve bio- logical diversity; for a detailed review see ÁVILA, ELIAS AND MEDEIROS (2000). Rocky shores in the Azores are usually covered by an intricate mixture of small-sized species of algae forming a characteristic algal turf (HAWKINS, BURNAY, NETO, CUNHA AND MARTINS, 1990; NETO, 1992; NETO AND TITTLEY, 1995). This turf is particularly effective for a number of small animal species, protecting them from wave exposure, excessive temperatures and desiccation (AZEVEDO, 1992). CHAPMAN (1955) was the first author to study the fauna asso- ciated to this algal turf, mainly com- posed of Corallina spp. and he was sur- prised by the great abundance of mol- luscs found in a sample of 10 x 10 cm, collected at Faial Island. AZEVEDO (1992) studied the molluscan species composi- tion, abundance, diversity, seasonal variations and the effect of differences in wave exposure on the high intertidal algal turf of the Azores, on a temporal scale at Sáo Miguel Island. He con- cluded that floristic composition and biomass of the algal turf are fundamen- tal for the molluscan communities in this particular habitat. According to NETO (1992), the “algal turf” is divided into two distinct zones, 68 differentiated by their species-composi- tion and morphology. The higher part is characterized by a “dense and short tangle forming a mat, almost impossible to separate into components” whereas the lower part is composed of frondose and larger algae. We follow her designa- tion and restrict most of this study to the upper part of the “algal turf”. The aim of this study is to incorpo- rate the vertical distribution of micro- molluscs in the general zonation pattern already described for the intertidal of the Azores. MATERIAL AND METHODS Between August 7 and 11, 1995, a 30 m long transect was examined on the gently sloping rocky shore of Lajes do Pico, near “Poca do Pano”, in a moder- ately exposed site (Fig. 1). The shore” profile was drawn, following the spirit leveling method (EMERY, 1961; HAWKINS AND JONES, 1992) and all elevations were related to Chart Datum, Azores (CD), by using sea-level at the time of predicted low-tide. Tidal range in the Azores is small (less than 2 m), for which reason the transect location was carefully chosen. The selected site presents a very gentle slope and, as a result, the transect was 30 m long, a distance very seldom found in Azorean intertidal shores. This procedure minimized problems derived from the blurring effect that occurs in the zonation of organisms, when tran- sects are made in sites with steeper slopes. Five quadrats of 25 x 25 cm were col- lected at 5, 10, 20, 25 and 30 m along one transect (Fig. 2). The highly uniform species composition of the algal turf and the large area used (25 x 25 cm instead of the usual 10 x 10 cm) (BuLLOCck, 1995) minimizes possible sampling problems derived from a single transect without replicates. After the littorinid zone that extended from 0 to 15 m along the tran- sect, and the barnacle zone (10-15 m), 3 quadrats of 25 x 25 cm were scraped from the “algal turf” at 20, 25 and 30 m and the material collected (all of the ÁVILA ET AL.: The molluscs of the intertidal algal turf in the Azores Graciosa Sáo Jorge Azores Archipelago sáo Migue! Atlantic Ocean Poca do Pano Figure 1. Azores archipelago (top), Pico island (bottom, left) and detail of location of Poga do Pano at Lajes do Pico village (Pico Island). Figura 1. Archipiélago de las Azores (arriba), isla de Pico (abajo, izquierda) y detalle de la localización de Poga do Pano y la villa de Lajes do Pico (isla de Pico). algae and contained sediment) put into labelled bags. In the laboratory, this material was washed several times and the animals removed from the algae. Samples were then labelled and pre- served in 70% ethanol. The live-col- lected molluscs were sorted, identified and counted under a binocular dissect- ing microscope. Dominant algae were identified and algal dry weight (g) was determined for each quadrat, after drying for 48 hours at 60 *C. Abundance of molluscs was expressed as density (n/m?) as well as n/100 g ADW (algal dry weight), where n is the number of specimens of the ith species in a quadrate. Species authorities and synonymy of mollusc species follow the CLEMAM database. 69 Iberus, 23 (1), 2005 0 5 10 15 Distance (m) SHA Littorina striata de Melarhapbe neritoides dh Chthamalus stellatus wo Algal turf “ LWST 20 23 30 Figure 2. Transect performed at Poga do Pano (Lajes do Pico, Pico island) and vertical distribution of rocky shore organisms. HWST: mean high water level at spring tides; LWST: mean low water level at spring tides. Figura 2. Transecto realizado en Poga do Pano (Lajes do Pico, isla de Pico) y distribución vertical de organismos de costa rocosa. HWST: nivel medio superior del agua en mareas de primavera: LWST: nivel medio inferior del agua en mareas de primavera. RESULTS Zonation: The “littoral fringe” is bound at the top by a littorinid zone that extends for 15 m along the transect, with a vertical range of 1.7 m, between 0.9 m and 2.6 m above chart datum. This is fol- lowed by a barnacle zone (Chthamalus stellatus (Poli)) with an extension of about 5 m along the transect (between 10 and 15 m), free of molluscs and with a vertical range of about 0.5 m (Fig. 2). After this, an algal turf, dominated by Corallina offic- inalis L., covers the rocky substrate entirely. No limpets were found in this zone, nor molluscs that were common elsewhere, like Stramonita haemastoma (Linnaeus, 1766) or Mitra cornea Lamarck, 1811 (HAWKINS, CORTE-REAL, PANNACCI- ULLI, WEBER AND BISHOP, 2000). Immedi- ately after LWST (mean low water level at spring tides), frondose algae (mainly Pterocladiella capillacea (S.G. Gmelin) and Enteromorpha muscoides (Clem.) Cre- mades, in Cremades et Perez-Cirera replace the coralline turf. Molluscs: A total of 15,275 specimens belonging to 19 species were found in the intertidal algal turf at “Poca do Pano” (Lajes do Pico) (Table I and Fig. 5). Five 7O species, the gastropods Omalogyra atomus (Philippi, 1841), Skeneopsis planorbis (Fabricius O., 1780), Alvania mediolittoralis Gofas, 1989 and Pisinna glabrata (Megerle von Múhlfeld, 1824) (=P. punctulum) and the bivalve Lasaea adansoni (Gmelin, 1791), account for 98% of the total num- ber of specimens, P. glabrata itself being responsible for 46% of all specimens (Table 1). No molluscs were collected in the middle of the barnacle zone. In quadrats 3 and 4, located at 20 and 25 m in the transect, three species dominated: L. adansoni, S. planorbis and P. glabrata (Table II). In the lowermost quadrat, lo- cated at LWST, P. glabrata was clearly the most abundant species, representing about 90% of all specimens in that quadrat. Species density and number of specimens /100g ADW (algal dry weight) generally decreased towards LWST, with the exception of O. atomus and Sinezona cingulata (Costa O. G., 1861) (=Schismope fayalensis Dautzenberg, 1889) (Figs. 3, 4). DISCUSSION The site studied broadly conforms to the zonation pattern described from the ÁVILA ET AL.: The molluscs of the intertidal algal turf in the Azores Table 1. Specific composition of the molluscan fauna inhabiting the intertidal algal turf and total number of specimens collected in each quadrat. Quadrats 1-5 collected at, respectively, 5, 10, 20, 25 and 30 m along transect (see Figure 2 for further details). Tabla I. Composición específica de la fauna de moluscos del cesped algal intermareal y número total de ejemplares recogido en cada cuadrante. Cuadrantes 1-5 obtenidos a 5, 10, 20, 25 y 30 m a lo largo del transecto, respectivamente (ver la Figura 2 para más detalles). — TaxaX Quadrats Alvania mediolittoralis Gotas, 1989 Bittium latreillii (Payraudeau, 1826) Bothryphallus ovummuscae (Gotas, 1990) Cardita calyculata (Linnaeus, 1758) Cingula trifasciata (Adams J., 1798) Gregariella semigranata (Reeve, 1858) (= Trichomusculus semigranatus (Reeve, 1858)) Lasaea adansoni (Gmelin, 1791) Littorina striata King and Broderip, 1832 Manzonia unifasciata Dautzenberg, 1889 Melarhaphe neritoides (Linnaeus, 1758) Odostomia sp. Omalogyra atomus (Philippi, 1841) Pisinna glabrata (Megerle von Múhlfeld, 1824) Rissoella cf. diaphana (Alder, 1848) Runcina cf. adriatica Thompson, 1980 Setia subvaricosa Gotas, 1990 Setia sp. Sinezona cingulata (Costa O. 6., 1861) Skeneopsis planorbis (O. Fabricius, 1780) Total O OOOO O Y OOOO OOOOOouomnN O rocky shores of the Azores by HAWKINS ET AL. (1990) (Caloura, Sáo Miguel Island) and NETO AND AZEVEDO (1990) for Flores Island (see also NETO, 1992, and MORTON ET AL., 1998). These authors divided the vertical zonation of the organisms into three main zones: an upper one, the splash and spray zone, dominated by lit- torinids, lichens and ephemeral (sea- sonal) algae; a middle zone located between HWST (mean high water level at spring tides) and LWST (mean low water level at spring tides), occupied by filter-feeding barnacles in its upper levels and by the algal turf in the lower levels; and the lowermost zone, characterized by the appearance of algal fronds. To this scheme, we may add now the vertical distribution of other molluscs besides the littorinids. In places where patellid limpets are absent, possibly due to 2 3 4 5 Total % 0 40 325 22 387 2,5 0 0 l 6 ] 0.0 0 | 0 0 ] 0.0 0 36 47 6 89 0.6 0 | 0 0 | 0.0 0 0 | 0 | 0.0 DAT | 4229 JE) 6 0 0 0 8 0.] 0 0 0 | | 0.0 3 0 0 0 8 0.] 0 0 3 0 3 0.0 0 9 35 156 287 1.9 DAA OSOS 6,993 45.8 0 3 0 0 3 0.0 0 0 20 0 20 0.1 0 0 0 25 25 0.2 0 0 2 12 14 0.] 0 2 ' 18 86 0.6 DA OA0OS 2 EZ 20.4 9 4246 9169 1,844 15275510010 human overexploitation (HAWKINS ET AL., 1990; HAWKINS ET AL., 2000), the algal turf, usually coralline dominated by Corallina, Jania, Amphiroa or Haliptylon spp. directly attached to the rocky sub- strate (NETO AND TITTLEY, 1995) extends higher in the intertidal zone, a situation also encountered by NETO (1992). In such places of the Azorean rocky shores, three very abundant micromolluscan species characterize and further define the algal turf located in the intertidal zone: Lasaea adansoni, Skeneopsis planorbis and Pisinna glabrata. In the transition from algal turf to algal fronds, Lasaea adansoni and Ske- neopsis planorbis virtually disappear, the only (very) abundant species being P. glabrata and, with less importance, Omal- ogyra atomus and Sinezona cingulata. Most of the species found in the inter- tidal algal turf at Lajes do Pico do not 71 Iberus, 23 (1), 2005 Table II. Density (n/m?) and number of specimens per 100 g algal dry weight (n/100g ADW) of the molluscan fauna inhabiting the intertidal algal turf at Lajes do Pico. For location of quadrats 1, 2, 3, 4 and 5, please refer to Figure 2. Tabla 1]. Densida (n/m?) y número de ejemplares por 100 g de peso seco de algas (n/100g ADW) de la fauna de moluscos del cesped algal intermareal en Lajes do Pico. Para la localización de los cuadranets 1 a 5, ver la Figura 2. Density (n/m?) n/1009 ADW Taxa A Quadrats 2 3 4 5 3 4 5 Alvania mediolittoralis Gotas, 1989 0 0 640 57 30% 208 618 57 Bittium latreillii (Payraudeau, 1826) 0 0 0 16 9% 0 2 16 Bothryphallus ovummuscae (Gotas, 1990) 0 0 16 0 0 5 0 0 Cardita calyculata (Linnaeus, 1758) 0 0 576 152 96 188 89 16 Cingula trifasciata (Adams J., 1798) 0 0 16 0 0 5 0 0 Rissoella cf. diaphana (Alder, 1848) 0 0 48 0 0 16 0 0 Lasaea adansoni (Gmelin, 1791) 0 0 28,976 38,672 16 91432 4,595 3 Littorina striata King and Broderip, 1832 32 9% 0 0 0 0 0 0 Manzonia unifasciata Dautzenberg, 1889 0 0 0 0 16 0 0 3 Melarhaphe neritoides (Linnaeus, 1758) 80 48 0 0 0 0 0 0 Omalogyra atomus (Philippi, 1841) 0 0 1536 560 2,496 500 67 407 Pisinna glabrata (Megerle von Múhlfeld, 1824) 0 O 18,384 68,944 24,560 5,984 8,192 4,008 Runcina cf. adriatica Thompson, 1980 0 0 0 320 0 0 38 0 Setia subvaricosa Gotas, 1990 0 0 0 0 400 0 0 65 Setia sp. 0 0 0 32 192 0 4 31 Sinezona cingulata (Costa 0. 6., 1861) 0 0 32 96 1,248 10 11 204 Skeneopsis planorbis (0. Fabricius, 1780) 0 0 17712 32,048 32 5,766 3,808 5 Gregariella semigranata (Reeve, 1858) 0 0 0 16 0 0 2 0 (= Trichomusculus semigranatus (Reeve, 1858)) Table II. Maximum densities (n/m?) recorded for the Azores. 1: Feteira, Faial Island; 2: Piscina de Santa Cruz, Flores Island, sheltered; 3: Ponta Delgada, Flores Island, exposed; 4: Caloura, Sáo Miguel Island, intertidal, sheltered; 5: Caloura, Sío Miguel Island, intertidal, exposed; 6: Caloura, Sao Miguel Island, infralittoral, sheltered; 7: llhéu de Vila Franca, Sio Miguel Island; 8: *Poga da Barra”, Lajes do Pico, Pico Island, intertidal, sheltered; 9: “Poca do Pano”, Lajes do Pico, Pico Island, intertidal, moderately exposed. Tabla III. Densidades máximas (n/m?) encontradas en las Azores. 1: Feteira, isla de Faial; 2: Piscina de Santa Cruz, isla de Flores, protegido; 3: Ponta Delgada, isla de Flores, expuesto; 4: Caloura, isla de Sáo Miguel, intermareal, protegido; 5: Caloura, isla de Sáo Miguel, intermareal, expuesto; 6: Caloura, isla de Sáo Miguel, infralitoral, protegido; 7: llhéu de Vila Franca, isla de Sáo Miguel; 8: “Poga da Barra”, Lajes do Pico, isla de Pico, intermareal, protegido; 9: “Poga do Pano”, Lajes do Pico, isla de Pico, inter- mareal, moderadamente expuesto. Chapman, 1955 Neto and Azevedo, 1990 Azevedo, 1991 Bullock, 1995 Ávila, 1998 — This work Alvania mediolittoralis 64 (2) 14 (4) 54,400 (7) 160(8) 5,200 (9) Lasaea adansoni 12,200 (1) 17,888 (3) 724 (4) 1,674,400 (7) 1,088 (8) 38,672 (9) Omalogyra atomus 68,046 (6) 16 (8) 2,496 (9) Pisinna glabrata 5,975 (4) 104,100 (7) 32 (8) 68,944 (9) Skeneopsis planorbis 2,000 (1) 4,304 (5) 129,400 (7) 16 (8) 32,048 (9) 2 ÁVILA ET AL.: The molluscs of the intertidal algal turf in the Azores Table IV. Geographical ranges of the most abundant molluscan species inhabiting the intertidal algal turf at Lajes do Pico (see ÁVILA, 2000 and references therein). np: non-planktotrophic type of development (it includes direct development and lecithotrophic development); br: brooding. Tabla IV. Distribución geográfica de las especies de moluscos más abundantes en el cesped algal interma- real de Lajes do Pico (ver ÁVILA, 2000 y las referencias allí citadas). np: desarrollo no planctotrófico (incluyendo desarrollos directo y lecitotrófico); br: desarrollo planctotrófico. Species Type of development Alvania mediolittoralis np Omalogyra atomus np Skeneopsis planorbis np Pisinna glabrata np Lasaea adansoni br extend their distribution to the sublit- toral. ÁviLA (2003) has established the molluscan vertical distribution between 3 and 30 m depths for the Azorean rocky shores covered by algal fronds. He found that the endemic rissoids Alvania angioyi van Aartsen, 1982, Manzonia unifasciata (Dautzenberg, 1889) and Rissoa guernel Dautzenberg, 1889, the also endemic trochid Gibbula delgadensis Nordsieck, 1982, the Macaronesian Anachis avaroides Nordsieck, 1975 and the small bivalve Parvicardium vroomi van Aartsen, Menkhorst and Gittenberger, 1984 were only abundant in shallow water (3 to 5-6 m), whereas Alvanía sleursi (Amati, 1987) was especially abundant below 20 m depth. Bittium latreillii (Payraudeau, 1826), Tricolia pullus azorica (Dautzenberg, 1889) and Jujubinus pseudogravinae Nord- sieck, 1973 were found in large numbers along the whole depth-range, albeit slightly more abundant in the lower levels (ÁviLa, 2003). Thus, as expected because of the different spatial architec- ture of algae, there is a marked difference between the molluscan species composi- tion of the algal turf and that present in algal fronds. In fact, none of the most abundant molluscan species of the algal turf is present in high numbers in algal fronds. The total number of specimens of Alvania mediolittoralis, Lasaea adansoni, Geographical range Azores, Madeira Scandinavia, British Isles, Bay of Biscay, Portugal, Mediterranean, Azores, Madeira, Canary Islands, Cape Verde and Ascension Island Scandinavia, British Isles, Bay of Biscay, Portugal, Mediterranean, Azores, Madeira, Canary Islands and Caribbean Azores, Canary Islands and Mediterranean Scandinavia, British Isles, Bay of Biscay, Portugal, Mediterranean, Azores, Madeira, Canary Islands, Cape Verde, Ascension Island, Saint Helena and Caribbean Omalogyra atomus, Pisinna glabrata and Skeneopsis planorbis collected in 51 quadrats (50 x 50 cm) between 3 and 30 m depth, was only 73 individuals, corre- sponding to about 0.20% of all specimens collected (ÁviLA, 2003). The only species that apparently extends its vertical distri- bution from the lower levels of the inter- tidal algal turf down to the shallow sub- littoral algal fronds (2-3 m depth) is the minute Omalogyra atomus, which is the most abundant species at 2-3 m depth throughout the year (AZEVvEDO, 1991). However, this species is very uncommon at greater depths (ÁviLa, 2003). The abundances of the algal-turf associated molluscan fauna found at Lajes do Pico are in the range of those found by other authors for other islands of the archipel- ago, being intermediate between the very high densities found by BULLOCK (1995) in a very sheltered place (Ilhéu de Vila Franca, Sáo Miguel Island) and the densi- ties found by NETO AND AZEVEDO (1990) and AZEVEDO (1991) (see Table III). Some species found in the quadrats are accidental (e.g. Botryphallus and Cingula) which live under intertidal boulders and not in algal turf. It is noteworthy that 4 out of the 5 most abundant molluscan species in the intertidal of the Azores (the minute Ske- neopsis planorbis and Omalogyra atomus, TAS Iberus, 23 (1), 2005 Distance (m) 1000 4------ 1004 10 Number per 100g algal dry weight 20 25 Distance (m) Alvania mediolittoralis Cardita calyculata Lasaea adansoni Omalogyra atomus Pisinna glabrata Sinezona cingulata A Skeneopsis planorbis 30 Figures 3, 4. Abundance of the molluscs collected in the algal turf at “Poga do Pano” (Lajes do Pico, Pico island). 3: n/m?; 4: n/100 g ADW. ADW: algal dry weight. Figuras 3, 4. Abundancia de moluscos recogidos en el cesped algal en “Poga do Pano” (Lajes do Pico, isla de Pico). 3: n/m?; 4: n/100 g ADW. ADW. peso seco de algas. the rissoid Alvania mediollitoralis and the anabathrid Pisinna glabrata) all have a non-planktotrophic type of develop- ment (ÁvILA, 2000). This type of devel- opment is usually associated with a restricted geographical range (SCHEL- TEMA, 1978; JABLONSKI, 1986), which clearly is not the case (with the excep- tion of A. mediolittoralis) (see Table IV). Perhaps the small size of these gastro- pod species is an advantage for disper- sal, as well as their location in the inter- tidal, therefore having higher possibili- ties of rafting. CONCLUSIONS In the studied algal turf located between the barnacle zone (above) and the frondose algae (below), three species of molluscs are very common: Lasaea 74 adanson1, Skeneopsis planorbis and Pisinna glabrata. In the transition from algal turf to frondose algae, Lasaea adansoni and Skeneopsis planorbis virtually disappear, the only (very) abundant species being P. glabrata and, with less importance, Omalogyra atomus and Sinezona cingulata. ACKNOWLEDGEMENTS We thank Manuel Campos Marques and Rui Pedro Ávila Marques for field assistance. We are also grateful to Frias Martins for correcting the manuscript. Pedro “Bué” Cerqueira and António Moniz (Seccáo de Geografia do Departa- mento de Biologia da Universidade dos Acores) are acknowledged for Figure 1. The author also thanks the comments of two anonymous referees. S.P. Ávila was supported by grant SFERH/BD /5115/2001. ÁVILA ET AL.: The molluscs of the intertidal algal turf in the Azores Y / Í Y a > a So ¿E 100 um Figure 5. Common molluscs of the intertidal zone in the Azores. A: Alvania mediolittoralis Gotas, 1989; B: Bothryphallus ovummuscae (Gofas, 1990); C: Manzonia unifasciata Dautzenberg, 1889; D: Cingula trifasciata (Adams J., 1798); E: Setía subvaricosa Gofas, 1990; E: Pisinna glabrata (Megerle von Muúhlfeld, 1824); G: Bittium latreillii (Payraudeau, 1826); H: Stramonita haemas- toma (Linnaeus, 1766); L, J: Skeneopsis planorbis (O. Fabricius, 1780; K: Melarhaphe neritoides (Linnaeus, 1758); L, M: Cardita calyculata (Linnaeus, 1758). Figura 5. Moluscos comunes en la zona intermareal de las Azores. A: Alvania mediolittoralis Gofas, 1989; B: Bothryphallus ovummuscae (Gofas, 1990); C: Manzonia unifasciata Dautzenberg, 1889; D: Cingula trifasciata (Adams J., 1798); E: Setia subvaricosa Gofas, 1990; F: Pisinna glabrata (Megerle von Múbhlfeld, 1824); G: Bittium latreillii (Payraudeau, 1826); H: Stramonita haemastoma (Linnaeus, 1766); L, J: Skeneopsis planorbis (O. Fabricius, 1780; K: Melarhaphe neritoides (Lin- naeus, 1758); L, M: Cardita calyculata (Linnaeus, 1758). ES Iberus, 23 (1), 2005 BIBLIOGRAPHY ÁviLA, S. P., 1998. Zonacáo intertidal de uma comunidade malacológica numa lagoa costeira localizada na costa Sul da ilha do Pico, Acores. Acoreana, 8(4): 436-486. ÁviLA, S. P., 2000. Shallow-water marine mol- luscs of the Azores: biogeographical rela- tionships. Arquipélago. Life and Marine Sci- ences. Supplement 2 (Part A): 99-131. ÁviLA, S. P., 2003. 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Proteccáo e conservacáo do meio marinho nos Acores. In Saldanha, L.,, Ré, P. and Martins, A. M. de F. (Eds.): Cen- tenaire de la Derniere Campagne Océanographique du Prince Albert de Monaco aux Acores á bord de L'Hirondelle. Acoreana, Suplemento 3: 107-122. SCHELTEMA, R. S., 1978. On the relation between dispersal of pelagic larvae and the evolution of marine prosobranch gastropods. In Battaglia, B. and Beardmore, J. A. (Eds.): Ma- rine Organisms: Genetics, Ecology, and Evo- lution. NATO Conference Series. Series IV: Marine Sciences. Plenum Press, New York. O Sociedad Española de Malacología Iberus, 23 (1): 77-82, 2005 Calyptraea capensis Tomlin, 1931 (Gastropoda, Calyp- traeidae), a valid species from South Africa Calyptraea capensís "Tomlin, 1931 (Gastropoda, Calyptraeidae), una especie válida de Sudáfrica Emilio ROLÁN* Recibido el 14-VII1-2004. Aceptado el 14-IV-2005 RESUMEN La especie del género Calypiraea de Sudáfrica considerada hasta la actualidad como C. chinen- sis se estudia y compara con otras especies de Africa occidental y meridional y de Europa. Se con- cluye que se trata de una especie diferente, cuyo nombre debe ser Calyptraea capensis Tomlin, 1931. ABSTRACT The species of the genus Calyptraea from South Africa up to now considered to be C. chinen- sis is studied and compared with other European and West and South African species. lt is con- cluded that it is a different species whose name should be Calyptraea capensis Tomlin, 1931. KEY WORDS: Calyptraeidae, Calyptraea capensis, Calyptraea chinensis, South Africa. PALABRAS CLAVE: Calyptraeidae, Calyptraea capensis, Calyptraea chinensis, Sudáfrica. INTRODUCTION The study of the genus Calyptraea in the Eastern Atlantic (ROLÁN, 2004) has shown the existence of three species. In this work it was mentioned that the South African species known as Calyp- traea chinensis Linné, 1758 could proba- bly be a different species. TOMLIN (1931) described C. capensis. Agreeing with Tomlin, TURTON (1932: 154) comments that the South African species called C. chinensis by several authors, should be called C. capensis. Nevertheless, this species has been recorded from South Africa in the most recent literature under the name C. chi- nensis (BARNARD, 1963; KENSLEY, 1973; KILBURN AND RIPPEY, 1982; STEYN AND Lussi, 1998, among others. In order to check if this species was dif- ferent from those previously described in ROLÁN (2004), material from the Natal Museum was examined and compared with that previously studied and illustrated from the West African coasts. The conclu- sion is that it is a different species and which should not be called C. chinensis but C. capensis. The diagnostic characters of this species are shown in the present work. Abbreviations: NMW: National Museum of Wales, Cardiff NM: Natal Museum, Pietermaritzburg sp: specimen with soft parts s: Shell e Museo de Historia Natural, Campus Universitario Sur. 15782 Santiago de Compostela. e-mail: emiliorolanGinicia.es M7 Iberus, 23 (1), 2005 RESULTS Family CALYPTRAEIDAE Lamarck, 1809 Genus Calyptraea Lamarck, 1799 Calyptraea capensis Tomlin, 1931 (Figs. 1-13, 17-19) Calyptraea capensis Tomlin, 1931. Annals of the Natal Museum, 6: p. 428, pl. 36, fig. 6. [Type locality: Jeffreys Bay, South Africa]. Calyptraea chinensis auct. non Linné, 1758. Type material: Holotype (NMW (1955.158.01006, Figs. 7, 8). Material studied (all from NM): W1093, 1 sp, S. of Cape St. Blaize, 34? 43.5” S 22” 09 E, 88 m, NMDP (Africana) St. A16562D; S8593, 8 s, False Bay, off Falk Bay (34* 08.5” S 18* 28” E), 18 m, sand with Pyura, NMDP CD29 (10.1V.1991); 7316, 1 s, Port Elizabeth; Amsterdam Neek, on Zwartkops River; B2887, 47 s, Pondoland Coast, H. Becker coll (1978); B2886, 13 s, Port Alfred, H. Becker coll. (1978); D2211, 2 sp, Algoa Bay; 1 sp, living inside outer lip of hermit crab shell Amalda obtusa, 49 m, W of Stilbaai, 34” 29” S 21” 16' E; V603, 2 sp, W of Stilbaai, (34? 29” S; 21” 16' E), 49 m, B2886, 13 s, Port Alfred; 6025, 16 s, Saldanha Bay; 7316, 1 s, Port Elisabeth, Amsterdam Nock, Zwartkops River; E6457, 3 s, Cape Agulhas, Struinbari, Caravan Park; S8121, 2 s, SW of Struis Bay (34” 46.6” S; 20? 09.4” E), 34 m, S3557, 1 s, False Bay (34* 11.2” S; 18” 35.8” E), 40 my; B8224, 5 s, E. Cape, off East London (33* 04.9” S; 27” 54” E), 70 m; A3454, 4 s, False Bay, Muizen- berg; B7690, 1 s, Port Elisabeth; B7677, 1 s, Port Elisabeth, Humewood Beach, S6353, 3 s, Agulhas Bank, W of Martha Point (34? 29.5” S; 20? 33.3” E, 28 m, V186, 3 s, 3 s, Agulhas Bank, W of Martha Point (34? 24.8” S; 20? 53.4 E, 31 m; C4677, 1 s, Transkei, of Qolora (32? 39.7" S; 28” 28.2' E), 50 m; B7870, 1 s, E. Cape, off East London (33* 01.0” S; 27” 57.3” E), 30 m,; B8466, 7 s, E. Cape, off East London (33* 06.2” S; 27” 52.4” E), 70 m; E8887, 2 s, Zululand, off Matigulu River mouth (29* 21.6' S; 31? 57.1” E), 300 m, S9109, 1 s, SW Cape, SE of Cape Infanta (34” 24.8” S; 20? 53.4” E), 31 m; C4309, 1 s, Transkei, off Stony Point (32* 32.8” S; 28” 38.2 E), 70 m; C3467, 1 s, Transkei, Kei River; S3624, 3 s, Cape, off Simonstown (34* 10.7” S; 18” 28.4” E), 31 m; E8940, 1 s, Zululand, off Matigulu River mouth (29? 22.2” S; 31? 57.2” E), 350 m, A3456, 4 sp, Fish Hock; 9068, 2 s, Jeffreys Bay; B7657, 2 s, Durban; A3455, 44 s, False Bay, Simonstown dredgings; B2887, 47 S, Pondoland Coast; S8443, 1 sp, False Bay, SE Seal Island (34* 11.5' S; 18? 37.1” E), 43 m; W1094, W1096, 2 sp with eggs, E. Cape, off East London (33* 06.8” S; 27” 51.4' E), 10 m. Description: The shell (Figs. 1-8) has been described in TOMLIN (1931) and KILBURN AND RIPPEY (1982). The color may be white, pink, light brown or violet, and the surface is usually smooth, but some shells from deeper water from Zululand have some spicules. Sometimes, its external surface is undulating towards the sutural depression. Protoconch (Figs. 12, 13) with about 1*/2 whorls, a diameter of about 900 um, and a nucleus with a diameter of 85-100 um, the protoconch surface is totally smooth. A few radial lines appear in the teleoconch of some shells, near the protoconch. Dimensions: up to 29.0 mm (in lot 9068). Soft parts: No live material was examined, but some lots had been pre- served in alcohol. Two males (10.3, 11.1 78 and 10.3 mm) with dry soft parts were hydrated in water with soap, and show that the animal (Figs. 17, 18) has a similar aspect to that of C. chinensis from Europe. Only the penis (Fig. 19) seems to be different: it was placed behind the right tentacle, being elongated, appar- ently flat, and placed on the dorsum of the animal and towards the posterior part. The tip is a little wider and finishes in a fine prolongation. Two females of 13 and 15 mm examined, have a small vestigial penis, elongated and sharp pointed without any wider part. Radula (Figs. 9-11) taenioglossate; rachidian tooth narrow and with a prominent central cusp on the edge, with 4, sometimes 5-6, smaller cusps at each side. Lateral tooth with a promi- nent and a little wider cusp at the con- ROLÁN: Calyptraea capensis Tomlin, 1931, a valid species from South Africa Figures 1-8. Calyptraea capensis. 1-3: 13.4 mm, Cape St. Blaize, 88 m (NM W1093); 4-6: 13.5 mm, Saldanha Bay (NM 6025); 7, 8: holotype, 10.2 mm (MNW). Figuras 1-8. Calyptraea capensis. 1-3: 13,4 mm, Cabo St. Blaize, 88 m (NM WI1093); 4-6: 13,5 mm, Bahía Saldanha (NM 6025); 7, 8: holotipo, 10,2 mm (MNW). YO Iberus, 23 (1), 2005 Figures 9-11. Radula of Calyptraea capensis. 9: female, specimen of 13.8 mm (NM W1093); 10, 11: males of 11.1 and 10.3 mm (NM S8593). Figures 12-16. Protoconchs. 12, 13: C. capensis, off East London; 14: C. chinensis, Vigo; 15: C. imexpectata, Guinea-Conakry; 16: C. africana, Ivory Coast. Figuras 9-11. Rádula de Calyptraea capensis. 9: hembra, ejemplar de 13,8 mm (NM W1093); 10, 11: machos de 11,1 and 10,3 mm (NM $8593). Figuras 12-16. Protoconchas. 12, 13: C. capensis, costa de East London; 14: C. chinensis, Vigo; 15: C. inexpectata, Guinea Conakry; 16: C. africana, Costa de Marfil. 80 ROLÁN: Calyptraea capensis Tomlin, 1931, a valid species from South Africa Figure 17-19. Drawings of soft parts. 17: detail of the head of a female with a 15 mm shell, ventral view; 18: a male of 10 mm with penis, dorsal view; 19: detail of the penis. Figuras 17-19. Dibujo de las partes blandas. 17: detalle de la cabeza de una hembra con una concha de 15 mm, en visión ventral; 18: macho de 10 mm con el pene, en visión dorsal; 19: detalle del pene. fluence of the internal and the external edges. Internal edge with few wide cusps (usually 1-2), while the external has between 5-7. Marginal teeth elon- gate, curved, sharp pointed, the internal ones with two edges with 4-6 cusps, while the external teeth have only 1-2 cusps on their internal part. Spawn: several spawns were exam- ined. They have a similar disposition to that of C. chinensis, the capsules being more irregular. A specimen of 15 mm diameter had a spawn of 12 capsules, each one between 2-3 mm, with between 20-60 embrionary larvae inside. Remarks: The holotype has an eroded protoconch but all other characters fit well with most of the studied material. Some variations were found in the numerous shells examined: most shells are depressed, but some are extremely elevated, even higher than wide. We could not determine if these elevated forms are a different species, due to the lack of protoconch in the specimens examined. The shell of C. capensis is rather dif- ferent from the West African species. C. chinensis is less pointed at the apex of the shell, and has external prominences more frequently. The penis is different, with a spoon shape and a wide prolon- gation in opposition. The rachidian and marginal teeth of the radula are very similar, but the lateral teeth have more cusps (3-4) on the internal side; the pro- toconch (Fig. 14) is wider, shorter (1 against 1!/2 whorls) and the nucleus larger; furthermore, the protoconch of C. capensis is smooth and the beginning of the teleoconch has some inconspicuous radial lines never present in C. chinensis. C. africana Rolán, 2004 has a larger shell, more depressed, a little ovoid, usually translucent white, frequently with elevated external prominences; the penis is bilobed at its extreme, with one of the lobes formed by two masses together, and a narrow worm-like fila- ment near the tip; the protoconch (Fig. 16) is rather similar, but the nucleus is somewhat smaller. The radula has more cusps in all the teeth. C. inexpectata Rolán, 2004 has a smaller shell, sometimes with a squa- mous sculpture, white or brown in colour, but never violet; the protoconch (Fig. 15) is smaller, with a wider nucleus and several evident sulcii at the begin- ning of the teleoconch. All the radular teeth have more cusps. There are no problems in separating the remaining South African species of Calyptraea: C. helicoidea Sowerby, 1883, has a subscalariform profile, with a narrow but distinct umbilicus and the dorsal surface has strong, narrow, rather oblique radial ribs. C. aurita Reeve, 1859 also has an umbilicus, the columella is widely reflexed, and the protoconch has clear radiating spiral lirae (see BARNARD, 1963: 73). 81 Iberus, 23 (1), 2005 ACKNOWLEDGEMENTS The author wishes to thank the fol- lowing: D. G. Herbert of the Natal Museum, who loaned the material studied in this work; Harriet Wood of the National Museum of Wales, Cardiff, for the information and photographs of the holotype of C. capensis; Jesús BIBLIOGRAPHY BARNARD, K. H., 1963 “1962”. Contribution to the knowledge of the South African marine Mollusca. Part III. Gastropoda: Proso- branchiata: Taenioglossa. Annals of the South African Museum, 47(1): 1-199. KENSLEY, B., 1973. Sea-shells of Southern Africa. Gastropods. Maskew Miller Ltd., Cape Town, 236 pp. KILBURN, R. AND RIPPEY, E., 1982. Sea Shells of Southern Africa. MacMillan South Africa, Jo- hannesburg, 249 pp. ROLÁN, E., 2004. The genus Calyptraea (Gas- tropoda, Caenogastropoda, Calyptraeidae) in the East Atlantic. Iberus, 22(2): 51-79. 82 Méndez of the Centro de Apoyo Cientí- fico y Tecnológico a la Investigación (CACTI) of Vigo University, who made the SEM micrographs, and Jesús S. Tron- coso of the Department of Ecology of the University of Vigo, for the digital optical photographs. STEYN, G. S. AND Lusst, M., 1998. Marine Shells of South Africa. Ekogilde, Hartebeespoort, 264 pp. TOMLIN, J. R. LE B., 1931. On South African ma- rine mollusca, with descriptions of new gen- era and species. Annals of the Natal Museum, 6: 415-450. TURTON, D. $. O., 1932. The marine shells of Port Alfred South Africa. Oxford University Press, London, 331 pp., 70 pls. O Sociedad Española de Malacología Iberus, 23 (1): 83-86, 2005 Notas breves Additional information on the biology of Argonauta argo (Cephalopoda: Octopoda) in the Mediterranean Sea from gastrointestinal contents of Risso's dolphin Aportación a la biología de Argonauta argo (Cephalopoda: Octopoda) en el mar Mediterráneo a partir del contenido gastroin- testinal del delfín de Risso Carmen BLANCO*, Ángeles RADUÁN* and Juan Antonio RAGA* Recibido el 27-VII1-2004. Aceptado el 14-IV-2005 KEY WORDS: Argonauta argo, Grampus griseus, cephalopod biology, Mediterranean. PALABRAS CLAVE: Argonauta argo, Grampus griseus, biología cefalópodos, Mediterráneo. INTRODUCTION Argonguta argo Linnaeus, 1758 is a cosmopolitan epipelagic octopod living in tropical and subtropical waters. It is sexually dimorphic, with dwarf males and females lodged in calcareous shells that serve as brood chambers (GUERRA, 1992). Despite its abundance, whole animals are rarely caught, so that their life cycle is not completely known. The reproduction of this species shows a protracted and continuous spawning which has been defined as asynchro- nous ovulation with monocycle spawn- ing reproductive strategy (ROCHA, GUERRA AND GONZÁLEZ, 2001). Living individuals of this species are rarely found in the Mediterranean Sea (BOLETZKY AND CENTELLES, 1979; BELLO AND RIZZI, 1990; POPPER, BARASH AND GALIL, 1990). Continuous spawning is believed to take place in the eastern Mediterranean Sea (LAPTIKHOVSKY AND SALMAN, 2003), but the life span and period of reproduction of the species continue to be unknown. The aim of this paper is to provide some data on the biology of this little known Mediter- ranean species by means of remains col- lected from the gastrointestinal contents of Risso's dolphins, Grampus griseus (Cuvier, 1812) (Cetacea: Delphinidae), a teuthivorous predator (KRUSE, CALD- WELL AND CALDWELL, 1999). MATERIAL AND METHODS The data analysed in this paper came from gastrointestinal contents of eleven Rissos dolphins, Grampus griseus, stranded on the west Mediter- ranean coast, between 40" 25' N, 00% 32* * Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, P. O. Box 22085, 46100 Valencia, Spain. E-mail: carmen.blancoCuv.es 83 Iberus, 23 (1), 2005 Ve Zea da Má as 61171 3,49 O A. argo =0— 6. griseus 3 0 10, AL A2 Figure 1. Annual distribution of specimens of Argonauta argo from gastrointestinal contents of Mediterranean Risso's dolphin. N: number of A. argo; n: number of dolphins. Figura 1. Distribución anual de los ejemplares de Argonauta argo obtenidos del contenido gastrointesti- nal del delfín de Risso en el Mediterráneo. W and 37* 35' N, 00% 45 E, collected from April 1987 to January 2003. All gas- trointestinal tracts were stored deep- frozen (-20%C) and their contents subse- quently washed through a 0.2 mm mesh sieve and preserved in 70% ethanol; glycerine was added to the preservative solution for cephalopod beaks. The beaks were identified according to SMALE, CLARKE, KLAGES AND ROELEVELD (1993). Cephalopod mantle length was estimated from hood length of lower beak (SMALE ET AL., 1993), due to the frequent rotten condition of the crest. Some underestimating of hood length and, hence, mantle length may be assumed because of the digestive action. RESULTS In the gastrointestinal contents of Risso's dolphins, 336 lower beaks of Argonauta argo were found, hood length range: 1.31-5.87 mm. Distribution through the time period for which data were available (see Figure 1) shows a higher number in early spring. The esti- mated mantle lengths are shown in Figure 2. Significant differences were found in the size of specimens through- out the year (nested ANOVA (Fos25 = 11.1, P < 0.001). The frequency distribu- tion of beak length (HL) in the months of maximum abundance, March and 84 April, is shown in Figure 3; significant length distribution differences were found between these two months (Kol- mogorov-Smirnov test, maximum dif- ferences between samples= 0.268, N (192, 99), P< 0.001), 25.6-28.7 mm and 31.8-35.0 mm being the estimated mantle length of modal class in March and April, respectively. In all available samples there were individuals whose mantle length was longer than 35 mm. An oviscapte with eggs (modal value size 1.22-1.42 mm length) was found in the intestine of a dolphin stranded in January; this value may be underesti- mated due to the possible shrinking action of preservation in alcohol. DISCUSSION The high number of beaks from A. argo gathered in this study, in contrast with the scarcity of their catches (see the Introduction), corroborates the effective- ness of teuthophagous predators as cephalopod collectors (BELLO, 1996). All remains of A. argo found in this study, apart from one collected in April, were ascribed to female specimens based on their size, since dwarf males only reach 10 mm of dorsal mantle length (GUERRA, 1992). In accordance with the smallest female size at maturity in the Mediterranean, i. e. 32 mm ML BLANCO ET AL.: Argonauta argo in gastrointestinal contents of Risso's dolphin 3 El March £ > > April LAS DORADO A Months EDO LAS E SA l, Beak length (HL mm) Figure 2. Annual length distribution of estimated mantle length (ML) of Argonauta argo from gas- trointestinal contents of Mediterranean Risso's dolphin. Box-and-whisker-plot (TUKEY, 1977). Figure 3. Frequency distribution of lower beak length (HL) of Argonauta argo in March and April. Figura 2. Distribución anual de la longitud estimada del manto (ML) de Argonauta argo a partir del contenido gastrointestinal del delfín de Risso en el Mediterráneo. Gráfico “Box-and-whisker” (TUKEY, 1977). Figura 3. Distribución de la frecuencia de la longitud de la mandíbula inferior (HL) de Argo- nauta argo en marzo y abril. (LAPTIKHOVSKY AND SALMAN, 2003), ma- ture females were present in all sampled seasons. Such a find and the occurrence of fertilized eggs in the brooding cham- ber from January, in addition to brood- ing females collected in the Adriatic in November (BELLO AND RIZZ1, 1990) and mature females found in the eastern Mediterranean Sea during winter (LAp- TIKHOVSKY AND SALMAN, 2003), show that the spawning period for Mediter- ranean A. argo extends longer than pre- viously believed (from May to October according to GUERRA, 1992). Differences BIBLIOGRAPHY BELLO, G., 1996. Teuthophagous predators as collectors of oceanic cephalopods: the case of the Adriatic Sea. Bollettino Malacologico, 32 (1- 4): 71-78. BELLO, G. AND RIZZI E., 1990. Comportamiento di tre femmine di Argonauta argo in acquario (Cephalopoda: Argonautidae). Atti della Societa Italiana di Scienze Naturali e del Museo Cívico di Storia Naturale di Milano, 131: 450-452. BOLETZKY, S. V. AND CENTELLES, J., 1979. Argo- nauta argo (Mollusca, Cephalopoda) dans la région de Banyuls-sur-mer. Vie et Milieu, 28- 29 (4): 659-660. GUERRA, A., 1992. Mollusca. Cephalopoda. In Ramos, M. A. et al. (eds.): Fauna Ibérica, vol. 1. Museo Nacional de Ciencias Naturales, CSIC, Madrid, 327 pp. in the size of specimens through the year, especially in the two consecutive months could indicate either seasonality or a preferential period of spawning time. ACKNOWLEDGEMENTS This study was supported by Con- sellería de Territorio y Vivienda, Gener- alitat Valenciana, and Dirección General de Conservación de la Naturaleza, Min- isterio de Medio Ambiente. KRUSE, S., CALDWELL, D. K. AND CALDWELL, M. C., 1999. Risso's dolphin Grampus griseus (G. Cuvier, 1812). In Ridgway, S. H. and Harrison, R. (eds.): Handbook of Marine Mammals, Vol. 6, pp. 183-212. Academic Press, London. LAPTIKHOVSKY, V. AND SALMAN, A., 2003. On reproductive strategies of the epipelagic oc- topods of the superfamily Argonautidea (Cephalopoda: Octopoda). Marine Biology, 142: 321-326. PopPPER, D., BARASH, A. AND GALIL, B. S., 1990. Argonauta argo — A rare occurrence off the shores of Israel. Israel Journal of Zoology, 37: 51-53. ROCHA, F., GUERRA, A. AND GONZÁLEZ, A. F,, 2001. A review of reproductive strategies in cephalopods. Biological Review, 76: 291-304. 85 Iberus, 23 (1), 2005 SMALE, M. J., CLARKE, M. R., KLAGES, T. W. TUKEY, J. W. 1977. Bob-and-whisker-Plot. In AND ROELEVELD, A. C., 1993. Octopod beak Explanatory data analysis, pp. 39-43. Addison- identification — resolution at a regional level Wesley, Massachusetts. (Cephalopoda, Octopoda: Southern Africa). South African Journal of Marine Science, 13: 269-293. 86 NORMAS DE PUBLICACIÓN * La revista /berus publica artículos de fondo, notas y monografías que versen sobre cualquiera de los aspectos relacio- nados con la Malacología. Se entiende por artículo un trabajo de investigación de más de 5 páginas de texto, meca- nografiadas, incluidas láminas, gráficos y tablas. Las notas son trabajos de menor extensión. 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Cuando un táxon aparezca por primera vez deberá citarse su autor y fecha de su descripción. En el caso de artículos sistemáticos, cuando se den las sinonimias de los táxones, éstas deberán citarse COMPLETAS, incluyendo en forma abreviada la publicación donde fueron des- critas, y la localidad tipo si es conocida entre corchetes, según el siguiente esquema (préstese especial cuidado a la pun- tuación): Dendrodoris limbata (Cuvier, 1804) Sinonimias Doris limbata Cuvier, 1804, Ann. Mus. H. N. Paris, 4 (24): 468-469 [Localidad tipo: Marsella]. Doris nigricans Otto, 1823, Nov. Act. Ac. Caes. Leop. Car., 10: 275. Dichas referencias no deberán incluirse en la lista de Bibliografía si es la única vez que se nombran en el texto. Si se incluyen una lista completa de referencias de un taxon inmediatamente tras éste, deberá seguirse el mismo esque- ma (sin incluir en Bibliografía las referencias que no se mencionen en otro lugar del texto). * Sólo los nombres en latín y los de táxones genéricos y específicos deberán llevar subrayado sencillo o preferentemente ir en cursiva. En ningún caso deberá escribirse una palabra totalmente en letras mayúsculas, ni siquiera el Título. Las unidades a utilizar deberán pertenecer al Sistema Métrico Decimal, junto con sus correctas abreviaturas. En artículos escritos en castellano, en los números decimales sepárese la parte entera de la decimal por una coma inferior (,), NUNCA por un punto (.) o coma superior (*). * Las referencias bibliográficas irán en el texto con minúsculas o versalitas: Fretter y Graham (1962) o FRETTER Y GRAHAM (1962). Si son más de dos autores se deberán citar todos la primera vez que aparecen en el texto [Smith, Jones y Brown (1970)] empleándose et al. las siguientes veces [Smith et al. (1970)]. Si un autor ha publicado más de un trabajo en un año se citarán con letras: (Davis, 1989a; Davis, 1989b). No deberá emplearse op. cit. La lista de refe- rencias deberá incluir todas las citas del texto y sólo éstas, ordenadas alfabéticamente. Se citarán los nombres de todos los autores de cada referencia, sea cual sea su número. Los nombres de los autores deberán escribirse, en letras minús- culas o VERSALITAS. No deberán incluirse referencias a autores cuando éstos aparezcan en el texto exclusivamente como autoridades de un taxon. Los nombres de las publicaciones periódicas deberán aparecer COMPLETOS, no abreviados. Cuando se citen libros, dése el título, editor, lugar de publicación, n* de edición si no es la primera y número total de páginas. Deberán evitarse referencias a Tesis Doctorales u otros documentos inéditos de difícil con- sulta. Síganse los siguientes ejemplos (préstese atención a la puntuación): Fretter, V. y Graham, A., 1962. British Prosobranch Molluscs. Ray Society, London, 765 pp. Ponder, W. F., 1988. The Truncatelloidean (= Rissoacean) radiation - a preliminary phylogeny. En Ponder, W. F. (Ed.): Prosobranch Phylogeny, Malacological Review, suppl. 4: 129-166. Ros, J., 1976. Catálogo provisional de los Opistobranquios (Gastropoda: Euthyneura) de las costas ibéricas. Miscelánea Zoolgica, 3 (5): 21-51. * Las gráficas e ilustraciones deberán ser originales y presentarse sobre papel vegetal o similar, con tinta china negra y ajustadas al formato de caja de la revista o proporcional a éste. Este formato es de 57 mm (una columna) o 121 mm (dos) de anchura y hasta 196 mm de altura, si bien se recomienda utilizar el formato a dos columnas. En caso de pre- parar figuras para que ocupen el total de una página, se ruega ajustar su tamaño para que puedan caber los pies de figura bajo ella. Si han de incluirse gráficas de ordenador, deberán imprimirse con impresora láser sobre papel de buena calidad. Las fotografías, bien contrastadas y sin retocar, deberán ajustarse siempre a los tamaños mencionados. Al componer fotografías sobre una hoja, procúrese que los espacios entre ellas sean regulares y que estén debidamente alineadas. Téngase en cuenta que incluir fotografías de distinto contraste en una misma página conlleva una pobre reproducción final. Las escalas de dibujos y fotografías deberán ser gráficas, y las unidades que se utilicen del sistema métrico decimal. Considérese la reducción que será necesaria a la hora de decidir el tamaño de las escalas o letras en las figuras, que no deberán bajar de los 2 mm. En figuras compuestas, cada parte deberá etiquetarse con letras mayús- culas, el resto de las letras deberán ser minúsculas. No deberán hacerse referencias a los aumentos de una determi- nada ilustración, ya que éstos cambian con la reducción, por lo que debe emplearse una escala gráfica. En su caso, se recomienda la utilización de mapas con proyección UTM. Cada figura, gráfica o ilustración deberá presentarse en hojas separadas y con numeración arábiga (1, 2, 3,...), sin separar “Figuras” y “Láminas”. Los pies de figura, en una hoja aparte, deberán acompañarse de su traducción al inglés (o español si el inglés es la lengua del trabajo). Utilícese el esquema siguiente: Figura 1. Neodoris carví. A: animal desplazándose; B: detalle de un rinóforo; C: branquia. Las abreviaturas empleadas en las ilustraciones deberán incluirse en la hoja de pies de figura. Los autores interesados en incluir láminas en color deberán abonarlas a precio de coste (30.000 ptas por página). Por lo demás, deberán ajustarse a los mismos requisitos que los indicados para las figuras. Si se pretende enviar gráficas o ilustraciones en formato digital es IMPRESCINDIBLE contactar previamente con el Editor e Las Tablas se presentarán en hojas separadas, siempre con numeración romana (1, IL, [IL...). Las leyendas se inclui- rán en una hoja aparte acompañándose de una traducción al inglés. Deberán evitarse las tablas particularmente com- plejas. Se recomienda reducir el número y extensión de ilustraciones, láminas o tablas al mínimo necesario. * Los artículos que no se ajusten a las normas de publicación serán devueltos al autor con las indicaciones de los cam- bios necesarios. e El Comité Editorial comunicará al autor responsable del trabajo la fecha de recepción del trabajo y la fecha de envío a revisión. Cada original recibido será sometido a revisión por al menos dos investigadores. El Comité Editorial, a la vista de los informes de los revisores decidirá sobre la aceptación o no de cada manuscrito. El autor recibirá en cada caso copia de los comentarios de los revisores sobre su artículo. En caso de aceptación, el mismo Comité Editorial, si lo considera conveniente, podrá solicitar a los autores otras modificaciones que considere oportunas. Si el trabajo es aceptado, el autor deberá enviar una copia impresa del mismo corregida, acompañada por una versión en disco flexi- ble (diskette), utilizando procesadores de texto en sus versiones de DOS o Macintosh. La fecha de aceptación figura- rá en el artículo publicado. * Las pruebas de imprenta serán enviadas al autor responsable, EXCLUSIVAMENTE para la corrección de erratas, y deberán ser devueltas en un plazo máximo de 15 días. Se recomienda prestar especial atención en la corrección de las pruebas. * De cada trabajo se entregarán gratuitamente 50 separatas. Aquellos autores que deseen un número mayor, deberán hacerlo constar al devolver las pruebas de imprenta, y NUNCA POSTERIORMENTE. El coste de las separatas adi- cionales será cargado al autor. INSTRUCTIONS TO AUTHORS e Iberus publishes research papers, notes and monographs devoted to the various aspects of Malacology. Papers are manuscripts of more than 5 typed pages, including figures and tables. Notes are shorter papers. Monographs should exceed 50 pages of the final periodical, and will be published as Supplements. Authors wishing to publish monographs should contact the Editor. Manuscripts are considered on the understanding that their contents have not appeared or will not appeared, elsewhere in substantially the same or any abbreviated form. + Manuscripts and correspondence regarding editorial matters must be sent to: Serge Gofas, Editor de Publicaciones, Departamento de Biología Animal, Universidad de Málaga, Campus de Teatinos, s/n, 29071, Málaga, Spain. + Manuscripts may be written in any modern language. * When a paper exceeds 20 pages, extra pages will be charged to the author(s) at full cost. + Manuscripts must be typed double spaced (including the references, figure captions and tables) on one side on A-4 (297x210 mm) with margins of at least 3 cm. An original and two copies must be submitted, together with a floppy disk containing the article written with a DOS or Macintosh word processor. When a paper has joint authorship, one author must accept responsability for all correspondence. * The authors must include a list of at least 4 possible referees; the Editor can choose any others if appropriate. * Papers should conform the following layout: First page. This must include a concise but informative title, with mention of family of higher taxon when appropri- atte, and its Spanish translation. It will be followed by all authors? names and surnames, their full adress(es), an abstract (and its Spanish translation) not exceeding 200 words which summarizes not only contents but results and conclusions, and a list of Key Words (and their Spanish translation) under which the article should be indexed. Following pages. These should content the rest of the paper, divided into sections under short headings. Whenever possible the text should be arranged as follows: Introduction, Material and methods, Results, Discussion, Conclusions, Acknowledgements and References. Unusual abbreviations used in the text must be grouped in one alphabetic sequence after the Material and methods section. * Notes should follow the same layout, without the abstract. + Footnotes and cross-references must be avoided. The International Codes of Zoological and Botanical Nomenclature must be strictly followed. The first mention in the text of any taxon must be followed by its authori- ty including the year. In systematic papers, when synonyms of a taxon are given, they must be cited IN FULL, includ- ing the periodical, in an abbreviate form, where they were described, and the type localities in square brackets when known. Follow this example (please note the punctuation): Dendrodoris limbata (Cuvier, 1804) Synonyms Doris limbata Cuvier, 1804, Ann. Mus. H. N. Paris, 4 (24): 468-469 [Type locality: Marseille). Doris nigricans Otto, 1823, Nov. Act. Ac. Caes. Leop. Car., 10: 275. These references must not be included in the Bibliography list, except if referred to elsewhere in the text. I£a full list of references of the taxon is to be given immediately below it, the same layout should be followed (also excluding those nowhere else cited from the Bibliography list). Only Latin words and names of genera and species should be underlined once or be given in ¿talics. No word must be written in UPPER CASE LETTERS. SI units are to be used, together with their appropriate symbols. In Spanish manuscripts, decimal numbers must be separated with a comma (,), NEVER with a point (.) or upper comma (*). » References in the text should be written in small letters or SMALL CAPITALS: Fretter and Graham (1962) or FRETTER AND GRAHAM (1962). The first mention in the text of a paper with more than two authors must include all of them [Smith, Jones 82 Brown (1970)], thereafter use et al. [Smith et al. (1970)]. Ifan author has published more than one paper per year, refer to them with letters: (Davis, 1989a; Davis, 1989b). Avoid op. cit. The references in the reference list should be in alphabetical order and include all the publications cited in the text but only these. ALL the authors of a paper must be included. These should be written in small letters or SMALL CAP- ITALS. The references need not be cited when the author and date are given only as authority for a taxonomic name. Titles of periodicals must be given IN FULL, not abbreviated. For books, give the title, name of publisher, place of publication, indication of edition if not the first and total number of pages. Keep references to doctoral theses or any other unpublished documents to an absolute minimum. See the following examples (please note the punctuation): Fretter, V. and Graham, A., 1962. British Prosobranch Molluscs. Ray Society, London, 765 pp. Ponder, W. F., 1988. The Truncatelloidean (= Rissoacean) radiation - a preliminary phylogeny. In Ponder, W. F. (Ed.): Prosobranch Phylogeny, Malacological Review, suppl. 4: 129-166. Ros, J., 1976. Catálogo provisional de los Opistobranquios (Gastropoda: Euthyneura) de las costas ibéricas. Miscelánea Zoológica, 3 (5): 21-51. * Figures must be original, in Indian ink on draughtsman's tracing paper. Keep in mind page format and column size when designing figures. These should be one column (57 mm) or two columns (121 mm) wide and up 196 mm high, or be proportional to these sizes. “Two columns format is recomended. It is desirable to print figures with their legend below, so authors are asked to take this into account when preparing full page figures. If computer generated graph- ics are to be included, they must be printed on high quality white paper with a laser printer. Photographs must be of good contrast, and should be submitted in the final size. When mounting photographs in a block, ensure spacers are of uniform width. Remember that grouping photographs of varied contrast results in poor reproduction. Take account of necessary reduction in lettering drawings; final lettering must be at least 2 mm high. In composite draw- ings, each figure should be given a capital letter; additional lettering should be in lower-case letters. A scale line is recomended to indicate size, magnification ratio must be avoided as it may be changed during printing. UTM maps are to be used if necessary. Figures must be submitted on separate sheets, and numbered with consecutive Arabic num- bers (1, 2, 3,...), without separating “Plates” and “Figures”. Legends for Figures must be typed in numerical order on a separate sheet, and an Spanish translation must be included. Follow this example (please note the punctuation): Figure 1. Neodoris carvi. A: animal crawling; B: rinophore; C: gills. If abbreviations are to be used in illustrations, group them alphabetically after the Legends for Figures section. Authors wishing to publish illustrations in colour will be charged with additional costs (30,000 ptas, 180 euros per page). They should be submitted in the same way that black and white prints. If the authors want to send Figures in digital format, CONTACT the Editor first. e Tables must be numbered with Roman numbers (1, II, TIL...) and each typed on a separate sheet. Headings should be typed on a separate sheet, together with their English translation. Complex tables should be avoided. As a general rule, keep the number and extension of illustrations and tables as reduced as possible. e Manuscripts that do not conform to these instructions will be returned for correction before reviewing. * Authors submitting manuscripts will receive an acknowledgement of receipt, including receipt date, and the date the manuscript was sent for reviewing. Each manuscript will be critically evaluated by at least two referees. Based of these evaluations, the Editorial Board will decide on acceptance or rejection. Anyway, authors will receive a copy of the referees* comments. If a manuscript is accepted, the Editorial Board may indicate additional changes if desirable. Acceptable manuscripts will be returned to the author for consideration of comments and criticism; a finalized man- uscript must then be returned to the Editor, together with a floppy disk containing the article written with a DOS or Macintosh word processor. Dates of reception and acceptance of the manuscript will appear in all published articles. * Proofs will be sent to the author for correcting errors. At this stage no stylistic changes will be accepted. Pay special attention to references and their dates in the text and the Bibliography section, and also to numbers of Figures and Tables appearing in the text. * Fifty reprints per article will be supplied free of charge. Additional reprints must be ordered when the page proofs are returned, and will be charged at cost. NO LATER orders will be accepted. LA SOCIEDAD ESPAÑOLA DE MALACOLOGÍA Junta Directiva desde el 14 de noviembre de 2000 Presidente Emilio Rolán Mosquera Vicepresidente Diego Moreno Lampreave Secretario Luis Murillo Guillén Tesorero Jorge J. Otero Schmitt Avda. de las Ciencias s/n, Campus Universitario, 15706 Santiago de Compostela, España Editor de Publicaciones Gonzalo Rodríguez Casero Apartado 156, 33600, Mieres del Camino, Asturias, España Bibliotecario Rafael Araujo Armero Museo Nacional de Ciencias Naturales, CSIC, c/ José Gutierrez Abascal 2, 28006 Madrid, España Vocales Ramon M. Álvarez Halcon Benjamín Gómez Moliner Eugenia María Martínez Cueto-Felgueroso Jesús Souza Troncoso José Templado González La Sociedad Española de Malacología se fundó el 21 de agosto de 1980. La sociedad se registró como una aso- ciación sin ánimo de lucro en Madrid (Registro N* 4053) con unos estatutos que fueron aprobados el 12 de diciembre de 1980. Esta sociedad se constituye con el fin de fomentar y difundir los estudios malacológicos mediante reuniones y publicaciones. A esta sociedad puede pertenecer cualquier persona o institución interesada en el estudio de los moluscos. SEDE SOCIAL; Museo Nacional de Ciencias Naturales, c/ José Gutierrez Abascal 2, 28006 Madrid, España. CUOTAS PARA 2005: Socio numerario (en España): 40 euros (en Europa) 40 euros (fuera de Europa): 48 euros Socio estudiante (en España): 23 euros (en el extranjero): 29 euros Socio Familiar: (sin recepcion de revista) 4 euros Socio Protector: (mínimo) 48 euros Socio Corporativo (en Europa): 48 euros (fuera de Europa): 54 euros INSCRIPCIÓN: 6 euros, además de la cuota correspondiente. A los socios residentes en España se les aconseja domiciliar su cuota. Todos los abonos deberán enviarse al Tesorero (dirección reseñada anteriormente) el 1 de enero de cada año. Los abonos se harán sin recargos para la sociedad y en favor de la Sociedad Española de Malacología y no de ninguna persona de la junta directiva. Aque- llos socios que no abonen su cuota anual dejarán de recibir las publicaciones de la Sociedad. Los bonos de ins- cripción se enviarán junto con el abono de una cuota anual al Tesorero. A los residentes en el extranjero se les ruega que abonen su cuota mediante giro postal en euros (internatio- nal postal money orders in euros sent to the Treasurer). Members living in foreing countries can deduce 6 euros if paid before 15 April. Cada socio tiene derecho a recibir anualmente los números de /berus, Reseñas Malacológicas y Noticiarios que se publiquen. UI UOAIAN ÍNDICE Iberus | 23 (1) 2005 YADAV, R. P, TIWARI, S. AND SINGH, A. Toxic effects of taraxerol extracted from Codiaeum varie- gatum stem-bark on target vector snail Lymnaea acuminata and non-target fish loxicidad del taraxerol extraído del tallo de Codiaeum variegatum sobre el caracol Lymnaea ACUIMINALA Y SODIO PECES A 1-13 MÁRQUEZ, R., ARRÉBOLA, J. R. Y DELGADO, R. Un avance sobre la composición y microestruc- tura de la concha de /berus gualtierianus morfotipo gualtierianus (Linnaeus, 1758) (Gastro- poda: Helicidae) Notes on the composition and microstructure of the shell of Iberus gualtierianus morphotype gualtierianus (Linnaeus, 1758) (Gastropoda: Helicidae) ici 15-24 SINGH, D., YADAV, R. P. AND SINGH, A. Changes in phospholipid and lipid peroxidation 15% due to latex of Croton tiglium in freshwater snail Lymnaea acuminata Cambios en los niveles de peroxidación de fosfolípidos y lípidos por efecto del latex de Croton tiglium sobre el molusco dulceacuícola Lymnaea acuminata ooo cicioccccccnoos 25:34 GIORDANO, D., BOTTARI, T. AND RINELLI, P. Cephalopod assemblages caught by trawling along the Southern Tyrrhenian Sea (Central Mediterranean) Asociaciones de cefalópodos capturados por la pesca de arrastre comercial en el Tirreno meridio- nal (Mediterráneo central) 0 do AI aO Ea EN 33-42 SÁNCHEZ-SANTOS, A. Onchidoris neapolitana (Delle Chiaje, 1844) (Gastropoda: Nudibranchia: Onchidorididae): una nueva especie de molusco para la fauna andaluza Onchidoris neapolitana (Delle Chiaje, 1844) (Gastropoda: Nudibranchia: Onchidorididae): a new molluscan species for the Andalousian fauna ooo .... 43-48 RAINES, B. AND PIZZINL M. Contribution to the knowledge of the family Caecidae: 16. Revision of the Caecidae of Easter Island (Chile) (Caenogastropoda: Rissooidea Gray J. E., 1847) Contribución al conocimiento de la familia Caecidae: 16. Revisión de los Caecidae de la Isla de Pascua (Chile) (Caenogastropoda: Rissooidea J. E. Gray 1847) ici 49-65 ÁVILA, S. P, SANTOS, A. C., PENTEADO, A. M., RODRIGUES, A. M., QUINTINO, L. AND MACHA- DO, M. I. The molluscs of the intertidal algal turf in the Azores Los moluscos del cesped algal intermareale en Azores iii 67-76 ROLÁN, E. Calyptraea capensis Tomlin, 1931 (Gastropoda, Calyptraeidae), a valid species from South Africa j Calyptraea capensis Tomlin, 1931 (Gastropoda, o una especie válida de SUAAICA da AU 77-82 Notas breves BLANCO, C., RADUÁN, A. AND RAGA, J. A. Additional information on the biology of Argonauta argo (Cephalopoda: Octopoda) in the Mediterranean Sea from gastrointestinal contents ef Risso's dolphin Aportación a la biología de Argonauta argo (Cephalopoda: Octopoda) en el mar Mei a partir del contenido gastrointestinal del delfín de RissO lll 83-86 ISSN 0212-3010