Structure-function studies of egg proteins of invasive molluscs,
plagues and vectors of parasites

Grupo de trabajo

Principal Investigator

Heras, Horacio

Investigator
h-heras@med.unlp.edu.ar

Dreon, Marcos Sebastián

Investigator
msdreon@gmail.com

Ituarte, Santiago

Investigator
santiago.ituarte@gmail.com

Pasquevich, M. Yanina

Investigator

mypasquevich@med.unlp.edu.ar

Brola, Tabata Romina

Research Fellow
tabatabrola@gmail.com

Giglio, Matiás Leonel

Research Fellow
matiasg_sp@hotmail.com

Chiumiento, Ignacio

Research Fellow
igchiumiento@gmail.com

Pavia, Ignacio

Becario
ignaciopavia18@gmail.com

Collaborators from other institutions

Jian-Wen Qiu
Hong Kong Baptist University, Republica Popular de China.

Ceolín, Marcelo
INIFTA -Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadasa (CONICET - CCT- La Plata - UNLP). La Plata. Provincia de Buenos Aires. Argentina.

Fernández, Patricia
Cátedra de Patología de la Facultad de Ciencias Veterinarias. UNLP. La Plata. Provincia de Buenos Aires. Argentina.

Line overview

Objetives

Our group is focused on classifying, identifying and studying the structure and functions of unknown egg proteins of invasive snails, agricultural plague and parasite vectors. We endeavour to understand the snail reproductive strategies from a biochemical perspective and, at the same time, to select some of these new proteins with potentials as bioactive compounds to exploit its application in Biomedicine.

Importance of the study of snail proteins

The knowledge of the structure and protein function is of key relevance for the understanding of all the biological processes. Gastropod molluscs are a source of bioactive molecules commonly used in Pharmacology and Medicine, since they have an outstanding diversity of compounds as reflection of their long evolutionary history. However, proteins of mollusc eggs are very little investigated. Our research group is studying them taking as a model those of Pomacea canaliculata (Lamarck, 1822), also called ampularia, an aquatic snail, worldwide invader which has turned out to be a plague for rice fields. Its uncontrolled expansion entailed the advance of a vector nematode parasite, responsible for human eosinophilic meningoencephalitis in Asia and Latin America. Its eggs strongly coloured by pigmented proteins almost have no predators.

Questions expected to answer

What kind of proteins do the eggs of this snail have?
Which are the properties of these proteins and what role do they play in the defence mechanism of the embryos?
Doe they have any application as tools in Biomedicine?

Employed methodology

Our group uses a multidisciplinary approach with collaborations of other research groups of our country and abroad in order to answer these questions, usually employing a combination of methodologies such as biochemical, molecular biology, biophysics, cellular biology, histopathological and bioassays with laboratory animals. Some of the techniques used are described as follows:
• Determination of amino acid sequence.
• Study of biochemical activity of inhibitors, lectins, enzymes isolated from the eggs.
• Study of structure/function of proteins through dispersion of angle x-rays and biophysical techniques.
• Microbiological studies of inhibition of bacterial growth.
• In vitro studies of toxicity and interaction with cultured tumour cells.
• Basic immunological studies.
• Toxicity bioassays in murine model.
• Use of optical and electronic microscopy for histopathological studies, effects on the nervous and digestive systems of potential predators.

Main results

We studied the proteome of egg fluid and demonstrated that P. canaliculata has achieved a fascinating antipredatory defence mechanism where perivitellins play a central role, providing the embryos with one of the best defences in animals at biochemical level. We characterize major perivitellins (PcOvo y PcPV2) that participate in the defence as follows:
PcOvo caroprotein provides photoprotective and antioxidant compounds which at the same time give an intense colour. Its striking colouring would warn potential predators of the presence of various protein toxins of perivitellin fluid surrounding the embryo. PcOvo also has associated an activity as protease inhibitor of gastrointestinal tract of the predator, limiting the digestion of egg nutrients (antidigestive defence). Orally administered, it diminishes the growth rate in rats and impedes nutrient acquisition.

PcPV2 is lethal for rodents and is the first protein toxin described within an egg. Its structure differs from all other animal toxins since it combines a lectin subunit that recognizes specific sequences of membrane sugars, and a subunit that forms pores in the membrane. Despite the fact that there is no equal in animals, it is similar to some toxins type AB described in seeds and to the botulinum toxin. Whether this novel defence system is present in the eggs of other species remains unknown.
We continue studying its properties and mechanism of action. However, we study the major egg protein ofPomacea scalaris so-called scalarin (PsSC) that provides the eggs with a striking rose-salmon colour that could function as a warning signal. We demonstrated that it is a powerful lectin that shows, nevertheless, many structural and functional similarities with PcOvo, that it is not.
We are studying its defence function as toxin lectin on the digestive system and its potential as biomarker of tumour cells. Other gastropod lectins have impact on medical practice and are commercialized to improve the pharmacokinetics of glycoprotein drugs or in the diagnosis of diseases such as tumour markers.
The structure and function of these three proteins are very resistant to high temperatures, to a very range of pH and to gastrointestinal digestion, what allows reaching biologically active intestines, highlighting even more its biotechnological application.

Evolutional and ecological implications

The selection pressure exerted by predators and hostile environment on the eggs would lead to the acquisition of new properties to endure the air development of an aquatic animal. At biochemical level it seems that these adaptations would involve a new type of perivitellins that besides being a reserve protein they are multifunctional compounds provided with very efficient defences against predation together with protective properties of the embryo facing hostile environment conditions.
The presence of eggs protected with proteins of these characteristics has not been found in other members of the animal kingdom, but it is similar to the plant defences against herbivory.

Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails.
Giglio, M. L.; Ituarte, S.; Milesi, V.; Dreon, M.S.; Brola, T. R.; Caramelo, J.; Ip, J. C. H.; Maté, S.; Qiu, J. W.; Otero, L. H.; Heras, H.
2020. Journal of structural biology. Amsterdam: ACADEMIC PRESS INC ELSEVIER SCIENCE, vol. 211, n° 2, ISSN 1047-8477
doi.org/10.1016/j.jsb.2020.107531

Apple snail egg perivitellin coloration, as a taxonomic character for invasive Pomacea maculata and P. canaliculata, determined by a simple method.
Pasquevich, M. Y.; Heras, H.
2020. Biological invasions: SPRINGER, - vol. 22, n° 7, p. 2299-2307. ISSN 1387-3547
doi.org/10.1007/s10530-020-02255-z

A highly stable, nondigestible lectin from Pomacea difusa unveils clade-related protection systems in apple snail eggs.
Brola, T. R; Dreon, M. S; Qiu, J. W; Heras, H.
2020. Journal of experimental biology: COMPANY OF BIOLOGISTS LTD, 2020 - ISSN 0022-0949
doi.org/10.1242/jeb.231878

Novel Role for Animal Innate Immune Molecules: Enterotoxic Activity of a Snail Egg.
Giglio, Matías L.; Ituarte, Santiago; Ibañez, Andrés E.; Dreon, Marcos S.; Prieto, Eduardo; Fernández, Patricia E.; Heras, Horacio.
2020. MACPF-Toxin. Frontiers in immunology. Frontiers. vol. 11
doi.org/10.3389/fimmu.2020.00428

Egg perivitelline fluid proteome of a freshwater snail: Insight into the transition from aquatic to terrestrial egg deposition
Ip, Jack C.H.; Mu, Huawei; Zhang, Yanjie; Heras, Horacio; Qiu, Jian.
2020. JOHN WILEY & SONS LTD, - vol. 34, n° 7, ISSN 0951-4198
doi.org/10.1002/rcm.8605

Hemocyanin of the Caenogastropod Pomacea canaliculata exhibits evolutionary differences among gastropod clades.
Chiumiento, IR., Ituarte, S., Sun, J., Qiu, J-W., Heras, H. and Dreon, MS. >
2020. Plos one. PUBLIC LIBRARY SCIENCE, 2020, vol. 15, n° 1, ISSN 1932-6203
doi.org/10.1371/journal.pone.0228325

Understanding the Transition from Water to Land: Insights from Multi-omic Analyses of the Perivitelline Fluid of Apple Snail Eggs.
Ip, J, Mu, H., Zhang, Y., Sun, J., Heras, H., Chu, FH, Qiu, J-W.
2019. J. Proteomics. 194 (1): 79-88
doi.org/10.1016/j.jprot.2018.12.014

Non-digestible proteins and protease inhibitors: Implications for defense of the colored eggs of freshwater apple snails.
Ituarte, S., Brola, TR., Dreon, MS., Sun, J., Qiu, J-W., Heras, H.
2019. Can. J. Zool. 97: 558–566
doi.org/10.1139/cjz-2018-0210

Genomic signatures of divergence and invasiveness in apple snails.
Sun, J, H Mu, JCH Ip, R Lib, T Xub, N Kumagai, Y Yusa, A Accorsi, A Sánchez Alvarado, E Ross, A Castro-Vazquez, IA Vega, H Heras, S Ituarte, B Van Bocxlaer, KA Hayes, RH Cowie, Z Zhao, Y Zhang, P-Y Qian, J-W Qiu.
2019. Molecular Biology and Evolution 36(7):1507-1520
doi.org/10.1093/molbev/msz084

Egg perivitelline fluid proteome of a freshwater snail (Caenogastropoda): insight into the transition from aquatic to terrestrial egg deposition.
Ip JCH, Mu, H., Zhang, Y., Heras, H., Chu, KH. and Qiu, J-W.
2019. Rap. Comm. Mass Spectrometry (2019 online) 2020 impreso 37(4): e8605
doi.org/10.1002/rcm.8605

Biosynthesis in the Albumen Gland-Capsule Gland Complex Limits Reproductive Effort in the Invasive Apple Snail Pomacea canaliculata.
Cadierno, M. P; Saveanu, L; Dreon, M. S; Martín, P. R; Heras, H.
2018. Biological bulletin. MARINE BIOLOGICAL LABORATORY, vol. 235, n° 1, p. 1-11. ISSN 0006-3185
doi.org/10.1086/699200

Validation by qPCR of reference genes for reproductive studies in the invasive apple snail Pomacea canaliculata.
Cadierno, M. P; Dreon, M. S; Heras, H.
2018. Malacologia. Philadelphia: INST MALACOL, vol. 62, p. 163-170. ISSN 0076-2997
doi.org/10.4002/040.062.0105

A lectin of a non-invasive apple snail as an egg defense against predation alters the rat gut morphophysiology.
Ituarte, S; Brola, T. R; Fernández, P. E; Mu, H; Qiu, J; Heras, H; Dreon, M. S.
2018. Plos one. PUBLIC LIBRARY SCIENCE, vol. 13, n° 6, ISSN 1932-6203
doi.org/10.1371/journal.pone.0198361

AmpuBase: a transcriptome database for eight species of apple snails (Gastropoda: Ampullariidae).
Ip, J; Mu, H; Chen, Q; Jin, S; Ituarte, S; Van Bocxlaer, B; Ganmanee, M; Huang, X; Qiu, J.
2018. BMC Genomics. 19: 179-187 ISSN 1471-2164
doi.org/10.1186/s12864-018-4553-9

Egg perivitelline fluid of the invasive snail Pomacea canaliculata affects mice gastrointestinal function and morphology.
Giglio, M. L; Garro, C; Caviedes Vidal, E; Heras, H.
2018. Peerj, Inc., ISSN 2167-9843
doi.org/10.7717/peerj.5314

An integrated proteomic and transcriptomic analysis of perivitelline fluid proteins in a freshwater gastropod laying aerial eggs.
Mu, H; Sun, J; Heras, H; Chu, K.H; Qiu, J-W.
2017. J. Proteomics. 155: 22-30
dx.doi.org/10.1016/j.jprot.2017.01.006

Apple snail perivitellin precursor properties help explain predator´ feeding behavior.
Cadierno, M.P; Dreon, M.S; Heras, H. Physiol.
2017. Biochem. Zool. 90, 4: 461-470
dx.doi.org/10.1086/691526

The influence of energy, nutritional value and noxiousness in sex- and size-biased predation by Snail Kites in southern South America.
Cadierno, M.P; Burela, S; Dreon, M.S; Martin, P; Heras, H.
2017. EMU. 117, 4: 382-387
doi.org/10.1080/01584197.2017.1338113

Dataset for the proteomic and transcriptomic analyses of perivitelline fluid proteins in Pomacea snail eggs.
Mu, H; Sun, J; Heras, H; Chu, K.H; Qiu, J-W.
2017. Data in Brief. 15: 203-207
dx.doi.org/10.1016/j.dib.2017.09.020

Convergent evolution of plant and animal embryo defences by hyperstable non-digestible storage proteins.
Pasquevich, M.Y; Dreon, M.S; Qiu, J-W; Mu, H; Heras, H.
2017. Scientific Reports. 7: 15848
dx.doi.org/10.1038/s41598-017-16185-9

The eggs of the apple snail Pomacea maculata are defended by indigestible polysaccharides and toxic proteins.
Giglio, M. L; Ituarte, S; Pasquevich, M. Y; Heras, H.
2016. Can. J. Zool. (2016)94, 11: 777-785
dx.doi.org/10.1139/cjz-2016-0049

Effects of sewage discharges on lipid and fatty acid composition of the freshwater Patagonian bivalve Diplodon chilensis.
Rochetta, I; Pasquevich, M. Y; Heras, H; Rios De Molina, M. C; Luquet, C. M.
2014. Mar. Pollut. Bull. (2014) 79:211-219
dx.doi.org/10.1016/j.marpolbul.2013.12.011

Insights into embryo defenses of the invasive apple snail Pomacea canaliculata: egg mass ingestion affects rat intestine morphology and growth.
Dreon, M. S; Fernández, P. E; Gimeno, E; Heras, H.
2014. PLoS Neglect. Trop. Dis. (2014). 8, 6: e2961
dx.doi.org/10.1371/journal.pntd.0002961

The major egg reserve protein from the invasive apple snail Pomacea maculata is a complex carotenoprotein related to those of Pomacea canaliculata and Pomacea scalaris.
Pasquevich, M. Y; Dreon, M, S; Heras, H.
2014. Comp. Biochem. Physiol. (2014) 169B: 63-71
dx.doi.org/10.1016/j.cbpb.2013.11.008

¿Por qué hay huevos que nadie quiere comer?
Heras, H. 2014. Ciencia Hoy 23,137:41-49. (Trabajo de divulgación)
cienciahoy.org.ar/2014/04/por-que-hay-huevos-que-nadie-quiere-comer/

Effect of crude oil petroleum hydrocarbons on protein expression of the prawn Macrobrachium borellii.
Pasquevich, M. Y; Dreon, M. S; Gutiérrez Rivera, J. N; Vázquez Boucard, C; Heras H.
2013. Comp. Biochem. Physiol C. (2013) 157: 390-396
dx.doi.org/10.1016/j.cbpc.2013.03.006

Study of biochemical biomarkers in freshwater prawn Macrobrachium borellii (Crustacea: Palaemonidae) exposed to organophosphate fenitrothion.
Lavarías, S; García, C; Crespo, R; Pedrini, N; Heras H.
2013. Ecotoxicol. Environ. Saf. (2013) 96:10-16
dx.doi.org/10.1016/j.ecoenv.2013.05.040

Novel animal defenses against predation: A snail egg neurotoxin combining lectin and pore-forming chains that resembles plant defense and bacteria attack toxins.
Dreon, M. S; Frassa, M. V; Ceolín, M; Ituarte, S; Qiu, J. W; Sun, J; Fernández, P. E; Heras, H.
2013. PLoS One (2013) 8, 5: e63782
dx.doi.org/10.1371/journal.pone.0063782

Vitellogenin and lipovitellin from the prawn Macrobrachium borellii as hydrocarbon pollution biomarker.
García, C. F; Heras, H.
2012. Mar. Poll. Bull. (2012) 64: 1631-1636
dx.doi.org/10.1016/j.marpolbul.2012.05.027

First proteome of the egg perivitelline fluid of a freshwater gastropod with aerial oviposition.
Sun, J; Zhang, H; Wang, H; Heras, H; Dreon, M; Ituarte, S; Ravasi, T; Qian, P; Qiu, J. W.
2012. J. Proteome Res. (2012) 11: 2440-2448
dx.doi.org/10.1021/pr3003613

Agglutinating activity and structural characterization of scalarin the major egg protein of the snail Pomacea scalaris.
Ituarte, S; Dreon, M. S; Ceolin, M; Heras, H. (d’Orbigny, 1832).
2012. PLoS ONE (2012) 7(11): e50115
dx.doi.org/10.1371/journal.pone.0050115

Antioxidant response and oxidative stress levels in Macrobrachium borellii (Crustacea: Palaemonidae)exposed to the water-soluble fraction of petroleum.
Lavarías, S; Heras, H; Pedrini, N; Turnier, H; Ansaldo, M.
2011. Comp. Biochem. Physiol. (2011) 153 C: 415-421
dx.doi.org/10.1016/j.cbpc.2011.02.002

Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade).
Pasquevich, M. Y; Dreon, M. S; Lavarías, S; Heras, H.
2011. Comp. Biochem. Physiol. (2011) 160 B: 201-207
dx.doi.org/10.1016/j.cbpb.2011.08.006

Isolation and characterization of two vitellins from eggs of the spider Polybetes pythagoricus (Araneae: Sparassidae).
Laino, A; Cunningham, M. L; Heras, H; García, C. F.
2011. Comp. Biochem. Physiol. 158 B (2011) 142-148
dx.doi.org/10.1016/j.cbpb.2010.11.001

In vitro lipid transfer between lipoproteins and midgut-diverticula in the spider Polybetes pythagoricus.
Laino, A; Cunningham, M; Heras, H; García, C. F.
2011. Comp. Biochem. Physiol. (2011) 160 B: 181-186
dx.doi.org/10.1016/j.cbpb.2011.08.003

Structure and stability of the neurotoxin PV2 from the eggs of Pomacea canaliculata.
Frassa, V; Ceolin, M; Dreon, M. S; Heras, H.
2010. Biochim.Biophys.Acta. 1804 (2010) 1492-1499
dx.doi.org/10.1016/j.bbapap.2010.02.013

The Role of the Proteinase Inhibitor Ovorubin in Apple Snail Eggs Resembles Plant Embryo Defenses Against Predation.
Dreón, M. S; Ituarte, S; Heras, H.
2010. PLoS ONE. (2010) 5,12: e15059
dx.doi.org/10.1371/journal.pone.0015059

Carbohydrates and glycoforms of the major egg perivitellins from Pomacea apple snails (Architaenioglossa: Ampullariidae).
Ituarte, S; Dreón, M. S; Pasquevich, M. Y; Fernández, P. E; Heras, H.
2010. Comp. Biochem. Physiol. (2010) 157: 66-72
dx.doi.org/10.1016/j.cbpb.2010.05.004

Structure and stability of crustacean lipovitellin: Influence of lipid content and composition.
García, C. F; Cunningham, M; Soulages, J. L; Heras, H; Garda, H. A.
2010. Comp. Biochem. Physiol. 155 (2010) 126-131
dx.doi.org/10.1016/j.cbpb.2009.10.010

First insight into the lipid uptake, storage and movilization in arachnids. Role of midgut diverticula and lipoproteins.
Laino, A; Cunningham, M; García, C. F; Heras, H.
2009. J. Insect Physiol. 55 (2009) 1118–1124
dx.doi.org/10.1016/j.jinsphys.2009.08.005

Partial characterization of a malonyl-CoA-sensitive carnitine o-palmitoyltransferase I from Macrobrachium borellii. (Crustacea: Palaemomidae).
Lavarías, S; Pasquevich, Y; Dreon, M. S; Heras, H.
2009. Comp. Biochem. Physiol. 152 (2009) 364-369
dx.doi.org/10.1016/j.cbpb.2009.01.004

Isolation and characterization of a novel perivitellin from the eggs of Pomacea scalaris (Mollusca, Ampullariidae).
Ituarte, S; Dreon, M. S; Ceolin, M; Heras, H.
2008. Mol. Reprod. Dev. 75 (2008) 1441-1448
dx.doi.org/10.1002/mrd.20880

First egg protein with a neurotoxic effect on mice.
Heras, H; Frassa, V; Fernández, P. E; Galosi, C. M; Gimeno, E; Dreon, M. S.
2008. Toxicon 52 (2008) 481-488
dx.doi.org/10.1016/j.toxicon.2008.06.022

Global shape and pH stability of ovorubin, an oligomeric protein from the eggs of Pomacea canaliculata.
Dreón, M. S; Ituarte, S; Ceolín, M; Heras, H.
2008. FEBS J. 275 (2008) 4522-4530
dx.doi.org/10.1111/j.1742-4658.2008.06595.x

Embryo lipoproteins and yolk lipovitellin consumption during embryogenesis in Macrobrachium borellii (Crustacea: Palaemonidae).
García, F; Cunningham, M; Garda, H; Heras, H.
2008. Comp. Biochem. Physiol.151 (2008) 317-322
dx.doi.org/10.1016/j.cbpb.2008.07.015

Astaxanthin binding and structural stability of apple snail carotenoprotein ovorubin.
Dreon, M. S; Ceolin, M; Heras, H.
2007. Arch. Biochem. Biophys. 460 (2007) 107-112
dx.doi.org/10.1016/j.abb.2006.12.033

Effect of the water-soluble fraction of petroleum on microsomal lipid metabolism of Machrobrachium borellii (Arthropoda: Crustacea).
Lavarías, S; García, F; Pollero, R. J; Heras, H.
2007. Aquatic Toxicol. 82 (2007) 265-271
dx.doi.org/10.1016/j.aquatox.2007.02.017

Egg carotenoproteins of neotropical ampullariidae (gastropoda: arquitaenioglossa).
Heras, H; Dreon, M. S; Ituarte, S; Pollero, R. J.
2007. Comp. Biochem. Physiol. 146C (2007) 158-167
dx.doi.org/10.1016/j.cbpc.2006.10.013

Apple Snail perivitellins. Multifunctional egg proteins¨. In: Biology and management of invasive apple snails.
Heras, H; Dreon, M.S; Ituarte, S; Pasquevich, M.Y; Cadierno, M.P.
2017. Philippine Rice Research Institute. Joshi, R.C., Cowie,R., and Sebastian, L.S. eds, Nueva Ecija. Maligaya, Philippines 99-118. ISBN: 978-621-8022-25-6

Métodos de control del desarrollo de floraciones cianobacterianas en ambientes acuáticos.
Bauzá, L; Giannuzzi, L; Petcheneshsky, T; Hansen, M; Benítez, R; De Titto, E.
2017. Revisión actualizada. Ciudad autónoma de Buenos Aires: Ministerio de Salud de la Nación. p. 125-146. ISBN 978-950-38-0255-7

Changes in carbohydrate expression in the cervical spinal cord of mice intoxicatedwith perivitellin PV2 from Pomacea canaliculata. In: Poisoning by plants, mycotoxins and related toxins.
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

¿Por qué hay huevos que nadie quiere comer?
Heras, H.
2014. Ciencia Hoy 23,137:41-49. (Trabajo de divulgación)
See article

“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.