[Doi 10.1007%2 f978 94-007-6389-0] gopalakrishnakone, p.; corzo, gerardo; de lima, maria elena; die -- spider venoms -_

1 3Reference
Toxinology
SpiderVenoms
P. Gopalakrishnakone Editor-in-Chief
Gerardo Corzo
Maria Elena de Lima
Elia Diego-García Editors

Toxinology
Editor-in-Chief
P. Gopalakrishnakone

In recent years, the field of toxinology has expanded substantially. On the one hand
it studies venomous animals, plants and micro organisms in detail to understand
their mode of action on targets. While on the other, it explores the biochemical
composition, genomics and proteomics of toxins and venoms to understand their
three interaction with life forms (especially humans), development of antidotes and
exploring their pharmacological potential. Therefore, toxinology has deep linkages
with biochemistry, molecular biology, anatomy and pharmacology. In addition,
there is a fast-developing applied subfield, clinical toxinology, which deals with
understanding and managing medical effects of toxins on human body. Given the
huge impact of toxin-based deaths globally, and the potential of venom in gener-
ation of drugs for so-far incurable diseases (for example, diabetes, chronic pain),
the continued research and growth of the field is imminent. This has led to the
growth of research in the area and the consequent scholarly output by way of
publications in journals and books. Despite this ever-growing body of literature
within biomedical sciences, there is still no all-inclusive reference work available
that collects all of the important biochemical, biomedical and clinical insights
relating to toxinology.
Composed of 12 volumes, Toxinology provides comprehensive and authoritative
coverage of the main areas in toxinology, from fundamental concepts to new
developments and applications in the field. Each volume comprises a focused and
carefully chosen collection of contributions from leading names in the subject.
Series Titles
1. Biological Toxins and Bioterrorism
2. Clinical Toxinology in the Asia Pacific and Africa
3. Spider Venoms
4. Scorpion Venoms
5. Marine and Freshwater Toxins
6. Venom Genomics and Proteomics
7. Snake Venoms
8. Evolution of Venomous Animals and Their Venoms
9. Microbial Toxins
10. Plant Toxins
11. Toxins and Drug Discovery
12. Clinical Toxinology in Australia, Europe, and Americas
More information about this series at http://www.springer.com/series/13330

Editor-in-Chief
Gerardo Corzo • Maria Elena de Lima
Elia Diego-García
Editors
Spider Venoms
With 111 Figures and 34 Tables

Editor-in-Chief
Venom and Toxin Research Programme
Department of Anatomy
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
Editors
Gerardo Corzo
Department of Molecular Medicine and
Bioprocesses
The Biotechnology Institute, National
Autonomous University of Mexico (UNAM)
Cuernavaca, Morelos, Mexico
Maria Elena de Lima
Departamento de Bioquímica e Imunologia
Laborato´rio de Venenos e Toxinas Animais
Instituto de Cieˆncias Biolo´gicas
Universidade Federal de Minas Gerais
Belo Horizonte, MG, Brazil
Elia Diego-García
Veerle, Belgium
ISBN 978-94-007-6388-3 ISBN 978-94-007-6389-0 (eBook)
ISBN 978-94-007-6390-6 (print and electronic bundle)
DOI 10.1007/978-94-007-6389-0
Library of Congress Control Number: 2015960445
# Springer Science+Business Media Dordrecht 2016
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt
from the relevant protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, express or implied, with respect to the material contained herein or for any errors
or omissions that may have been made.
Printed on acid-free paper
This Springer imprint is published by SpringerNature
The registered company is Springer Science+Business Media B.V. Dordrecht

Series Preface
The term TOXIN is derived from the Greek word Toeikov and is defined as a
substance derived from tissues of a plant, animal, or microorganism that has a
deleterious effect on other living organisms. Studying their detailed structure,
function, and mechanism of action as well as finding an antidote to these toxins is
the field of TOXINOLOGY, and the scientists are called TOXINOLOGISTS.
In recent years, the field of toxinology has expanded substantially. On the one
hand, it studies venomous animals, plants, and microorganisms in detail to under-
stand their habitat, distribution, identification, as well as mode of action on targets,
while on the other, it explores the biochemical composition, genomics, and prote-
omics of toxins and venoms to understand their interaction with life forms (espe-
cially humans), the development of antidotes, and their pharmacological potential
for drug discovery. Therefore, toxinology has deep linkages with biochemistry,
molecular biology, anatomy, pharmacology, etc. In addition, there is a fast devel-
oping applied subfield, clinical toxinology, which deals with understanding and
managing medical effects of venoms and toxins on the human body following
envenomations. Given the huge impact of envenomation-based deaths globally and
the potential of venom in the generation of drugs for debilitating diseases (e.g.,
diabetes, chronic pain, and cancer), the continued research and growth of the field is
imminent.
Springer has taken the bold initiative of producing this series, which is not an
easy target of producing about 12 volumes, namely, biological toxins and bioter-
rorism, clinical toxinology, scorpion venoms, spider venoms, snake venoms,
marine and freshwater toxins, toxins and drug discovery, venom genomics and
proteomics, evolution of venomous animals and their toxins, plant toxins, and
microbial toxins.
Singapore P. Gopalakrishnakone
M.B.B.S., Ph.D., F.A.M.S., D.Sc.
Editor-in-Chief
v

Acknowledgments
I would like to sincerely thank the section editors of this volume, Gerardo Corzo,
Maria Elena de Lima, and Elia Diego-García for the invaluable contribution of their
expertise and time and the authors who obliged with my request and provided a
comprehensive review on the topics.
Springer provided substantial technical and administrative help by many indi-
viduals at varying levels, but special mention should go to Mokshika Gaur, Sarah
Mathews, Meghna Singh, and Audrey Wong for their tireless effort in bringing
these volumes to reality.
Singapore P. Gopalakrishnakone
M.B.B.S., Ph.D., F.A.M.S., D.Sc.
Editor-in-Chief
vii

Volume Preface
Spider venoms are a great and extensive source of bioactive compounds, and as
such form a boundless and bountiful area awaiting us to discover and explore
it. Springer’s Toxinology handbook series offers assistance in entering this vast
and still largely uncharted territory, guiding through this tremendous space in – and
hopefully for the enthusiastic reader-scientist also over – unprecedented ways.
Through biochemical characterization, structure-function studies, proteomics,
bioinformatics, molecular biology, transcriptomics, and genomics of various spider
species, our knowledge concerning venom components, toxins, and their mode of
action has increased considerably over the years. It is by virtue of dedicated
scientists that new toxins are discovered and that new insights arise, leading the
way towards the investigation of their pharmacological effects and, hopefully, as a
consequence, arriving at the discovery of venom components as new drug
candidates.
The Spider Venom volume contains 20 chapters, each one revealing different
aspects of and perspectives on the current scientific state of the art and research
progress of spider venoms. Its authors are scientists, experts in their subdomain. We
aimed to present the enthusiastic reader-scientist, students, and other people inter-
ested in this fascinating subject with a general work of spider venoms, with every
chapter reflecting a description of the specialists’ work or offering an overview of a
particular aspect. Furthermore, their contributions are the fruit of diverse interna-
tional collaborations, reflecting that scientific investigation today is a worldwide
trade.
The first part of Spider Venom includes contributions regarding the wide diver-
sity of spider venom components and depicts some of their biological effects (i.e.,
antimicrobial, ion channel modulators, insecticides, including peptide and
nonpeptide toxins), and emphasizes those spiders of public health importance.
The second part covers transcriptomes, proteomes (and peptidomics), bioinformat-
ics, and molecular dynamics. The last part describes antimicrobial, insecticidal
toxins, envenomation, and the medical potential of spider venoms.
As editors, we endeavored to include all the necessary information to yield a
general and comprehensive work, containing those essential facts that can aid and
ix

accompany the enthusiastic reader in their navigation through unexplored domains.
To accomplish this, reviews, historical data, and all recent scientiﬁc spider venom
publications (peptides, toxins, transcripts, genes, transcriptomes, proteomes, in
silico analysis, molecular dynamics, medical potential, and insecticidal potential)
were included. The wealth of references assists in widening the vista on spider
venom research and related topics.
We offer our gratitude to the editor-in-chief, Professor Gopalakrishnakone of the
National University of Singapore, for presenting us the opportunity to contribute to
Springer’s Toxinology handbook series, by coordinating this Spider Venom volume.
We are deeply indebted to the academic reviewers for their invaluable comments to
improve the quality of the current work, and to all authors who kindly accepted the
invitation to contribute to this volume. Furthermore, we greatly appreciate the
assistance of Springer’s editorial team, in particular Audrey Wong, Sarah Mathews,
and Meghna Singh.
We hope that the Spider Venom volume will be useful to the enthusiastic reader-
scientist with an interest in spider toxinology and venom research, whether she or
he is a student, educator, aspiring or established scientist, or seasoned expert.
Finally, this book tries to be a nearly complete guide that we hope will inspire
fruitful research in various parts of the world.
February 2016 Elia Diego-García
Veerle, Belgium
Gerardo Corzo
Department of Molecular Medicine and Bioprocesses
The Biotechnology Institute
National Autonomous University of Mexico (UNAM)
Maria Elena de Lima
Belo Horizonte, MG, Brazil
x Volume Preface

Contents
Part I Venoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1 The Nonpeptide Low Molecular Mass Toxins from
Spider Venoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Paulo Cesar Gomes and Mario Sergio Palma
2 The Venom of Australian Spiders . . . . . . . . . . . . . . . . . . . . . . . . . 21
David T.R. Wilson
3 Venom of Cupiennius salei (Ctenidae) . . . . . . . . . . . . . . . . . . . . . . 47
Lucia Kuhn-Nentwig, Johann Schaller, Stefan Sch€urch, and
Wolfgang Nentwig
4 Phoneutria nigriventer Venom and Toxins: A Review . . . . . . . . . . 71
Maria Elena de Lima, Suely Gomes Figueiredo, Alessandra Matavel,
Kenia Pedrosa Nunes, Carolina Nunes da Silva, Flávia de Marco
Almeida, Marcelo Ribeiro Vasconcelos Diniz, Marta Nascimento do
Cordeiro, Maria Stankiewicz, and Paulo Sérgio Lacerda Beira˜o
5 The Venom from Lasiodora sp.: A Mygalomorph
Brazilian Spider . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Carolina Campolina Rebello Horta, Maria Chatzaki, Bárbara Bruna
Ribeiro Oliveira-Mendes, Anderson Oliveira do Carmo, Flávia de
Faria Siqueira, and Evanguedes Kalapothakis
6 Pain-Modulating Peptides in Spider Venoms: Good and Evil . . . . 121
Diochot Sylvie
7 Studying the Excitatory and Inhibitory Neurotransmissions with
Spider Venoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
José Luiz Liberato and Wagner Ferreira dos Santos
8 Phoneutria nigriventer Venom: Action in the
Central Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Maria Alice da Cruz-Ho¨ﬂing, Juliana Carvalho Tavares, and
Catarina Rapoˆso
xi

Part II Genes, Transcriptomes, and Bioinformatics . . . . . . . . . . . . 203
9 In Silico Modeling of Spider Toxins: Bioinformatics,
Molecular Docking, and Molecular Dynamics . . . . . . . . . . . . . . . . 205
Moacyr Comar Jr, Vanildo Martins Lima Braga, and Débora de
Oliveira Lopes
10 Spider Transcriptomes from Venom Glands: Molecular
Diversity of Ion Channel Toxins and Antimicrobial Peptide
Transcripts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Elia Diego-García, Camila Takeno Cologna, Juliana Silva Cassoli,
and Gerardo Corzo
11 Peptidome and Transcriptome Analysis of the Toxin-Like
Peptides in the Venom Glands of Tarantula
Grammostola rosea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Tadashi Kimura and Tai Kubo
Part III Medical and Insecticidal . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
12 Spider Venom and Drug Discovery: A Review . . . . . . . . . . . . . . . 273
Alessandra Matavel, Georgina Estrada, and Flávia De Marco Almeida
13 Anticancer Potential of Spider Venom . . . . . . . . . . . . . . . . . . . . . . 293
Elaine Maria de Souza-Fagundes, Betania Barros Cota, and Flávia De
Marco Almeida
14 Hippasa Spider: Biology, Envenomation, Toxin Proﬁles, and
Biological Functions – A Review . . . . . . . . . . . . . . . . . . . . . . . . . . 313
S. Nagaraju
15 Recent Insights in Latrodectus (“Black Widow” Spider)
Envenomation: Toxins and Their Mechanisms of Action . . . . . . . 333
Osmindo Rodrigues Pires Jr, Wagner Fontes, and Mariana S. Castro
16 Antimicrobial, Insecticides, Analgesics, and Hyaluronidases from
the Venom Glands of Brachypelma Spiders . . . . . . . . . . . . . . . . . . 345
Herlinda Clement, Guillermo Barraza, Estefania Herrera,
Francia García, Elia Diego-García, Elba Villegas, and Gerardo Corzo
17 Antimicrobial Peptides in Spider Venoms . . . . . . . . . . . . . . . . . . . 361
Daniel M. Santos, Pablo. V. Reis, and Adriano M.C. Pimenta
18 Structural Diversity and Basic/Acidic Residue Balance of Active
Cysteine-Rich Insecticidal Peptides from Spiders . . . . . . . . . . . . . 379
Francia García, Elba Villegas, Ernesto Ortiz, and Gerardo Corzo
xii Contents

19 Identifying Insect Protein Receptors Using an Insecticidal
Spider Toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
Mireya Cordero, M. Anwar Hossain, Nayely Espinoza,
Veronica Obregon, Mariel Roman, Samantha Navarro, Laura Lina,
Gerardo Corzo, and Elba Villegas
20 Loxosceles and Loxoscelism: Biology, Venom, Envenomation,
and Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Ceila Maria Sant’Ana Malaque, Olga Meiri Chaim, Marlene Entres,
and Katia Cristina Barbaro
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Contents xiii

Editor-in-Chief
Venom and Toxin Research Programme
Department of Anatomy
Yong Loo Lin School of Medicine
National University of Singapore
Singapore
antgopal@nus.edu.sg
P. Gopalakrishnakone, M.B.B.S., Ph.D., F.A.M.S.,
D.Sc., is presently professor of anatomy and chairman of
the Venom and Toxin Research Programme at Yong Loo
Lin School of Medicine, National University of Singa-
pore. He is also a consultant to the Defence Science
Organization in Singapore and adjunct senior research
scientist at the Defence Medical Research Institute. Professor Gopalakrishnakone is
an honorary principal fellow at the Australian Venom Research Unit, University of
Melbourne, Australia.
His research studies include structure function studies, toxin detection, biosen-
sors, antitoxins and neutralization factors, toxinogenomics and expression studies,
antimicrobial peptides from venoms and toxins, and PLA2 inhibitors as potential
drug candidates for inflammatory diseases. The techniques he employs include
quantum dots to toxinology, computational biology, microarrays, and protein chips.
Prof. Gopalakrishnakone has more than 160 international publications, 4 books,
about 350 conference presentations, and 10 patent applications.
He has been an active member of the International Society on Toxinology (IST)
for 30 years and was president from 2008 to 2012. He is also the founder president
of its Asia Pacific Section, a council member, as well as an editorial board member
of Toxicon, the society’s official journal.
His research awards include the Outstanding University Researcher Award from
the National University of Singapore (1998); Ministerial Citation, NSTB Year 2000
Award in Singapore; and the Research Excellence Award from the Faculty of
Medicine at NUS (2003).
xv

His awards in teaching include Faculty Teaching Excellence Award 2003/4 and
NUS Teaching Excellence Award 2003/4. Professor Gopalakrishnakone also
received the Annual Teaching Excellence Award in 2010 at both university and
faculty levels.
xvi Editor-in-Chief

Editors
Dr. Gerardo Corzo
Department of Molecular Medicine and Bioprocesses
The Biotechnology Institute
National Autonomous University of Mexico (UNAM)
Cuernavaca, Morelos
Mexico
Dr. Gerardo Corzo was born in Chiapas, Mexico, and
he studied biochemical engineering at the Metropolitan
University Campus Iztapalapa (Mexico, 1986),
obtained a master’s degree at the Institute of Biomed-
ical Research-UNAM (Mexico, 1993), and achieved a
Ph.D. at Oklahoma State University (USA, 1997). After
3 years of postdoctoral training at the Suntory Institute for Bioorganic Research
(Osaka, Japan), he became research associate at the same institute where he focused
on the peptide chemistry of arachnid venoms. In 2004, he moved to the Institute of
Biotechnology-UNAM as a Full Professor. He has maintained a long interest in the
discovery of natural products from arthropods, and in the recombinant expression
of cysteine-rich venom peptides and enzymes for therapeutic uses. Dr. Corzo has
published 78 peer-review articles and 8 patents. He currently sustains a strong
collaboration with Mexican pharmaceutical industries to which he had transferred
the intellectual property of two patents.
xvii

Dr. Maria Elena de Lima
Belo Horizonte
MG, Brazil
Dr. Maria Elena de Lima was born in Sacramento,
state of Minas Gerais (MG), Brazil. She is a Full Pro-
fessor of Biochemistry at Universidade Federal de
Minas Gerais, UFMG (MG, Brazil). She is graduated
in Biological Sciences by Universidade Federal de
Uberlândia (MG, Brazil), and holds a master’s in
Biochemichemistry by Universidade Federal de Minas Gerais and a Ph.D. in
Neuroscience by Aix Marseille University, Marseilles, France. She has been work-
ing on venoms and toxins since her master thesis, specially focused on those from
arthropods, including spiders, scorpions, among others. Her main focus is the
biochemical and pharmacological studies of the venoms and their toxins, selecting
those with therapeutic potential. She has been advisor of more than 50 master and
doctoral students. At Universidade Federal de Minas Gerais she was adjoint-dean of
research, president of the Ethical Committee for Investigation Involving Human
Being, the head of the Biochemical and Immunology Department, and the coordi-
nator of the Post Graduation Program of Biochemistry and Immunology. At
present, she is a member of the university council of UFMG.
Dr. de Lima has more than 80 published papers in indexed international journals,
five filled patents, and was editor-in-chief of the book Animal Toxins: State of the
Art – Perspectives in Health and Biotechnology published by UFMG’s editor. She
published about 10 book chapters, besides being editor of three scientific journals.
She has received awards for her scientific work, among them the “Santos Dumong
Medal” attributed by the governor of Minas Gerais state. She is a member of the
Brazilian Society of Toxinology, the Brazilian Society of Biochemistry and Molec-
ular Biology (SBBq), and the International Society on Toxinology (IST). She was
the president of the Brazilian Society of Toxinology for 4 years, having coordinated
two Congress of Toxinology, of which one of them was the World Congress of the
IST held at Recife, Pernambuco, Brazil, in 2009. She appreciates very much the
scientific interaction with many colleagues in the world.
xviii Editors

Dr. Elia Diego-García
Veerle, Belgium
Dr. Elia Diego-García is a molecular biologist and
toxinologist, specializing in the study of transcripts
and genes and the potential of toxins as ion channel
modulators. She graduated as a biologist with honors
from the Faculty of Biology, Universidad Michoacana
de San Nicolás de Hidalgo (UMSNH), Mexico. She
began her scientific career in plant tissue culture and
genetic transformation, and obtained her master’s in Biochemistry in 1998 from the
Universidad Nacional Autońoma de México (UNAM). Dr. Diego-García started a
Ph.D. under the professional guidance of Professor Emeritus Dr. Lourival
D. Possani at the Department of Molecular Medicine and Bioprocesses, Biotech-
nology Institute, UNAM. Her research was mainly focused on the characterization
of arachnid venom compounds and the genomic organization of toxin genes. She
received her Ph.D. in Biomedical Sciences in 2005. She continued her research
projects at UNAM as a postdoctoral research associate (2005–2007) and was
awarded the “Scholarships Programme for Young Professors and Researchers
from Latin America Universities” grant by the Coimbra Group in 2006.
In 2007, Dr. Diego-García entered as a postdoctoral fellow into the internation-
ally acknowledged research group of Professor Dr. Jan Tytgat at the Katholieke
Universiteit Leuven (KU Leuven), Belgium. She held this position until 2014 and
was involved in various projects using venom glands and venoms to search for new
compounds, combining transcriptomic, proteomic, and genomic analysis from
spiders and other animal species. She searched for new compounds that are
potential medicinal drugs (ion channel modulators and other biological activities).
Dr. Diego-Garcıá has published 20 scientific manuscripts in international reviewed
academic journals. She was an academic advisor for several Master’s and Ph.D.
students at UNAM and KU Leuven. She is currently an independent researcher
collaborating with the academic sector for venom and venom gland research
projects.
Editors xix

Contributors
Katia Cristina Barbaro Laboratory of Immunopathology, Butantan Institute, Saõ
Paulo, SP, Brazil
Guillermo Barraza Departamento de Medicina Molecular y Bioprocesos,
Instituto de Biotecnología, UNAM, Cuernavaca, Morelos, Mexico
Paulo Sérgio Lacerda Beiraõ Laborato´rio de Membranas Excitáveis,
Departamento de Bioquímica e Imunologia, Instituto de Cieˆncias Biolo´gicas,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Juliana Silva Cassoli Laborato´rio de Venenos e Toxinas Animais, Departamento
de Bioquimica e Imunologia, Instituto de Cieˆncias Biolo´gicas - Universidade
Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Mariana S. Castro Laboratory of Toxinology, Department of Physiological
Sciences/IB, University of Brasilia, Brasilia-DF, Brazil
Laboratory of Biochemistry and Protein Chemistry, Department of Cell Biology/
IB, University of Brasilia, Brasilia-DF, Brazil
Olga Meiri Chaim Department of Cell Biology, Laboratory of Extracellular
Matrix and Venom Biotechnology, Federal University of Paraná, Curitiba, PR,
Brazil
Maria Chatzaki Department of Molecular Biology and Genetics, Democritus
University of Thrace, Alexandroupoli, Greece
Herlinda Clement Departamento de Medicina Molecular y Bioprocesos, Instituto
de Biotecnología, UNAM, Cuernavaca, Morelos, Mexico
Camila Takeno Cologna Laboratory of Mass Spectrometry, Department of
Chemistry, University of Liege, Liege, Belgium
Moacyr Comar Jr Campus Centro-Oeste, Federal University of Saõ Joaõ Del
Rei, Divino´polis, MG, Brazil
Mireya Cordero Laboratorio de Estructura-Funcioń e Ingeniería de Proteínas,
Centro de Investigacioń en Biotecnología, Universidad Autońoma del Estado de
Morelos, Cuernavaca, Morelos, Mexico
xxi

Gerardo Corzo Department of Molecular Medicine and Bioprocesses, The
Biotechnology Institute, National Autonomous University of Mexico (UNAM),
Betania Barros Cota Chemistry of Bioactive Natural Products, Rene Rachou
Research Center/Fiocruz Foundation, Belo Horizonte, MG, Brazil
Maria Alice da Cruz-Ho¨fling Department of Biochemistry and Tissue Biology,
Institute of Biology, State University of Campinas (UNICAMP), Campinas, State
of Saõ Paulo, Brazil
Carolina Nunes da Silva Departamento de Bioquímica e Imunologia, Instituto de
Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Flávia de Faria Siqueira Departamento de Biologia Geral, Universidade Federal
de Minas Gerais, Belo Horizonte, MG, Brazil
Maria Elena de Lima Departamento de Bioquímica e Imunologia, Laborato´rio de
Venenos e Toxinas Animais, Instituto de Cieˆncias Biolo´gicas, Universidade Fed-
eral de Minas Gerais, Belo Horizonte, MG, Brazil
Flávia De Marco Almeida Department of Biochemistry and Immunology,
Biological Sciences Institute, Federal University of Minas Gerais, Belo Horizonte,
MG, Brazil
Débora de Oliveira Lopes Campus Centro-Oeste, Federal University of Saõ Joaõ
Del Rei, Divino´polis, MG, Brazil
Elaine Maria de Souza-Fagundes Department of Physiology and Biophysics,
Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
Elia Diego-García Veerle, Belgium
Marcelo Ribeiro Vasconcelos Diniz Centro de Pesquisa e Desenvolvimento Prof.
Carlos Diniz, Fundaçaõ Ezequiel Dias, Belo Horizonte, MG, Brazil
Anderson Oliveira do Carmo Departamento de Biologia Geral, Universidade
Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Marta Nascimento do Cordeiro Centro de Pesquisa e Desenvolvimento Prof.
Carlos Diniz, Fundaçaõ Ezequiel Dias, Belo Horizonte, MG, Brazil
Wagner Ferreira dos Santos Neurobiology and Venoms Laboratory, Biology
Department, College of Philosophy, Sciences and Literature, University of Saõ
Paulo, Ribeiraõ Preto, SP, Brazil
Marlene Entres Department of Health, Center for Poisoning Control, Parana,
Curitiba, PR, Brazil
Nayely Espinoza Laboratorio de Estructura-Funcioń e Ingeniería de Proteínas,
xxii Contributors

Georgina Estrada Centro de Investigacion Cientifica de Yucatan, Merida,
Yucatan, Mexico
Suely Gomes Figueiredo Departamento de Cieˆncias Fisiolo´gicas, Centro
Biomédico, Universidade Federal do Espírito Santo, Vito´ria, ES, Brazil
Wagner Fontes Laboratory of Biochemistry and Protein Chemistry, Department
of Cell Biology/IB, University of Brasilia, Brasilia-DF, Brazil
Francia García Departamento de Medicina Molecular y Bioprocesos, Instituto de
Biotecnología, UNAM, Cuernavaca, Morelos, Mexico
Paulo Cesar Gomes Department of Biology/CEIS/Institute of Biosciences of Rio
Claro, University of Saõ Paulo State (UNESP), Rio Claro, SP, Brazil
Estefania Herrera Departamento de Medicina Molecular y Bioprocesos, Instituto
de Biotecnología, UNAM, Cuernavaca, Morelos, Mexico
Carolina Campolina Rebello Horta Departamento de Biologia Geral,
Departamento de Biologia Geral, Programa de Po´s-Graduaçaõ em Cieˆncias
Biolo´gicas: Fisiologia e Farmacologia, Instituto de Cieˆncias Biolo´gicas,
M. Anwar Hossain Department of Microbiology, University of Dhaka, Dhaka,
Bangladesh
Evanguedes Kalapothakis Departamento de Biologia Geral, Universidade Fed-
Tadashi Kimura Molecular Neurophysiology Group, Biomedical Research Insti-
tute, National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba, Ibaraki, Japan
United Graduate School of Drug Discovery and Medical Information Sciences,
Gifu University, Gifu, Japan
Division of Biotechnology, The Institution of Professional Engineers, Japan (IPEJ),
Tokyo, Japan
Laboratory for Drug Discovery, and Glycoscience and Glycotechnology Research
Group, Biotechnology Research Institute for Drug Discovery, National Institute of
Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
Tai Kubo Molecular Neurophysiology Group, Biomedical Research Institute,
National Institute of Advanced Industrial Science and Technology (AIST),
Tsukuba, Ibaraki, Japan
Molecular Profiling Research Center for Drug Discovery, National Institute of
Advanced Industrial Science and Technology (AIST), Tokyo, Japan
United Graduate School of Drug Discovery and Medical Information Sciences,
Gifu University, Gifu, Japan
Contributors xxiii

Lucia Kuhn-Nentwig Institute of Ecology and Evolution, University of Bern,
Bern, Switzerland
José Luiz Liberato Neurobiology and Venoms Laboratory, Biology Department,
College of Philosophy, Sciences and Literature, University of Saõ Paulo, Ribeiraõ
Preto, SP, Brazil
Vanildo Martins Lima Braga Campus Centro-Oeste, Federal University of Saõ
Joaõ Del Rei, Divino´polis, MG, Brazil
Laura Lina Laboratorio de Estructura-Funcioń e Ingeniería de Proteínas, Centro
de Investigacioń en Biotecnología, Universidad Autońoma del Estado de Morelos,
Ceila Maria Sant’Ana Malaque Vital Brazil Hospital, Butantan Institute, Saõ
Paulo, SP, Brazil
Alessandra Matavel Research and Development Division, Ezequiel Dias
Foundation, Belo Horizonte, MG, Brazil
S. Nagaraju Department of Studies and Research in Biochemistry, Tumkur
University, Tumkur, Karnataka, India
Samantha Navarro Laboratorio de Estructura-Funcioń e Ingeniería de Proteínas,
Wolfgang Nentwig Institute of Ecology and Evolution, University of Bern, Bern,
Switzerland
Kenia Pedrosa Nunes Department of Cell and Regenerative Biology, School of
Medicine and Public Health, University of Wisconsin, Madison, WI, USA
Veronica Obregon Laboratorio de Estructura-Funcioń e Ingeniería de Proteínas,
Bárbara Bruna Ribeiro Oliveira-Mendes Departamento de Biologia Geral,
Ernesto Ortiz Departamento de Medicina Molecular y Bioprocesos, Instituto de
Biotecnología, UNAM, Cuernavaca, Morelos, Mexico
Mario Sergio Palma Department of Biology, CEIS, Laboratory of Structural
Biology and Zoochemistry, Sao Paulo, State University (UNESP), Institute of
Biosciences, Rio Claro, SP, Brazil
Adriano M. C. Pimenta Departamento de Bioquímica e Imunologia,
xxiv Contributors

Osmindo Rodrigues Pires Jr Laboratory of Toxinology, Department of Physio-
logical Sciences/IB, University of Brasilia, Brasilia-DF, Brazil
Catarina Rapoˆso Department of Biochemistry and Tissue Biology, Institute of
Biology, State University of Campinas (UNICAMP), Campinas, State of Saõ Paulo,
Brazil
Pablo. V. Reis Departamento de Bioquímica e Imunologia, Universidade Federal
de Minas Gerais, Belo Horizonte, MG, Brazil
Mariel Roman Laboratorio de Estructura-Funcioń e Ingeniería de Proteínas,
Daniel M. Santos Departamento de Bioquímica e Imunologia, Universidade Fed-
Stefan Sch€urch Department of Chemistry and Biochemistry, University of Bern,
Bern, Switzerland
Johann Schaller Department of Chemistry and Biochemistry, University of Bern,
Bern, Switzerland
Maria Stankiewicz Department of Biophysics, Faculty of Biology and Environ-
ment Protection, Nicolaus Copernicus University, Torun, Poland
Diochot Sylvie Institut de Pharmacologie Moléculaire et Cellulaire, CNRS
UMR7275, Université de Nice-Sophia Antipolis, Valbonne, France
Juliana Carvalho Tavares Department of Physiology and Biophysics, Federal
University of Minas Gerais (UFMG), Belo Horizonte, MG, Brazil
Elba Villegas Laboratorio de Estructura-Funcioń e Ingeniería de Proteínas, Centro
de Investigacioń en Biotecnología, Universidad Autońoma del Estado de Morelos,
David T. R. Wilson Centre for Biodiscovery and Molecular Development of
Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook
University, Smithfield, QLD, Australia
Contributors xxv

The Nonpeptide Low Molecular Mass
Toxins from Spider Venoms 1
Paulo Cesar Gomes and Mario Sergio Palma
Contents
Introduction ....................................................................................... 5
The Low-Molecular-Mass Defensive Compounds in the Arthropods ...................... 5
The Spider Venoms ........................................................................... 6
Low-Molecular-Mass Toxins ................................................................. 7
Acylpolyamines ................................................................................... 8
The Structure of the Nephilinae Acylpolyamine Toxins .................................... 9
Biological Activity ............................................................................ 10
Acylpolyamines as Neuroprotective Agents ................................................. 12
Nucleoside Toxins ................................................................................ 12
Bis(agmatine)oxalamide .......................................................................... 14
β-Carboline Toxins ............................................................................... 14
Diazenaryl Organometallic Toxins .............................................................. 15
Dioxopiperidine Compounds ..................................................................... 15
Conclusion and Future Directions ............................................................... 16
Cross-References ................................................................................. 17
References ........................................................................................ 17
Abstract
Spiders occupy most of the ecological niches of the planet, revealing a huge
adaptive plasticity, reﬂected in the chemical diversity of their venom toxins. The
spiders are distributed throughout the planet, adapting themselves to many
P.C. Gomes
Department of Biology/CEIS/Institute of Biosciences of Rio Claro, University of Sa˜o Paulo State
(UNESP), Rio Claro, SP, Brazil
e-mail: pccesar@rc.unesp.br
M.S. Palma (*)
Department of Biology, CEIS, Laboratory of Structural Biology and Zoochemistry, Sao Paulo,
State University (UNESP), Institute of Biosciences, Rio Claro, SP, Brazil
e-mail: mspalma@rc.unesp.br
P. Gopalakrishnakone et al. (eds.), Spider Venoms, Toxinology,
DOI 10.1007/978-94-007-6389-0_14
3

different environments, to form the largest taxonomic group of organisms with a
diet exclusively carnivorous. The organic low-molecular-mass compounds pre-
sent in spider venoms are used both for defensive purposes and to paralyze/kill
their preys. Among the low-molecular-mass organic compounds present in
spider venoms, the most common ones are free organic acids, amino acids,
biogenic amines, and neurotransmitters. These compounds were also used in
the course of evolution as substrates for the biosynthesis of novel spider toxins,
which were neglected by the toxinology during a long time, mainly due to the
difficulties to isolate and to assign the chemical structures of very low abundant
compounds. However, the recent technological advances in the spectroscopic
techniques used for structural analysis of small molecules allowed the structural
elucidation of many of these toxins in spider venoms, permitting the identifica-
tion of at least six families of low-molecular-mass toxins in spider venoms:
(i) acylpolyamines, (ii) nucleoside analogs, (iii) bis(agmatine)oxalamide,
(iv) the betacarboline alkaloids, (v) organometallic diazenaryl compounds, and
(vi) dioxopiperidinic analogs. Investigations of structure/activity relationship of
these toxins revealed that some of them have been identified both as interesting
tools for chemical investigations in neurobiology and as potential models for the
rational development of novel drugs for neurotherapeutic uses, as well as for
developing specific insecticides.
List of Abbreviations
13C Carbon-13
1H Hydrogen-1
ALS Amyotrophic lateral sclerosis
AMPA α-Amino-3-hydroxy-5-methylisoxasole-4-propionic acid
CID Collisional-induced dissociation
CNS Central nervous systems
COSY Homonuclear correlation spectroscopy
dqf COSY Double-quantum-filter-COSY
ESI-MS Electron spray ionization mass spectrometry
FRIT-FAB Continuous-flow fast atom bombardment
FTX Funnel web toxin
GABA Gamma-aminobutyric acid
Glu-R Glutamate receptor
HMBC Heteronuclear multiple bond coherence
HMQC Heteronuclear multiple quantum coherence
HPLC High-performance liquid chromatography
HRMS High-resolution mass spectrometry
JSTX Joro spider toxin
KA Kainic acid
kDa Kilodalton
L-Arg-3,4 L-Arginyl-3,4-spermidine
LC-MS Liquid chromatography mass spectrometry
LMM Low molecular mass
4 P.C. Gomes and M.S. Palma

MALDI-TOF Matrix-assisted laser desorption/ionization time of ﬂight
MS/MS Tandem mass spectrometry
nACh-R Nicotinic acetylcholine receptor
NMDA N-Methyl- D-aspartate
NMR Nuclear magnetic resonance
NOESY Nuclear Overhauser enhancement spectroscopy
THβC Tetrahydro-β-carbolines
Introduction
The Low-Molecular-Mass Defensive Compounds in the Arthropods
In recent decades, a growing interest in the study of venomous organisms, mainly in
the wide variety of non-polypeptide organic toxins found in their venoms, has been
observed. These toxins serve as sources of basic tools for understanding the
functioning of the central and peripheral nervous system, cardiovascular physiol-
ogy, some aspects of the hormonal regulation, activation of the complement and
immune systems, mechanisms of pain, allergic processes, blood coagulation, and
hemostasis regulation. The study of the structure/activity relationship of these
toxins has inspired the development of novel drugs for different diseases,
as well as resulted in alternative treatments for some types of envenoming
(Menez et al. 2006).
The success of arthropods to colonize the Earth is generally attributed to the
extraordinary chemical versatility of these animals. They produce substances for
many different proposals: venoms to paralyze and/or kill their preys, repellent
compounds to ward off their enemies, and pheromones for social communication
and reproduction, among other functions. These substances are generally produced
by integumental glands, originated by evolution of specialized regions of the
epidermis (Eisner and Meinwald 1995).
Many different natural products are produced and secreted by arthropods under
different situations. An interesting example is the defensive system of pseudoscor-
pion Mastigoproctus giganteus, which ﬂushes a mixture of acetic and caprylic acids
against their enemies, where the caprylic acid acts as lipophilic agent on the
epicuticle (lipid coated) of the offender insect, facilitating the transport of acetic
acid through the wax layer that protects the offender, causing his death, a simple
strategy that has allowed the pseudoscorpion to survive for 400 Ma. Many arthro-
pods use other types of chemical defenses, to detain their abusers (Attygalle
et al. 1993).
While the defensive secretions of arthropod compounds are based on well-
characterized chemical structures, such as aliphatic acids, aldehydes, phenols, and
quinones, there are many cases in which compounds have a complex chemical
composition, such as in the case of the Dytiscidae beetle Abedus herberti, which
produces a mixture of pregnanes, including the deoxycorticosterone as the main
component of the mixture of defensive structures (Lokensgard et al. 1993).
1 The Nonpeptide Low Molecular Mass Toxins from Spider Venoms 5

Among the defensive compounds from arthropods, the wide structural diversity
of alkaloids must be emphasized as an exquisite example of chemical creativity of
the nature. During decades it was believed that the alkaloids were produced
exclusively by the secondary metabolism of plants; however, recently there have
been many reports of arthropodan alkaloids, used as defensive compounds, such as
acetogenins, simple aromatics, quinones, and isoprenoids. Apparently these types
of alkaloids are produced by the classic major pathways; however, some species of
millipedes, coccinellid beetles, and spiders have been reported as very creative
alkaloid chemists that use biosynthetic pathways, not well understood up to now.
There are evidences that some of these arthropods have the ability to sequester
ready-made defensive compounds from plants and even from animal sources, using
these compounds as substrates for semisynthesis of a wide range of structurally
innovative compounds used in different defensive situations (Meinwald and Eisner
1995; Eisner and Meinwald 1995).
The Spider Venoms
Belonging to the phylum Arthropoda, spiders (order: Araneae) occupy most of the
ecological niches of the planet, revealing a huge adaptive plasticity, which is
reflected in the chemical diversity of their toxins. Generally the structures and
modes of action of toxins from spider venoms are in closer relationship with the
mode of hunting and prey selection by the spiders, which in turn are reflecting the
characteristic biology of these animals. The spiders are distributed throughout our
planet, having adapted theirselves to all natural environments, with the exception
of the air and the open sea, to form the largest taxonomic group of organisms with
a diet exclusively carnivorous (Coddington and Levi 1991).
Depending on the type of ecosystem where the spider lives, it adjusts its
behavior to optimize prey capture for obtaining food. According to the feeding
habits and hunting strategy, spiders can be divided into two groups: (a) the
orb-weaving spiders that expend all their life on the webs, which are also used
for prey capture, and (b) the wandering spiders that may occupy the most varied
environments from the surface of an aquatic environment and can inhabit the most
hospitable places of the Earth, with great behavioral adaptation to hunt the most
varied types of prey (Foelix 1996).
Despite the large number of known spider species, only a small number have
been investigated up to now in relation to the composition and properties of their
venoms. The majority of toxins already identified in spider venoms are peptides and
proteins rich in disulfide bonds. In combination with the low-molecular-mass
toxins, peptides appear to represent the major toxic arsenal of spider venoms. The
proteins include both neurotoxins and high-molecular-mass enzymes (proteases,
hyaluronidases, sphingomyelinases, isomerases, and phospholipases) (Rash and
Hodgson 2002).
There is a wide variation of composition of the constituents of spider venoms
according to the type of prey available in each season and preying strategy

adopted by the spiders. Considerable heterogeneity in the composition between
the venoms of individuals of the same species has also been observed, according
to factors such as age, sex, size of animals, diet, and geographic distribution. Since
the primary purpose of spider venoms is to paralyze their preys, many components
of these venoms have as main targets the central and/or peripheral nervous
system, modulating the generation and propagation of action potentials on mul-
tiple molecular sites as axons, synapses, or neuromuscular junctions (Escoubas
et al. 2008).
Low-Molecular-Mass Toxins
The organic low-molecular-mass compounds present in spider venoms are used
both for defensive purpose and to paralyze and/or kill their preys, affecting
the synaptic transmission and blocking the functioning of ion channels of the
prey animals (Grishin 1994). Among the low-molecular-mass organic
compounds present in spider venoms, the most common ones are free acids
such as citric and lactic; glucose; free amino acids; biogenic amines such as
diaminopropane, putrescine, cadaverine, spermine, and spermidine; and
neurotransmitters such as aspartate, glutamate, serotonin, histamine, γ-butyric
acid, dopamine, and epinephrine (Escoubas et al. 2000; Palma and
Nakajima 2005). Some of these compounds act synergistically with the low-
molecular-mass nonpeptide toxins, activating on ion channels before toxin
actions. Some free amino acids, polyamines, and neurotransmitters are used as
building blocks for the synthesis of the low-molecular-mass toxins (Palma and
Nakajima 2005).
The most of low-molecular-mass toxins from spider venoms are potent antag-
onists of neurotransmitter receptors, which apparently exhibit a high speciﬁcity for
L-glutamate receptors (Antonov et al. 1989). These toxins have enormous potential
to be used as tools for neurochemical studies, as well as models for the develop-
ment of novel drugs of neurotherapeutic applications (McCormick and Meinwald
1993).
Recently, different experimental protocols such as bidimensional NMR
spectroscopy, HRMS, and MS/MS have been combined for the structural elucida-
tion of toxins in complex matrices, such as spider venoms. Direct analysis by NMR
of the crude venom through 1H, dqf COSY (HMQC), (HMBC), and NOESY,
complemented by analyses of mass spectrometry under conditions of CID, allowed
the structural characterization of the main low-molecular-mass components of
the venoms from different types of spiders. The use of spectroscopic strategies
applied to the toxinology of spiders in the recent years allowed the achievement of
an overall view of the panel of these toxins in spider venoms.
Among these compounds, the most important types are (i) acylpolyamines,
(ii) nucleoside analogs, (iii) bis(agmatine)oxalamide, (iv) the betacarboline
alkaloids, (v) organometallic diazenaryl compounds, (vi) and dioxopiperidinic
analogs.

Acylpolyamines
Until the 1980s, it was widely accepted that the most neurotoxins from spider and
wasp venoms were basically proteins and peptides; however, the discovery of
acylpolyamines in the venoms of orb-weaving spiders and parasitic wasps
completely changed this viewpoint.
The acylpolyamines are neurotoxic compounds occurring only in the venom
glands of spiders at a picomolar level (Palma et al. 1998). More than 100 chemical
structures of acylpolyamines have been elucidated, which constitute a family of
closely related toxins. The most well-characterized chemical structures of
acylpolyamine toxins among the Araneidae spiders are those from the
orb-weaving spiders belonging to the Nephilinae subfamily and from the Argiope
genus (Aramaki et al. 1987).
The acylpolyamine toxins were the first low-molecular-mass neurotoxins from
spider venoms to have their chemical structures elucidated. These toxins have been
isolated mainly from the venoms of spiders of the order Araneidae; these toxins
block postsynaptic potential at the level of glutamate receptors of the neuromus-
cular junctions of crustaceans (Hagiwara and Byerly 1981; Kawai et al. 1982);
some of these toxins also cause paralysis in insects (Grishin 1994).
The venoms from the Nephilinae orb-weaving spiders were thoroughly investi-
gated; thus, the acylpolyamine toxins from this group of spiders are by far the most
well known. Until the 1990s, the standard procedures for elucidating the structure
of these toxins required an extensive purification from a huge amount of venom,
followed by the use of traditional chemical protocols (hydrolysis and derivatization,
amino acid analysis by Edman degradation chemistry, and 1
H and 13
C-NMR).
Between 1985 and 1990, about 17 different structures were elucidated with this
experimental approach from the venom of the spider Nephila clavata (Aramaki
et al. 1987). A very sensitive methodology for the direct detection of these toxins in
venom extracts was developed by using online microcolumn HPLC continuous
flow (FRIT) FAB LC/MS and high-energy CID methods with sodium-attached
molecular ions, to produce very effective information about the structures of this
class of toxins in Nephila clavipes and Nephilengys borbonica. The venom of the
Brazilian garden spider Nephilengys cruentata was characterized by using a com-
bination of HPLC/MS, MALDI-TOF, and MALDI-sector-type mass spectrometry
(Palma et al. 1997, 1998).
The general structures of the acylpolyamine toxins (1) may be separated into
four parts as represented in Fig. 1: a lipophilic aromatic acyl moiety (part I), a
linker amino acid residue (part II), the polyamine backbone chain (part III), and
the backbone tail (part IV). The aromatic acyl group and the polyamine backbone
constitute mandatory parts of these compounds, shared by all known toxins of this
class, whereas the linker amino acid(s) and the tail constitute optional parts,
occurring only in some toxins. The Araneidae orb-weaving spiders biosynthesize
acylpolyamine toxins containing both the mandatory and optional structural parts,
while the toxins from funnel-web, trap-door, and tarantula spiders generally present
only the two mandatory structural parts (Schambacher et al. 1973).

Each one of these structural parts is built from simple chemical building blocks,
which in turn may be connected to each other, offering the possibility to create a
natural combinatorial chemistry strategy by the Nephilinae spiders. This strategy is
the result of the evolution maximizing the efﬁciency of preying. It also reﬂects the
plasticity of this group of spiders to diversify their venom arsenals according to the
different prey availability under different ecological niches (Palma and Nakajima
2005).
The acylpolyamines are present in the venom of spiders of different taxonomic
genus and ecological niches and exhibit high structural similarity between their
molecules, like those from the venoms of spiders of the genus Argiope (compounds
(2) and (3) in Fig. 2) and Nephila (compounds (4) and (5) in Fig. 2).
The Structure of the Nephilinae Acylpolyamine Toxins
The structural parts of Nephilinae acylpolyamine toxins are constituted by chemical
blocks described as follows: (I) the aromatic lipophilic head part from indole acetic
acid, 4-hydroxyindole acetic acid, or even 2,4-dihydroxy phenyl acetic acid; (II) the
linker amino acid part, an asparagine residue, or a dipeptide asparaginyl-ornithine;
(III) the polyamine backbone part that may be constituted by simple chemical
blocks, such as the polyamines cadaverine, putrescine, and diaminopropane, or by
the amino acid residues of glycine, alanine, asparagine, and ornithine; and (IV) the
backbone tail part that is either putreanine or arginine and/or ornithine; sometimes
even a glycine residue may be used to build this tail.
HO
OH
(1)
O
Mandatory parts
Aromatic
Acyl group
(Part I)
(Part II)
Linker
Amino acid
(Part IV)
Backbone
tail
Optional parts
Polyamine backbone
(Part III)
O
O
O
H
N
N
H
NH2
NH2
NH2
NHO
N
H
N
H
H
N
H
N
Fig. 1 Structural parts of the acylpolyamine toxins from spider venoms

Depending on the different combinations of these chemical blocks, the poly-
amine backbone chains are classiﬁed into seven different subtypes. Considering the
polyamine chain (part III) as reference for the biosynthesis of these toxins, it is
possible to select one of the seven subtypes to combine with the linker group (part II)
to produce a complete polyamine backbone. The asparagine residue is the most
commonly used linker group in this backbone. However, nature has already tried the
dipeptide asparaginyl-ornithine, instead of the single amino acid residue as linker
group. This backbone, in turn, may be connected to one of the known chromophores
(2,4-dihydroxyphenyl acetic acid, 4-hydroxyindole acetic acid, or even with indole
acetic moiety), creating molecules with different hydrophobicity. Optionally
a tail (part IV) may be attached to the polyamine chain in a single unit or
sometimes in tandem mode. Potentially the combinations of three chromophores,
two types of amino acid linkers, seven polyamine backbone subtypes, and nine
options of tails offer 378 different structural possibilities (Palma and Nakajima
2005).
Biological Activity
Most of the known acylpolyamine toxins are neurotoxic compounds, acting as
antagonists of different subtypes of ionotropic glutamate (Glu) receptors (Rs),
while some of these toxins also may act on nicotinic acetylcholine receptors
(Kawai and Nakajima 1993).
Acylpolyamines interact strongly with the neuromuscular junctions of insects,
which generally are rich in different types of ionotropic Glu-Rs; however, there is
relatively high structural similarity between these insect receptors and their homo-
logues in vertebrates. The vertebrate Glu-Rs are divided into subclasses based on
Argiope lobata
Argiope aperta
Nephila clavata
Nephila maculata
Argiotoxin
(Arg 636)
SPIDER SPECIES TOXIN STRUCTURE
HO
HO
HO
CONH2
CONH2
CONH2
OH NH
NH2
NH2
AGEL-489 (7, R=H)
AGEL-505 (8, R=OH)
NH2
NH2
NH2H
NH
NH2O
O
O
O
O
O O
O
O
R
O
H
N
H
N
H
HN
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
N
N
H H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
N
H
(2)
(3)
(4)
(5)
N
OH
OH
OH
Nephila Toxin 3
(NSTX 3)
Joro spider Toxin 3
(JSTX 3)
a-Agatoxin-489 (AGEL 489);
a-Agatoxin-505 (AGEL 505)
Fig. 2 Examples of acylpolyamine toxins isolated from spider venoms

their responses to exogenous ligands: N-methyl-D-aspartate (NMDA)-dependent,
α-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA)-dependent, and
kainate (KA)-dependent receptors (Collingridge and Lester 1989). Meanwhile, the
invertebrate Glu-Rs are classified into four subtypes: (i) quisqualate receptors that
gate cation channels (qGlu-R) (AMPA receptors), (ii) ibotenate receptors that gate
chloride channels, (iii) a purported KA receptor, and (iv) a purported NMDA
receptor (Collingridge and Lester 1989; Mellor and Usherwood 2004). The
AMPA- and KA-dependent subtypes are involved in synaptic pathways of central
signaling, playing different roles in conjunction with each other.
A general model of binding between acylpolyamines and Glu-Rs was proposed,
in which electrostatic interactions between the protonated amino groups of the
toxins and the negatively charged amino acid residues in the interior of the receptor
ion channel contribute to the binding. Thus, the toxin enters and plugs the open
cation channels gated by a Glu-R, inhibiting the ion flow through the channel (Choi
et al. 1995; Mellor and Usherwood 2004). Despite this, due to the complex
properties of these toxins and the diversity of cellular responses, the interactions
between the acylpolyamines and the most types of ion channels are still not
completely understood at the molecular level.
The amino acid-containing polyamine toxins generally act on neuromuscular
junctions of invertebrates, causing reversible noncompetitive inhibition of
quisqualate sensitive Glu-Rs. However, there are different selectivities and speci-
ficities for different subtypes of glutamate receptors. The non-amino acid-
containing acylpolyamine toxins generally occur as selective and reversible,
noncompetitive inhibitors of NMDA-Glu-Rs from the mammalian brain, in the
venoms of funnel-web spiders, trap-door spiders, and some tarantulas (Willians
1997; Parks et al. 1991). The presence of basic amino acid residues or a positively
charged moiety in the terminal region (“tail”) of the polyamine chain determines
the potential to block irreversibly Glu-Rs (Palma et al. 1998; Jackson and
Usherwood 1988).
The spider toxin JSTX-3 was reported to block the postsynaptic action of Glu-Rs
in mammalian central neurons. The use of recombinant expression of AMPA/KA-
Glu-Rs in Xenopus oocytes permitted the use of electrophysiological measurements
of this preparation, to demonstrate that JSTX-3 acts as a specific blocker of the
receptor subunit at level of Glu-R1, Glu-R3, Glu-R4, and Glu-R1/3, with a recti-
fying current–voltage (I–V) relationship. The toxin did not affect the Glu-R1/2,
Glu-R2/3, and Glu-R6. Later, the use of site-directed mutagenesis permitted iden-
tification of the transmembrane domain position responsible for the interaction of
Glu-R with JSTX-3 (Palma et al. 1997; Estrada et al. 2007).
It is well known that the acylpolyamine toxins cause paralysis in spider’s prey
that may last from several hours to many days, depending on the composition of the
toxin cocktail present in the venom (Manzoli-Palma et al. 2006). It was reported
that the paralytic activity may be strongly potentiated by the presence of Zn+2
ions,
which naturally occur in high concentrations in spider venoms. The acylpolyamine
toxins can form structural complexes with some metal ions due to the nitrogen-
crowded conformation assumed by the polyamine backbone that permits the

chelation of metal ions. In turn, these metal ions are transported by the toxin until
the binding site region within the Glu-R (Manzoli-Palma et al. 2006). Next to the
acylpolyamine binding site, another binding site specific for Zn+2
ions does exist.
Once occupied by its ligand, this neighbor site induces conformational changes in
the receptor, promoting in turn the interaction with the binding site of the
acylpolyamines and potentiating the paralytic action (Manzoli-Palma et al. 2006;
Stone 1995).
Acylpolyamines as Neuroprotective Agents
Cerebral ischemia may cause excessive activation of excitatory synapses, followed
by sustained influx of calcium (Ca2+
) ions (mediated by Glu-Rs); consequently, the
intracellular concentrations of Ca2+
ions are elevated, contributing in turn to
neuronal death. The use of acylpolyamines as neuroprotective agents is generally
associated with the occurrence of long-term ischemia in the brain, as observed in
stroked patients or as a result of brain damage (Kawai 2005; Schurr 2004).
Epilepsy is a chronic neurological disorder affecting about 1 % of the world’s
population. The response to therapy is generally good, but up to 30 % of patients
cannot achieve acceptable seizure control despite adequate trials with potentially
effective antiepileptic agents. In order to develop new antiepileptic therapeutic
strategies, it is important to understand the basic mechanisms involved in epileptic
discharges. Many diseases and neuronal disorders are caused by receptor and ion
channel dysfunction. Currently, the excitatory amino acid receptors represent
promising targets for the development of novel drugs to treat epilepsy. There are
evidences indicating that the Nephilinae acylpolyamines block nonselectively the
AMPA and NMDA-Glu-Rs, producing a synergic anticonvulsant effect. The
acylpolyamine JSTX-3 was reported to present anti-epileptogenic action due, at
least in part, to the inhibitory action of this toxin on the cationic currents evoked by
NMDA receptor activation (Kwan and Brodie 2000; Salamoni et al. 2005).
Nucleoside Toxins
Nucleosides are relatively common in arthropod venoms, playing toxic roles in the
envenoming processes; sometimes these compounds are esterified to one or two
sulfate groups, becoming very potent paralytic toxins. They were initially identified
in the venom of the grass spider Hololena curta. The first chemical structure of a
nucleoside toxin completely assigned was named HF-7 (compound (6) in Fig. 3); it
is a bisulfated glyconucleoside presenting the ability of blocking kainate Glu-Rs, in
addition to weakly blocking L-type of calcium channels. Sulfated guanosine deriv-
atives (compounds (7) and (8) in Fig. 3) were also characterized from the venom of
hobo spider Tegenaria agrestis venom. The toxins 2,5-disulfated guanosine and
2-sulfated guanosine [compounds (9) and (10), in Fig. 3] were recently identified in

the venoms of three species of Loxosceles spiders: L. arizonica, L. deserta, and the
well-known brown recluse L. reclusa (Schroeder et al. 2008).
The major components of the low-molecular-mass fractions of the venom of the
spider Latrodectus menavodi were found to be adenosine, guanosine, inosine, and
2,4,6- trihydroxypurine (compounds (11), (12), (13), (14) in Fig. 4 Horni
et al. 2001).
Inosine was also identified as low-molecular-mass component from the venom
of the colonial spider Parawixia bistriata; this compound presented pro-convulsant
action in rats. Despite nucleosides being considered as anticonvulsant compounds
and/or neuroprotective agents, studies have indicated that the injection of low
O
O
O
O
(6)
N
N
N
NH
OH
HO
OAc
NH2HO3
SO
H3
C
O OH
HO
OH
Me
Me
OSO3
H
(8)
OH
H
NN
N
NH
O
O
O
O
HO3
SO
OH
O
O
(10)
O
N
N
N
NH
NH2HO
OSO3
H
O
O
(9)
OH
N
N
N
NH
NH2HO3
SO
OSO3
H
O
(7)
O
O
O
N
N
N
NH
OH
OH
O
OH
NH2HO3
SO
Fig. 3 Nucleosides toxins identified in spider venoms: HF-7 (6) isolated from the grass spider
Hololena curta; sulfated guanosine derivatives (7) and (8) characterized of the hobo spider
Tegenaria agrestis; sulfated nucleosides 2,5-disulfated guanosine and 2-sulfated guanosine (9)
and (10), identified in the three species from the genus Loxosceles
Fig. 4 Nucleoside structures of the venom of the spider Latrodectus menavodi: adenosine (11),
guanosine (12), inosine (13), and 2,4,6-trihydroxypurine (14)

concentrations of inosine into rat cortex caused epileptiform discharges and sei-
zures; it has been suggested that this nucleoside may play a role in the initiation of
seizures. However, at high concentrations, this compound may play anticonvulsant
action (Lewin and Bleck 1981; Rodrigues et al. 2004).
Bis(agmatine)oxalamide
N,N-Bis(4-guanidinobutyl)oxalamide [compound (15), Fig. 5] was isolated from the
venom of the fishing spider Plectreurys tristis (Plectreuridae), which is a species
native to Mexico and the Southwestern USA. The mechanism of action of this toxin
is unknown, but it is used as a prey paralyzing agent by the spider (Quistad et al. 1993).
b-Carboline Toxins
Tetrahydro-β-carboline (THβC) compounds are endogenous in some animals and
generally are found at trace levels in mammalian brains. These alkaloids act on
various aspects of the neurotransmission modulation and are neurotoxic since they
constitute a family of high-affinity ligands of the benzodiazepine receptors, which
is a subtype of GABA receptor. The THβC compounds are structurally related to
the serotonin molecules, and because of this structural similarity, these alkaloids are
capable of binding to multiple receptors, such as benzodiazepinic, imidazolynic,
and serotonergic types. Trypargine, a β-carboline isolated from the skin of the
African frog Kassina senegalensis, has been studied since the 1980s; it is known
to cause an action-related inhibition of Na+
and Ca2+
ion current when applied
in internal surface of squid axonal membranes and also plays a modulatory action in
5-hydroxytryptamine-like receptors. Alkaloidal toxins such as 1-3-
guanidinopropyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline and 1-4-guanidinob-
utoxy-6-hydroxy-1,2,3,4-tetrahydro-β-carboline, known as PwTx-I and PwTx-II,
respectively, are THβC compounds isolated from the venom of the colonial spider
Parawixia bistriata (compounds (16) and (17) in Fig. 6). These compounds are used
as toxins for killing/paralyzing the preys of the colonial spiders.
H
N
H2N N
H
NH
O
O (15)
NH
HN
NH
NH2
Fig. 5 Chemical structure of bis(agmatine)oxalamide (15), isolated from the venom of the fishing
spider Plectreurys tristis

Meanwhile, the indolylalkaloid toxin, known as NWTx-I, was isolated from the
oily droplets of the web of the spider N. clavipes [compound (18), Fig. 6]. These
compounds are part of the chemical weaponry to kill/paralyze the arthropod preys
of the orb-weaving spiders and are also neurotoxic, convulsive, and lethal to rats.
Apparently, these toxins promote the activation of Ca+2
ion (by an unknown
mechanism) (Cesar-Tognoli et al. 2011).
Diazenaryl Organometallic Toxins
The Nephilinae orb-weaving spiders are predators which use their orb webs as part
of the strategy for prey capture. The web of Nephila clavipes generally is covered
by adhesive droplets containing different types of toxins, directly involved with
prey paralysis/killing without need of venom injection by the spider. These droplets
contain small vesicles ﬁlled with solutions of low-molecular-mass nonpeptide
compounds, which act as part of the cocktail of paralytic/killing arsenal of this
spider. Most of the compounds already identiﬁed within these droplets are
neurotransmitters, such as N-acetyltaurine, 4-aminobutyramide, glycine, betaine,
choline, and putrescine (Cesar-Tognoli et al. 2011).
Recently, an organometallic 1-(diazenylaryl) ethanol compound from the web of
the spider N. clavipes, which presents a potent lethal action against the spiders’
prey, was characterized [compound (19) in Fig. 7] (Marques et al. 2004).
Dioxopiperidine Compounds
The compound hydroxyl-hydrazyl-dioxopiperidine [1,10-(1-hydroxyhydrazine-1,2-
diyl)bis(oxy)bis(4-hydroxy-2,6-dioxopiperidine-4-carboxylic acid)], generically
named nigriventrine, was isolated and structurally characterized from the hydrophilic
fraction of the venom from the “armed” spider Phoneutria nigriventer [compound
Fig. 6 Structures of beta-carboline toxins from orb-weaving spiders: PwTx-I (16), isolated from
the venom of the spider P. bistriata; NWTx-I toxin (17) isolated from the venom of the spider
N. clavipes; PwTx-II (18), isolated from the web of the spider P. bistriata

(20) in Fig. 8]. It is a novel natural compound not previously reported among the
venoms of arthropods. The dioxopiperidine moiety is uncommon among the low-
molecular-mass nonpeptide compounds from animal venoms. It has already been
reported as a basic building block of analgesic, antianxiety, and antipsychotic syn-
thetic drugs (Gittos 1989). This was the ﬁrst report of a natural compound of animal
origin presenting this type of chemical structure. The neuroactivity of nigriventrine in
rat brain was investigated through monitoring the pattern of expression of c-Fos
protein. This protein is an inducible transcription factor, which is an important tool
and well-established marker to identify activated neurons in the autonomous or central
nervous system after physical, chemical, and/or biological stimuli. This assay revealed
that nigriventrine acted in seven different brain regions: the motor cortex, sensory
cortex, piriform cortex, median preoptic nucleus, dorsal endopiriform nucleus, lateral
septal nucleus, and hippocampus. This is the ﬁrst type of low-molecular-mass toxin
reported in the venom of the “armed” spider P. nigriventer and must be more deeply
investigated in the near future. Electrophysiological studies were performed in prep-
arations of rat brain hippocampal CA1 region suggesting that nigriventrine is a potent
blocker of NMDA-R1, with anti-epileptogenic properties (Gomes et al. 2011).
Conclusion and Future Directions
Generally the structures and modes of action of toxins from spider venoms are in
close relationship with spider’s biology, mode of hunting, and prey selection. The
use of large orbital webs associated with social cooperation between the individuals
N
OHH3C
(19)
Fe
NH
Fig. 7 Structure of
1-(diazenylaryl) ethanol (19)
isolated from the web of the
spider N. clavipes
HO
O
HO
N
N
H
N
O
O
O
O
OH
OH
O
OH
O (20)
O
N
Fig. 8 Structure of the nigriventrine (20) isolated from the venom of the armed spider Phoneutria
nigriventer

of some spider species for preying and feeding, in addition to the evolutionary
position of other species between wandering spiders and those truly orb-weaving
ones, challenges our knowledge to find novel and interesting toxins in venom of
these spiders.
The actions of nonpeptide low-molecular-mass compounds present in spider
venoms usually complement those actions of the protein/peptide toxins, being used
as chemical tools for both defensive purposes and paralysis/death of the spiders’
prey. Many of these compounds have toxic functions within these venoms,
performing well-defined roles in the envenoming processes caused by spider
bites. Many of these low-molecular-mass compounds are neurotoxins, which play
their roles by blocking ion channels and/or their associated receptors.
For many years, these toxins were neglected by the toxinology, mainly due to the
difficulties to isolate and to assign the chemical structures of very low abundant
compounds. However, the recent technological advances in the spectroscopic
techniques used for structural analysis of small molecules allowed the structural
elucidation of many of these toxins in spider venoms. This paved the way for the
chemical synthesis of these molecules, providing them in amounts enough for
physiological and pharmacological studies. Investigations of structure/activity rela-
tionship of these toxins revealed that some of them have been identified both as
interesting tools for chemical investigations in neurobiology and as potential
models for the rational development of novel drugs for neurotherapeutic uses, as
well as for developing specific insecticides.
Cross-References
▶ Identifying Insect Protein Receptors Using an Insecticidal Spider Toxin
▶ Spider Venom and Drug Discovery: A Review
▶ Studying the Excitatory and Inhibitory Neurotransmissions with Spider Venoms
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The Venom of Australian Spiders
2
David T. R. Wilson
Contents
Introduction ....................................................................................... 22
Clinical Implications of Spiders of Medical Significance in Australia ......................... 23
Australian Funnel-Web Spiders (Hexathelidae) ............................................. 23
Redback and Widow Spiders (Theridiidae) .................................................. 24
Other Australian Spiders ...................................................................... 26
Australian Spider Venom Components .......................................................... 28
Australian Funnel-Web Spiders (Hexathelidae) ............................................. 29
The δ-HXTX-1 Family ....................................................................... 29
The ω-HXTX-1 Family ....................................................................... 32
The ω-HXTX-2 Family ....................................................................... 34
The κ-HXTX-1 Family ....................................................................... 34
The ω/κ-HXTX-1 Family ..................................................................... 35
The U1-HXTX-1 Family ...................................................................... 37
The U2-HXTX-1 Family ...................................................................... 37
The Venom of Redback Spiders (Latrodectus hasselti) ..................................... 38
The Venom of Mouse Spiders (Missulena spp.) ............................................. 39
The Venom of Australian Tarantulas (Theraphosidae) ...................................... 40
The Venom of Other Australian Spider Species ............................................. 41
Conclusions and Future Directions .............................................................. 42
Cross-References ................................................................................. 42
References ........................................................................................ 43
Abstract
Australia is home to an estimated 10,000 species of spider, including species
from the Latrodectus genera and Atracinae family, two of the four widely
recognized medically significant spider groups. It is predicted in excess of
D.T.R. Wilson (*)
Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of
Tropical Health and Medicine, James Cook University, Smithfield, QLD, Australia
e-mail: david.wilson4@jcu.edu.au
P. Gopalakrishnakone et al. (eds.), Spider Venoms, Toxinology,
DOI 10.1007/978-94-007-6389-0_21
21

5,000 spider bite cases occurring annually in Australia, predominantly by spiders
that have not shown any medical relevance. Bites by medically relevant spiders
are rare, and of those treatment with antivenom is rarer. Despite extensive
publicity and rumor, there is no conclusive evidence that the venom of any
Australian spiders is responsible for necrotic arachnidism. The complexity and
diversity of spider venoms, combined with potent activity on a range of targets in
mammalian and insect systems, have attracted interest in the potential of spider
venoms as a source of insecticidal and therapeutic leads. The venom of species
of Australian funnel-web spider has received the most attention for study, with
more than 75 venom peptides identified from nine toxin families. Recent work
has identified venom peptides from the venom of Australian tarantulas with
potential as insecticidal and therapeutic leads. This chapter provides an overview
of spiders in Australia and their medical and clinical importance and provides a
current comprehensive review of the published toxins from Australian spider
venoms.
Introduction
Spiders (Arthropoda: Arachnida: Araneae) constitute the most successful venom-
ous creature, in terms of speciation and distribution, on the planet and, with the
possible exception of predatory beetles, are the most prevalent terrestrial predators
(King and Hardy 2013). More than 45,000 species are currently described (World
Spider Catalog, version 16.5 (2015)), and estimates predict there are more than
150,000 extant species in total (Coddington and Levi 1991). Australia is thought to
be home to 10,000 of these species (Nicholson et al. 2006). Consequently, human
interaction with spiders is common, and the number of spider bites in Australia is
estimated to exceed 5,000 cases annually (Isbister and White 2004). Worldwide
there are four widely recognized groups of spiders that are significantly medically
important: members of the Araneomorphae genera Latrodectus, Loxosceles, and
Phoneutria and the genera belonging to the mygalomorph family, Hexathelidae.
Australia is home to two of these groups, namely, Latrodectus and Hexathelidae.
While these spiders are widely recognized, other spiders have been identified as
potentially medically important (e.g., mouse spiders, Missulena spp.), or rumored
to be clinically important (e.g., white-tailed spiders, Lampona spp., and huntsman
spiders, Neosparassus spp.). The Australian tarantulas have been responsible for a
number of bites, but records have shown little effect in humans but more significant
effects, including death, in canines.
More recently, work on spider venoms has focused more on the large number
and variety of individual molecules present and their potential as bioinsecticides
or therapeutic drug leads. Predictions estimate the number of bioactive
peptides collectively in spider venoms to exceed ten million, and presently
only approximately 0.01 % of this diversity has been characterized (Klint
et al. 2012).
22 D.T.R. Wilson

This chapter will provide an overview of spiders in Australia and their medical
and clinical importance and provide a current comprehensive review of the
published toxins from Australian spider venoms.
Clinical Implications of Spiders of Medical Significance
in Australia
Australian Funnel-Web Spiders (Hexathelidae)
The Australian funnel-web spiders (Araneae: Mygalomorphae: Hexathelidae:
Atracinae) are a group of relatively large, highly venomous primitive spiders
found primarily along the southeast coast of mainland Australia and Tasmania,
with isolated pockets in South Australia and far north Queensland (see Fig. 1a). A
recent revision (2010) of the taxonomy divided the Atracinae into three genera and
35 characterized species: Atrax (three species), Hadronyche (31 species), and
Illawarra (one species) (Gray 2010). They are arguably the world’s most venomous
spiders, with the male Sydney funnel-web spider (Atrax robustus) (see Fig. 2a, b)
responsible for 13 human fatalities prior to the introduction of an antivenom in 1980
(Nicholson et al. 2006). Completely unrelated to the American funnel-web or grass
spider (Agelenopsis aperta), the Australian funnel-web spiders are relatively large
and are typically highly aggressive when provoked (see Fig. 2a). Of particular
interest with the venom is the “selectivity” toward primates, causing only very mild
symptoms in other mammals. The reason is still unknown, but proposed ideas from
work that showed a purified fraction of nonimmune rabbit serum as an effective
antitoxin against male A. robustus venom suggest the presence of endogenous
inactivating factors in the form of immunoglobulin G (IgG) in the plasma of
non-primates that bind to the δ-hexatoxin-1 (δ-HXTX-1) peptide family (the active
toxins in the venom) or a general nonspecific binding to immunoglobulins due to
the highly basic nature of the toxins (Nicholson et al. 2006). Also of interest are the
gender-related differences in venom activity observed for some of the species,
including A. robustus. Only the venom of the male spider has been responsible
for fatalities. Bites are relatively rare, only contributing to ~1 % of the total number
of spider bites reported in Australia (Isbister and Gray 2002), and severe enven-
omation is observed to occur in 10–25 % of funnel-web spider bites (Isbister and
Gray 2004b; Miller et al. 2000). The clinical symptoms of severe envenomation
include localized pain, salivation, sweating, vomiting, piloerection, lacrimation,
skeletal muscle fasciculation, and disturbances in respiration, blood pressure, and
heart rate, followed by severe hypotension. Death can occur due to respiration and
circulatory failure or from increased intracranial pressure resulting from cerebral
edema (Miller et al. 2000).
No deaths have been recorded from Australian funnel-web envenomation since
the introduction of a purified rabbit IgG antivenom, raised against the venom
of male A. robustus, by Prof. Struan Sutherland in 1980 (Nicholson et al. 2006).
2 The Venom of Australian Spiders 23

The antivenom has also been reported in case studies to reverse the envenomation
syndrome of other species of funnel-web spiders, including H. formidabilis,
H. versuta, H. infensa, H. cerberea, H. nimoola (previously H. sp.7), and
H. macquariensis (previously H. sp.14) (Gray 2010; Miller et al. 2000). In vitro
studies showed the ability of funnel-web spider antivenom to reverse and neutralize
venom from male and female specimens of several species of Hadronyche, Atrax,
and Illawarra (Graudins et al. 2002a).
Redback and Widow Spiders (Theridiidae)
The widow, or comb-footed, spiders (Araneae: Araneomorphae: Theridiidae),
termed theridiids, can be considered the most clinically relevant spiders in the
world. This is due to a worldwide distribution of the primary clinically signiﬁcant
genus, Latrodectus. In Australia, the most infamous theridiid is the redback spider,
WA
NT
SA
QLD
TAS
NSW
VIC
Latrodectus hasselti
WA
NT
SA
QLD
TAS
NSW
VIC
Missulena spp.
WA
NT
SA
QLD
TAS
NSW
VIC
Illawarra wisharti
Hadronyche spp.
Atrax spp.
WA
NT
SA
QLD
TAS
NSW
VIC
Theraphosidae
a c
b d
Fig. 1 Distribution maps of Australian spiders. (a) Funnel-web spiders (Atrax spp., Hadronyche
spp., and Illawarra wisharti), (b) redback spider (Latrodectus hasselti), (c) mouse spiders
(Missulena spp.), (d) Australian tarantulas (Theraphosidae)
24 D.T.R. Wilson

Latrodectus hasselti (Fig. 2e). However, other Australian Theridiidae spiders from
the genera Steatoda and Archaearanae have also shown clinical relevance (Isbister
and Gray 2003c). In Australia alone, a gross approximation estimates that there are
in excess of 5,000 bites by theridiid spiders per year (Isbister and White 2004). The
true incidence of envenomation by these spiders worldwide is largely unknown.
Some studies exist for envenomation incidence in particular countries.
The clinical symptoms experienced from envenomation by spiders of the genus
Latrodectus are collectively termed latrodectism. These symptoms include local
and regional pain that can be prolonged for days, associated with diaphoresis,
Fig. 2 Photographs of Australian spiders. (a) Male Sydney funnel-web spider (Atrax robustus),
(b) female Sydney funnel-web spider (Atrax robustus), (c) female eastern mouse spider
(Missulena bradleyi), (d) male eastern mouse spider (Missulena bradleyi), (e) female redback
spider (Latrodectus hasselti), and (f) female northern tarantula (Phlogius crassipes) (Photographs
by Dr. David Wilson)

malaise, lethargy, nausea, vomiting, headache, fever, hypertension, and tremor, and
are responsible for significant morbidity and infrequent mortality (Isbister and Gray
2003c). In a prospective study of redback spider bites in Australia, the majority of
bites were shown to cause significant effects, with pain identified as the primary
symptom. Persistent pain was reported in 66 % of cases, and one-third experienced
severe pain that prevented sleep within the first 24 h (Isbister and Gray 2003b).
Envenomation by the genera Steatoda and Archaearanae was shown to exhibit
similar symptoms to latrodectism. In severe cases of envenomation by Steatoda
(“steatodism”), the clinical effects have been reported as almost indistinguishable
from latrodectism, although diaphoresis was not present. In cases of envenomation
by Archaearanae, the associated pain was reported as similar to latrodectism
(Isbister and Gray 2003c).
The treatment of bites by theridiid spiders is problematic and the subject of
significant controversy. Antivenom is only available in some countries, and clinical
practices vary worldwide. Australia has had access for more than 60 years to a
highly purified equine antivenom raised against the redback spider, L. hasselti. This
antivenom has been shown to prevent both in vitro and in vivo toxicity from
venoms of numerous Latrodectus species and α-latrotoxin, the primary toxic
component in the venom, in mice (Graudins et al. 2001). In addition, the redback
antivenom has been reported to have successfully treated a clinical case of
steatodism and demonstrated the ability to reverse the effects of Steatoda spp.
venom in vitro (Graudins et al. 2002b). The effectiveness of redback spider
antivenom in the clinical setting has come into question after three randomized
controlled trials in Australia and one in the USA. Two of the Australian studies
showed no evidence of a difference between administration of the antivenom
intravenously and intramuscularly. The third study demonstrated that the addition
of redback spider antivenom to standardized analgesia treatment of patients suffer-
ing latrodectism did not significantly improve pain or systemic effects. The results
of this study support the results of the only other placebo-controlled randomized
trial of widow spider antivenom, performed in the USA. Collectively, these studies
support the idea that widow spider antivenom may not be effective. Further and
larger studies involving different widow spiders and antivenom are required before
a definitive conclusion can be reached (Isbister et al. 2014).
Other Australian Spiders
The Australian mouse spiders (Araneae: Mygalomorphae: Actinopodidae) belong
to the genus Missulena and are primitive ground-burrowing spiders (see Fig. 2c, d).
The 16 known species (World Spider Catalog, version 16.5 (2015)) in Australia are
distributed across all states except Tasmania (see Fig. 1c). They are often confused
with the Australian funnel-web spiders (Isbister and Gray 2004b). Serious bites
from these spiders are rare, with only one report of a serious bite occurring in a
19-month-old child (Missulena bradleyi) (Isbister and Gray 2004b). The child
experienced a number of symptoms resembling those observed for Australian
26 D.T.R. Wilson

funnel-web spider bites (muscle fasciculation, dyspnea, hypertension, heavy per-
spiration, and tachycardia). The condition was reversed by administration of
Australian funnel-web spider antivenom (Isbister and Gray 2004b). Isbister and
Gray (2004b) reviewed confirmed mouse spider bite cases and identified 40 records
from three species (M. bradleyi, M. occatoria, and M. pruinosa) (Isbister and Gray
2004b). Minor local neurotoxic effects, including paresthesia, numbness, and
diaphoresis, were evident in six records of bites by M. bradleyi. Five cases reported
minor systemic effects (headache and nausea). Mouse spider bites were concluded
to have the potential to result in severe envenomation in rare cases and have been
concluded to not pose a major medical problem (Isbister and Gray 2004b).
A number of genera of Australian tarantulas (Araneae: Mygalomorphae:
Theraphosidae) (see Fig. 2f), referred to as theraphosids, are distributed across
the warmer tropical and temperate regions of the continent (Isbister et al. 2003) (see
Fig. 1d). Presently, the taxonomy of Australian theraphosids is incomplete and
makes definitive identification of specimens difficult. The current genera include
Coremiocnemis, Selenotholus, Selenotypus, and Selenocosmia (World Spider Cat-
alog, version 16.5 (2015)); however, recent references in the literature also refer to
Phlogiellus (Raven 2005) and Phlogius, a synonym replacing the Australian
Selenocosmia genera (Chow et al. 2015; Raven and Covacevich 2012; Raven
2005) (Dr. Robert Raven, personal communication). Bites and envenomation in
humans by these spiders are rare. Isbister et al. (2003) noted only nine confirmed
reports of human envenomation over the 25-year period from 1978 to 2002 (Isbister
et al. 2003). No reports of major effects were evident in any of the case reports.
Local pain was the most common symptom, and mild systemic effects were
reported in one case. Raven and Covacevich (2012) reported one further case by
Phlogius crassipes that resulted in pain and swelling, but no systemic effects
(Raven and Covacevich 2012). The venom of Australian theraphosids has shown
significant selectivity toward different mammalian systems (Isbister et al. 2003). In
contrast to the primate-specific activity of the Australian funnel-web spiders, case
studies of seven confirmed bites on canines (weighing up to ~50 kg weight) by
identified Australian theraphosids reported that the bites were rapidly fatal in all
cases and highlight the selectivity of the venom components to some mammalian
systems other than humans (Isbister et al. 2003). Given that bites to canines up to
the weight of a small human are rapidly fatal and that most bites to humans result in
local pain only, it has been concluded that the Australian theraphosids pose no
significant medical problem (Isbister et al. 2003).
A study of 750 definite spider bite cases over a 27-month period from three
Australian states showed that the most common spider bite encountered is from
members of the Sparassidae (huntsman) family (22.9 %), with members of the
Araneidae (orb weavers) second (21.4 %). Only 6 % of the total bites were
medically significant, and of the medically significant bites, 84 % were attributed
to the redback spider (Latrodectus hasselti), five bites were from Australian
funnel-web spiders (Atracinae family), and one bite was from an Araneidae
(Isbister and Gray 2002). An important note of significance from this study was
the occurrence of 16 % of the total bites by white-tailed spiders (Lamponidae

family), commonly attributed to and believed to cause necrotic arachnidism
(Isbister and Gray 2004a). No necrotic lesions were reported from any of the
definite spider bite cases.
Isbister and Hirst (2003) conducted a prospective study over 27 months on bites
from the Sparassidae family, the most prevalent source of spider bites in Australia
(Isbister and Gray 2002). The Sparassidae family (Araneae: Araneomorphae:
Sparassidae) are large spiders found on most continents in tropical and temperate
regions of the world. Bites were recorded from six genera: Isopeda, Isopedella,
Neosparassus, Heteropoda, Delena, and Holconia. Bites by these spiders
were predominantly characterized by immediate pain with a duration averaging
5 min, and associated with bleeding and/or puncture marks and local redness.
Severe pain was reported in a small number of cases, and the incidence of
local effects, including local redness and itchiness, and systemic effects was
less than for bites by other spiders. No clinical effects consistent with an enven-
omation syndrome were evident. The study concluded that bites from spiders of
the Sparassidae family cause only minor effects and these spiders are not danger-
ous to humans. It also showed that there are no differences between bites from
different genera within the family, refuting previous reports that Neosparassus
spp. can cause severe effects and should be considered dangerous (Isbister and
Gray 2002).
One clinically important aspect of spider bite in Australia that would be remiss
not to mention due to the debate and publicity it has received relates to necrotic
arachnidism. A number of Australian spider species have been suspected of causing
necrotic ulcers including black house spiders (Badumna spp.), wolf spiders (family
Lycosidae), and the most infamous suspects, white-tailed spiders (Lampona spp.)
(Isbister and Gray 2004a). In prospective studies of 750 spider bites (Isbister and
Gray 2002), 130 definite bites by white-tailed spider species (Isbister and Gray
2003a) and black house spider bites (Isbister and Gray 2004a), Isbister and col-
leagues showed that there was no evidence of necrotic arachnidism. Given the lack
of evidence of confirmed necrotic arachnidism in Australia, it is unlikely that
necrotic arachnidism is a real problem in Australia.
Australian Spider Venom Components
Research into the components of Australian spider venoms has focused on four
primary areas: identification and characterization of the primary toxic components
of clinically relevant venoms (Nicholson et al. 1996), discovery of insecticidal
components with potential commercial application (Hardy et al. 2013; Windley
et al. 2012), discovery of potential therapeutic leads (Chow et al. 2015), and use of
venom component fingerprinting as a taxonomic tool (Palagi et al. 2013; Wilson
and Alewood 2004, 2006). The identification and characterization of the primary
toxic components of clinically relevant venoms (Nicholson et al. 1996) have been
undertaken to understand the mechanism of action and develop and understand the
action of relevant antivenoms. As one of the most successful insect predators on the
28 D.T.R. Wilson

[Doi 10.1007%2 f978 94-007-6389-0] gopalakrishnakone, p.; corzo, gerardo; de lima, maria elena; die -- spider venoms -_

[Doi 10.1007%2 f978 94-007-6389-0] gopalakrishnakone, p.; corzo, gerardo; de lima, maria elena; die -- spider venoms -_

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