Structure-function studies of egg mollusk proteins
Director of Line
Heras, Horacio
Researchers
Dreon, Marcos Sebastián
Heras, Horacio
Ituarte, Santiago
Fellows
Cadierno, María Pilar
Pasquevich, María Yanina
Team of work Collaborators from other institutions
International
Qiu, Jian-Wen
Hong Kong University, Hong Kong. Republica Popular de China
National
Ceolín, Marcelo
INIFTA -Research Institute of Theoretical and Applied Physical Chemistry (CONICET - CCT- La Plata) UNLP. La Plata. Province of Buenos Aires. Argentina
Fernández, Patricia
Cathedra of Pathology, Faculty of Veterinary Science. UNLP. La Plata. Province of Buenos Aires. Argentina
Winik,Beatriz
National University of Tucumán. Province of Tucumán. Argentina
Summary line
Objetives
Our main goal is to contribute to the knowledge of gastropod reproductive biology, studying the egg proteins of ampullariid snails of the genus Pomacea.
Animal model: An Invasive Snail: pest and host of parasites
Our main model is the Apple snail Pomacea canaliculata (also called ampularia in Spanish and Apfelschnecke in German), which is very frequent in Argentinean rivers, springs and ponds. The natural range of the species spreads from Southern Buenos Aires province up to Mesopotamia, center and NOA provinces.
In the last three decades this snail has spread through Southeast Asia, North America, Spain and some Pacific islands, being listed within the 100 top invasive species.It became a serious pest of rice and taro fields as well as an intermediate host of Angiostrongylus cantonensis, the causative agent of eosinophilic encephalitis, and a potencially lethal desease.
The spread of the species is probably related to its high fecundity rate (a female lays about 10,000 pink eggs outside the water) and also to the unusual characteristics of the egg-proteins, which we study.
Despite being rich in proteins and carbohydrates, the eggs of this species are devoid of predators within the native range of the species (South America) and have only one or two occasional predators in Asia.
Experimental Approach
To study structure-function relationships in these proteins we combine classical biochemistry techniques with molecular biology, biophysics, histopathology and also bioassays with laboratory animals.
Major Results
Structural aspects of the perivitelins
The major glyco-lipoproteins present in the eggs are high molecular weight oligomers: ovorubin and PV2 in P. canaliculata and scalarin in P. scalaris. Ovorubin and scalarin are composed of three highly glycosylated subunits, and carry carotenoid pigments, i.e. they are carotenoproteins. PV2 is an octamer, composed of four disulfide linked heterodimers.
We have shown that these perivitellins are thermally stable; ovorubin is stable up to 95°C, PV2 up to 60°C and scalarin up to 80°C.
Particularly, ovorubin and scalarin are also stable in a wide pH range; ovorubin between pH 4.0 and 12.0 and scalarin between pH 2.0 and 10.0.
Finally, the three perivitellins are resistant to proteolitic digestion by pepsin (the major gastric protease in mammals) and trypsin (a highly conserved protease in both vertebrates and invertebrates).
Functional aspects related to embryo defenses against predation
Ovorubin
Given that ovorubin is a trypsin inhibitor, which is stable in a wide pH range and also resists proteolitic attack, a mammalian model was used to test ovorubin’s effects in vivo. It was found that the protein diminishes the growth rate in rats when administered orally.
These results indicate that ovorubin is part of the egg defenses by inhibiting the predator proteases, thus limiting the digestion of the egg proteins (antidigestive defense). This kind of defense system had not been reported in animals before, but is typical of plant defense against herbivores. Moreover, ovorubin binds a carotenoid molecule (astaxanthin) which provides the eggs a bright reddish coloration, probably working as a warning signal (aposematic coloration).
PV2
Functional studies with in vivo experiments showed that PV2 is a neurotoxin with lethal effect on mice, the first toxin to be reported in an egg, and in a freshwater mollusk. Moreover, PV2 reaches the intestine in a biologically active form (antinutritive defense).
Scalarin
The major perivitellin of P. scalaris eggs, may also be involved in egg antinutritive defense, since it resists proteolitic digestion. Similar to ovorubin, this protein binds a carotenoid pigment and gives the eggs a bright coloration which may function as a warning signal.
Evolutionary Implications
This antinutritive/antidigestive system would be the only animal defense reported so far, with no trade-off between aposematism and toxicity, since both components are encoded in the same macromolecule.
We have shown that these multifunctional perivitellins provide the embryo not only with reserve components, but also with antidigestive, antinutritive and, possibly, aposematic defenses. These defenses would account for the rapid expansion of the species in non-native environments, and also for the virtual absence of egg predators. It is notable in this sense that, when predating the adult female snails, the snail kite (Rostrhamus sociabilis) and other birds reject the gland that synthesizes the egg components.
It seems that before being consumed by the embryo, perivitellins endow the eggs with one of the most effective defense systems. This opens new perspectives for ecological and evolutionary studies of reproductive strategies.
Line
Book - Book Chapters Publications
Publications
Lavarías, S; Heras, H; Pedrini, N; Turnier, H; Ansaldo, M.
2011. Comp. Biochem. Physiol. 153 C: 415-421. doi:10.1016/j.cbpc.2011.02.002
Pasquevich, M. Y; Dreon, M. S; Lavarías, S; Heras, H.
2011. Comp. Biochem. Physiol. 160 B: 201-207. doi:10.1016/j.cbpb.2011.08.006
Frassa, V; Ceolin, M; Dreon, M. S; Heras, H.
2010. Biochimica and biophysica acta. Elsevier. vol. 1804, p. 1492-1499
Dreón, M. S; Ituarte, S; Heras, H.
2010. Plos one. Public Library Science. vol. 5, n 12, p. 15059-15068
Ituarte, S; Dreón, M. S; Pasquevich, M. Y; Fernández, P. E; Heras, H.
2010. Comparative biochemistry and physiology. part b, biochemistry & molecular biology. Elsevier Science Inc. vol. 157, p. 66-72
Ituarte, S; Dreon, M. S; Ceolin, M; Heras, H.
2008. Molecular reproduction and development.Wiley-Liss, vol. 75, p. 1441-1448
Heras, H; Frassa, V; Fernández, P. E; Galosi, C. M; Gimeno, E; Dreon, M. S.
2008. Toxicon. Elsevier, vol. 52, p. 481-488
Dreón, M. S; Ituarte, S; Ceolín, M; Heras, H.
2008. Febs journal. Blackwell Pub. on behalf of the Federation of European Biochemical Societies, vol. 275, n° 18, p. 4522-4530. ISSN 1742-464X
Dreon, M. S; Ceolin, M; Heras, H.
2007. Archives of biochemistry and biophysics. vol. 30, p. 359-365
Heras, H; Dreon, M. S; Ituarte, S; Pollero, R. J.
2007. Comparative biochemistry and physiology. toxicology & pharmacology. vol. 146, p. 158-167
Fernández, P. E; Frassa, V; Gimeno, E. J; Dreon, M; Heras, H.
2011. Edited by Riet-Correa, F., Pfister J., Schild A.L. and Panter K. CAB International, Oxfordshire, UK 482-498. ISBN 13 978 1 84593 833 8. Chapter 83
Publications
International
- Transferencia de lípidos in vitro entre lipoproteínas y divertículos intestinales en la araña Polybetes pythagoricus (Araneae, Sparassidae).
García, F; Laino, A; Heras, H; Herrera Hidalgo, J; Cunningham, M.
2011. Montenegro. Colombia. III Congreso Latinoamericano de Aracnología.
- Colorfull and nutritious but lethal and antidigestive. The multiple roles of egg proteins in the successful reproductive strategy of Pomacea canaliculata (Mollusca: Gastropoda)
2011. Hong Kong Baptist University, China. Invitado por el Dr. J-W Qiu, Department of Biology.
- Antinutritive perivitellins in Pomacea Perry, 1810: The case of P.scalaris (d Orbigny 1835) (Architaenioglossa, Ampullariidae).
Ituarte, S; Dreon, M. S; Heras, H.
2010. Quebec: Quebec. Resumen. Congreso. Physiomar 10.
- Biochemical defenses against predation of apple snail eggs resemble plant strategies against herbivory.
Dreon, M. S; Ituarte, S; Frassa, V; Heras, H.
2010. Quebec: Quebec. Resumen. Congreso. Physiomar 10.
- Changes in carbohydrate expression in the cervical spinal cord of mice intoxicated with perivitellin PV2 from Pomacea canaliculata.
Fernández P. E; Frassa, V; Gimeno, E; Dreon, M. S; Heras, H.
2009. João Pessoa, Brasil: João Pessoa, Brasil. Artículo Breve. Congreso.
8th International Symposium on Poisonous Plants (ISOPP 8). Federal University of Campina Grande.
- Structure-function relationships of ovorubin, a protease inhibitor from the eggs of Pomacea canaliculata.
Dreon, M. S; Ituarte, S; Ceolin, M; Heras, H.
2009. Buzios, Brasil. Resumen. Congreso. VII Iberoamerican Congress of Biological Physics. Sociedad Argentina de Biofísica.
- Changes in carbohydrate expression in the cervical spinal cord of mice intoxicated with perivitellin PV2 from Pomacea canaliculata.
Fernández P. E; Frassa, V; Gimeno, E; Dreon, M. S; Heras, H.
2009. João Pessoa, Brasil: João Pessoa, Brasil. Artículo Breve. Congreso.
8th International Symposium on Poisonous Plants (ISOPP 8). Federal University of Campina Grande.
- Structure-function relationships of ovorubin, a protease inhibitor from the eggs of Pomacea canaliculata.
Dreon, M. S; Ituarte, S; Ceolin, M; Heras, H.
2009. Buzios, Brasil. Resumen. Congreso. VII Iberoamerican Congress of Biological Physics. Sociedad Argentina de Biofísica.
- Perivitelinas resistentes a la digestión en Pomacea Perry,1810 (Architaenioglossa, Ampullariidae). ¿Un mecanismo antinutritivo de defensa?
Ituarte, S; Dreon, M. S; Ceolin, M; Heras, H.
2008. Valdivia, Chile: Valdivia, Chila. 2008. Resumen. Congreso. VII Congreso Latinoamericano de Malacología (CLAMA). Universidad Austral de Chile.
- Low Resolution Structure and pH stability of Ovorubin, a lipoglycocarotenoprotein from the eggs of Pomacea canaliculata.
Ituarte, S; Dreon, M. S; Ceolin, M; Heras, H.
2007. Montevideo, Uruguay: ICBP. Resumen. Conferencia. International Conference of Biophysics of Proteins (ICBP). ICBP.
- PH stability and Low Resolution Structure of Ovorubin, a protease inhibitor from the eggs of Pomacea canaliculata.
Ituarte, S; Dreon, M. S; Ceolin, M; Heras, H.
2007. Acapulco, Mexico: Latin American Protein Society Meeting. Resumen. Congreso. 2nd Latin American Protein Society Meeting. 2nd Latin American Protein Society Meeting.
- Structural characterization of thermal and urea denaturation of the Pv2 apple snail perivitellin.
Frassa, V; Dreon, M. S; Ceolin, M; Heras, H.
2007. Montevideo, Uruguay: ICBP. Resumen. Conferencia.
International Conference of Biophysics of Proteins (ICBP).
National
- Coloridas y nutritivas pero letales y antidigestivas. El rol múltiple de las proteínas de los huevos en la exitosa estrategia reproductiva de Pomacea.
Heras, H; Ituarte, S; Frassa, M. V; Dreon, M. S.
2011. Pto Madryn, Argentina. VIII Congreso Latinoamericano de Malacología (CLAMA).
- Effect of organophosphate fenitrothion on biochemical parameters of the crustacean Macrobrachium borellii.
Lavarías, M.S; García; Crespo, R; Pedrini, N; Heras, H.
2011. Potrero de los Funes, Argentina. 30 octubre - 2 Noviembre. XLVII Reunión SAIByBM.
- Hemagglutinating activity and structural stability of a snail egg-protein.
Ituarte, S; Dreon, M. S;Heras, H.
2011. Potrero de los Funes, Argentina. 30 octubre - 2 Noviembre. XLVII Reunión SAIByBM.
- Effect of water-soluble crude oil hydrocarbon exposure on protein expression of a freshwater crustacean.
Pasquevich, M. Y; Dreon, M. S; Heras, H.
2011. Potrero de los Funes, Argentina. 30 octubre - 2 Noviembre. XLVII Reunión SAIByBM
- Effects of urban discharge on lipid and fatty acid composition of Diplodon chilensis.
Yussoppone, M. S; Pasquevisch, Y; Heras, H; Luquet, C. M; Ríos de Molina, M. C; Rocchetta, I.
2011. Potrero de los Funes, Argentina. 30 octubre - 2 Noviembre. XLVII Reunión SAIByBM
- Rol de la neurotoxina PV2 en la defensa de los embriones de Pomacea canaliculata.
2011. La Plata, Argentina. Invitado por Dr. Catanesi, Instituto de Biología Celular IMBICE.
- Multifunctional perivitellins in Pomacea: Characteristic of aerial eggs.
Ituarte, S; Dreon, M. S; Heras, H.
2010. Tucumán: Tucumán. Resumen. Workshop. IV International workshop on the biology of Ampullariidae.
- Pomacea canaliculata perivitellins. Storage proteins or defensive molecules.
Heras, H; Ituarte, S; Frassa, V; Dreon, M. S.
2010. Tucumán: Tucumán. Resumen. Workshop. IV International workshop on the biology of Ampullariidae. INIBIOLP.
- PV2: a novel toxin isolated from the eggs of P.
Frassa, V; Dreon, M. S; Heras, H.
2010. Canaliculata. S.M. Tucumán: Argentina. 2010. Resumen. Workshop.
IV International workshop on the biology of Ampullariidae. INIBIOLP.
- The role of the proteinase inhibitor ovorubin in the apple snail eggs defenses against predation.
Dreon, M. S; Ituarte, S; Frassa, V; Heras, H.
2010. Tucumán: Tucumán. Resumen. Workshop. IV International Workshop on the Biology of Ampullariidae.
- Structure and stability of the snail neurotoxin PV2.
Frassa, V; Dreon, M. S; Ceolin, M; Heras, H.
2009. Tucumán. Resumen. Congreso.
XLV Reunión SAIB y BM. XLV Reunión SAIB y BM.
- First egg protein with a neurotoxic effect on mice.
Frassa, V; Fernández, P. E; Galosi, C. M; Gimeno, E; Dreon, M. S; Heras, H.
2008. Va. Carlos Paz, Argentina: Biocell. 2008. Resumen. Congreso.
XLIV Reunión SAIB y BM.
- Ovorubin as a protease inhibitor in Pomacea canaliculata eggs. Implications in the biochemical defenses of snail embryos.
Dreon, M. S; Ituarte, S; Ceolin, M; Heras, H.
2008. La Plata. Resumen. Congreso.
XXXVII Congreso Soc. Argentina de Biofísica. SAB.
- Inducción de la síntesis de vitelogeninas por efecto del plaguicida organofosforado fenitrothion sobre el camarón Macrobrachium borelii.
García, F; Cunningham, M; Pifano, M; Heras, H.
2008. Mar del Plata, Buenos Aires: Sociedad de Toxicología y Química Ambiental. Simposio SETAC Argentina. Resumen. Congreso.
Sociedad de Toxicología y Química Ambiental. Simposio SETAC Argentina.
Congress
Awards
- “IFS Jubilee Award 2006”
Premio otorgado a los investigadores sobresalientes de proyectos subsidiados por la Internacional Foundation for Science al Dr. Heras Horacio. Suecia – 2006.
Provincia de Buenos Aires - Argentina. Tel. 54-221-4824894 - Fax. 54-221-4258988 - email: info@inibiolp.org.ar