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- 6. NEUROMETHODS Series Editor Wolfgang Walz University of Saskatchewan Saskatoon, SK, Canada For other titles published in this series, go to www.springer.com/series/7657
- 8. Zebrafish Models in Neurobehavioral Research Edited by Allan V. Kalueff and Jonathan M. Cachat DepartmentofPharmacologyandNeuroscienceProgram, TulaneUniversityMedicalSchool,NewOrleans,LA,USA
- 9. Editors Allan V. Kalueff Department of Pharmacology and Neuroscience Program Tulane University Medical School 70112 New Orleans, LA, USA avkalueff@gmail.com Jonathan M. Cachat Department of Pharmacology and Neuroscience Program Tulane University Medical School 70112 New Orleans, LA, USA cachatj@gmail.com ISSN 0893-2336 e-ISSN 1940-6045 ISBN 978-1-60761-921-5 e-ISBN 978-1-60761-922-2 DOI 10.1007/978-1-60761-922-2 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010935582 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
- 10. Series Preface Under the guidance of its founders Alan Boulton and Glen Baker, the Neuromethods series by Humana Press has been very successful since the first volume appeared in 1985. In about 17 years, 37 volumes have been published. In 2006, Springer Science + Business Media made a renewed commitment to this series. The new program will focus on methods that are either unique to the nervous system and excitable cells or which need special consideration to be applied to the neurosciences. The program will strike a balance between recent and exciting developments like those concerning new animal models of disease, imaging, in vivo methods, and more established techniques. These include immunocytochemistry and electrophysiological technologies. New trainees in neurosciences still need a sound footing in these older methods in order to apply a crit- ical approach to their results. The careful application of methods is probably the most important step in the process of scientific inquiry. In the past, new methodologies led the way in developing new disciplines in the biological and medical sciences. For exam- ple, physiology emerged out of anatomy in the nineteenth century by harnessing new methods based on the newly discovered phenomenon of electricity. Nowadays, the rela- tionships between disciplines and methods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing also make it possible for scientists to download chapters or protocols selectively within a very short time of encountering them. This new approach has been taken into account in the design of individual volumes and chapters in this series. Wolfgang Walz v
- 12. Preface The use of animal models has become increasingly important for biomedical research over the past decade, enabling a better understanding of pathogenic pathways involved in a variety of human disorders. Within the realm of neurobehavioral research, animal mod- els have played a crucial role in the development of new insights and theories of brain pathogenesis. Animal models such as mice, hamsters, and rabbits have been utilized in a multitude of neurobehavioral studies, yielding experimental data that have lead researchers to a better comprehension of neurobiology. As scientific research progresses, investigators are attempting to identify more novel animal models to utilize in new avenues of neurobe- havioral research. Zebrafish (Danio rerio) have become increasingly popular in biomedical research. Research conducted on these aquatic vertebrates has generated considerable discoveries not only in the areas of genetics and embryology but also in fields such as cardiology, endocrinology, and neuroscience. Zebrafish are promising animal models because of their high genetic homology with humans and quantifiable behavioral and neuropathological phenotypes analogous to humans. The use of zebrafish to investigate the pathological mechanisms underlying neuropsy- chiatric disorders and behavior quantification is explored in depth in this book. The opening Chapter 1 is a comprehensive review of zebrafish behavior, ecology, taxonomy, reproduction, and genetics. This chapter emphasizes the need for continued experimenta- tion in cognition, behavior, and field-based studies, resulting in a more thorough under- standing of the zebrafish model. Critical to survival in a natural habitat and strongly influencing their behavior, the olfactory system in zebrafish is explored in Chapter 2. Zebrafish possess three distinct types of olfactory sensory neurons, which integrate with other areas of the brain to induce various physiological and behavioral effects in response to odors. Olfaction allows zebrafish to detect nearby food, predators, and mates, in addition to conveying information relating to spawning sites, reproduction, dangerous environments, and the distinction between self and kin. Advanced knowledge of the neurological basis of olfaction is key to a better understanding of zebrafish wild type and anxiety-related behavior. Chapter 3 focuses on the emergence of zebrafish as an effective model to study stress and anxiety. This chapter presents a concise introduction to anxiety-induced endocrine and behavioral responses in zebrafish. Since zebrafish possess all the classical vertebrate transmitters, and their neuroendocrine system yields robust cortisol responses to stress, zebrafish models enable greater insight into neural mechanisms associated with anxiety- related disorders. Furthermore, this chapter illustrates the importance of behavioral assays, genetic manipulation, pharmacological treatment, and video tracking for analysis of the phenomena involved in anxiety-related phenotypes. While zebrafish demonstrate promising potential in the field of anxiety and stress- related research, they have also emerged as valuable models in other areas of neurobe- havioral research. Chapter 4 describes how the effects of nicotine on processes such as learning, memory, and stress are similar to those exhibited by humans and rodents. The vii
- 13. viii Preface authors’ analysis suggests that zebrafish may present significant translational capabilities in research as a model for the behavioral effects of nicotine. Based on the establishment of zebrafish as a suitable model for behavioral research, Chapter 5 details the process for quantitative trait loci (QTL) mapping and how it attempts to discover the specific causative genes responsible for variations in complex behavioral traits in zebrafish. Because of the strides taken recently in the study of zebrafish behavior, QTL mapping would not only lead to a greater understanding of zebrafish activ- ity, but also strengthen its application as a genetic model. Chapter 6 explores the effects of alcohol on several strains of zebrafish. Like anxi- ety, alcoholism is a serious brain disease for which the pathogenic mechanisms are not well understood. Alcohol abuse in the world is on the rise, making a genetic model for the development of alcoholism vital. Using a noninvasive evaluation technique, the acute and chronic effects of ethanol on zebrafish were observed, clarifying the genetic factors involved in alcoholism. Along the same line, the authors of Chapter 7 explore the use of zebrafish as a model of drug dependence and relapse behaviors in humans. These robust reactions to nicotine and alcohol not only reinforce the use of zebrafish as a behavioral model of addiction but also strengthen the notion that zebrafish may be utilized to discover various genetic factors underlying drug dependence, withdrawal, and relapse. As previously mentioned, many neuroscientists seek to gain a more concrete under- standing of the pathogenic mechanisms that induce neurobiological disorders and behav- ior. However, in some cases, an error in the mechanism of the neural circuitry is not the only contributing cause of behaviors or diseases that are expressed. Chapter 8 exam- ines the impact of neurotoxic chemicals on the nervous system and their potential to increase susceptibility to neurodegenerative disorders. In this chapter, the authors uti- lize the high sensitivity of zebrafish to environmental changes to investigate the corre- lation between the influence of environmental neurotoxins and neurodegenerative dis- orders. This research analyzes alterations in the biogenic amine system following expo- sure to pesticides, as well as the detrimental effect of neurotoxins on the nervous system. Other experiments that examine the neural effects of environmental factors are explored in Chapter 9. This chapter analyzes predator-avoidance behavior exhibited by zebrafish, which is induced by external environmental factors such as alarm pheromone. The predator-avoidance behavior displayed by zebrafish is based upon learned recognition of external environmental cues. Exploration into the process of learned recognition in zebrafish will enable researchers to gain a more tangible understanding of the mechanisms that underlie cognitive processes of learning and memory. In Chapter 10, the authors discuss avoidance behavior in zebrafish. Similar to the learned recognition phenomenon, inhibitory avoidance paradigms provide insight into the learning and memory capabilities of zebrafish. While the behavioral phenotypes of small teleost fish have frequently been considered to be dominated by reflex and instinct, recent studies have suggested a more complex phenotype influencing emotional, social, and reproductive behavior. The authors employ new experimental models with zebrafish, and area to investigate learning and memory, and area of research that will contribute to a more comprehensive understanding of the zebrafish brain and behavior. Further exploring the zebrafish neurocognitive domain, Chapter 11 reviews previous studies on the spatial cognitive abilities of zebrafish. Mounting evidence, summarized in this chapter, demonstrates the capability of zebrafish to learn from visual cues that identify
- 14. Preface ix potential risk or reward. The application of these tests may serve as an insightful resource by which the spatial cognition of zebrafish can be illuminated. Finally, Chapter 12 describes common larval zebrafish behaviors. While the behavioral phenotypes of adult zebrafish are important to study in detail, the functionality of zebrafish larvae must be equally well understood in relation to its anatomical size and development. This chapter explores the scope of larval behavior, from movement to stimuli response to more complex behaviors such as swim bladder inflation, sleep, and social behavior. While a general repertoire may be established, specific behavioral tendencies are influenced by environmental factors such as temperature or nearby predators. Future experimentation is necessary to correlate the synergistic aspects of behavior and neurobiological development in zebrafish larvae. Overall, this book emphasizes the growing importance of zebrafish in neurobehavioral research. As a promising alternative to mammalian animal models, zebrafish yield robust physiological responses analogous to humans but do not possess the complex behavioral phenotypes exhibited by many other animal models. This book portrays an extensive, thorough perspective on the emergence of zebrafish as a robust animal model in neuro- science research. The contributors to this book are leading international scholars whose work spearheads innovative research projects in laboratories around the world. The themes discussed within this book, ranging from stress-related behaviors to learning and memory phenotypes, encompass a wide spectrum of the utility of zebrafish within neurobiolog- ical disciplines. This theoretical book, as vol. 52 of the Humana Press Neuromethods series, complements another book (“Zebrafish Neurobehavioral Protocols”, vol. 51) of this series, which focuses on practical laboratory applications of these concepts. Together, these two volumes will serve as a useful source for scientists new to the field, as well as established researchers seeking valuable insight into the growing utility of zebrafish in behavioral neuroscience. Allan V. Kalueff Jonathan M. Cachat
- 16. Contents Series Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii 1. Zebrafish Ecology and Behaviour . . . . . . . . . . . . . . . . . . . . . . . . . 1 Rowena Spence 2. Olfactory Behavior: Making Scents of a Changing World . . . . . . . . . . . . . 47 Kathleen E. Whitlock 3. Modeling Stress and Anxiety in Zebrafish . . . . . . . . . . . . . . . . . . . . . 73 Jonathan M. Cachat, Peter R. Canavello, Marco F. Elegante, Brett K. Bartels, Salem I. Elkhayat, Peter C. Hart, Anna K. Tien, David H. Tien, Esther Beeson, Sopan Mohnot, Autumn L. Laffoon, Adam M. Stewart, Siddharth Gaikwad, Keith Wong, Whitlee Haymore, and Allan V. Kalueff 4. Nicotinic Receptor Systems and Neurobehavioral Function in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Edward D. Levin 5. QTL Mapping of Behaviour in the Zebrafish . . . . . . . . . . . . . . . . . . . 101 Dominic Wright 6. Genetics of Ethanol-Related Behaviors . . . . . . . . . . . . . . . . . . . . . . 143 Cynthia A. Dlugos 7. Conditioned Place Preference Models of Drug Dependence and Relapse to Drug Seeking: Studies with Nicotine and Ethanol . . . . . . . . . . . . . . . 163 Caroline H. Brennan, Amit Parmar, Layla K.M. Kily, Arani Ananthathevan, Arti Doshi, and Salma Patel 8. Zebrafish Biogenic Amine Transporters and Behavior in Novel Environments: Targets of Reuptake Inhibitors and Pesticide Action as Tools for Neurotoxicology Research . . . . . . . . . . . . . . . . . . . . . . . . 181 Georgianna G. Gould 9. Learned Recognition by Zebrafish and Other Cyprinids . . . . . . . . . . . . . . 211 Brian D. Wisenden 10. Inhibitory Avoidance and Color Discrimination Learning in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Luciana Cofiel and Rosana Mattioli xi
- 17. xii Contents 11. Spatial Cognition in Zebrafish . . . . . . . . . . . . . . . . . . . . . . . . . . . 235 Joshua L. Haight and Joseph A. Schroeder 12. The Behavioral Repertoire of Larval Zebrafish . . . . . . . . . . . . . . . . . . . 249 Kandice Fero, Tohei Yokogawa, and Harold A. Burgess Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
- 18. Contributors ARANI ANANTHATHEVAN • Biological and Experimental Psychology Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, UK BRETT K. BARTELS • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA ESTHER BEESON • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA CAROLINE H. BRENNAN • Biological and Experimental Psychology Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, UK HAROLD A. BURGESS • Laboratory of Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA JONATHAN M. CACHAT • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA PETER R. CANAVELLO • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA LUCIANA COFIEL • Laboratório de Neurociências, Departamento de Fisioterapia, Universidade Federal de São Carlos, São Carlos, SP, Brazil CYNTHIA A. DLUGOS • Department of Pathology and Anatomical Sciences, School of Medicine and Biomedical Sciences, University of Buffalo/State University of New York, Buffalo, NY, USA ARTI DOSHI • Biological and Experimental Psychology Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, UK MARCO F. ELEGANTE • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA SALEM I. ELKHAYAT • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA KANDICE FERO • Laboratory of Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA ADAM M. STEWART • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA GEORGIANNA G. GOULD • Department of Physiology, University of Texas at Health Science Center at San Antonio, San Antonio, TX, USA JOSHUA L. HAIGHT • Behavioral Neuroscience Program, Connecticut College, New London, CT, USA PETER C. HART • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA WHITLEE HAYMORE • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA ALLAN V. KALUEFF • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA xiii
- 19. xiv Contributors LAYLA K.M. KILY • Biological and Experimental Psychology Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, UK AUTUMN L. LAFFOON • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA EDWARD D. LEVIN • Department of Psychiatry and Behavioral Sciences, Duke University Medical Center, Durham, NC, USA ROSANA MATTIOLI • Laboratório de Neurociências, Departamento de Fisioterapia, Universidade Federal de São Carlos, São Carlos, SP, Brazil SOPAN MOHNOT • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA AMIT PARMAR • Biological and Experimental Psychology Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, UK SALMA PATEL • Biological and Experimental Psychology Group, School of Biological and Chemical Sciences, Queen Mary University of London, London, UK JOSEPH A. SCHROEDER • Department of Psychology, Connecticut College, New London, CT, USA ROWENA SPENCE • University of St Andrews, St. Andrews Fife, Scotland SIDDHARTH GAIKWAD • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA ANNA K. TIEN • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA DAVID H. TIEN • Department of Pharmacology and Neuroscience Program, Tulane University Medical School, New Orleans, LA, USA KATHLEEN E. WHITLOCK • Centro de Genomicas Celular, Centro de Neurociencia de Valparaíso (CNV), Universidad de Valparaíso, Valparaíso, Chile BRIAN D. WISENDEN • Biosciences Department, Minnesota State University Moorhead, Moorhead, MN, USA KEITH WONG • Department of Pharmacology and Neuroscience Program, Tulane Uni- versity Medical School, New Orleans, LA, USA DOMINIC WRIGHT • IFM-Biology, Linköping University, Linköping, Sweden TOHEI YOKOGAWA • Laboratory of Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
- 21. Chapter 1 Zebrafish Ecology and Behaviour Rowena Spence Abstract The zebrafish is an important model organism in developmental genetics, neurophysiology and biomedicine, but little is known about its natural ecology and behaviour. It is a small, shoaling cyprinid, native to the flood-plains of the Indian subcontinent, where it is found in shallow, slow-flowing waters. Zebrafish are group spawners and egg scatterers, although females are selective with respect to sites for oviposition and males are territorial around such sites. Laboratory studies of zebrafish behaviour have encompassed shoaling, foraging, reproduction, sensory perception and learning. This chapter reviews these studies in relation to the suitability of the zebrafish as a model for studies in behavioural ecology. Key words: Model organism, social behaviour, morphology, ecology, reproduction, development (ontogeny), evolution (phylogeny), natural habitat, diet, social behaviour, reproductive behaviour, cognitive behaviour, genetics. 1. Introduction 1.1. The Zebrafish as a Model Organism The zebrafish, Danio rerio (Hamilton), is one of the most impor- tant vertebrate model organisms in genetics, developmental biol- ogy, neurophysiology and biomedicine (1–4). It has a number of attributes that make it particularly tractable to experimental manipulation. It is a small, robust fish, so large numbers can be kept easily and cheaply in the laboratory, where it breeds all year round. Females can spawn every 2–3 days and a single clutch may contain several hundred eggs. Generation time is short, typ- ically 3–4 months, making it suitable for selection experiments. Zebrafish eggs are large relative to other fish eggs (0.7 mm in diameter at fertilisation), and optically transparent, the yolk being sequestered into a separate cell. Furthermore, fertilisation A.V. Kalueff, J.M. Cachat (eds.), Zebrafish Models in Neurobehavioral Research, Neuromethods 52, DOI 10.1007/978-1-60761-922-2_1, © Springer Science+Business Media, LLC 2011 1
- 22. 2 Spence is external so live embryos are accessible to manipulation and can be monitored through all developmental stages under a dissect- ing microscope (5). Development is rapid, with precursors to all major organs developing within 36 h and larvae displaying food seeking and active avoidance behaviours within 5 days post fertil- isation, i.e. 2–3 days after hatching (5). As a popular aquarium species, the zebrafish has been used in developmental biology for many years (e.g. (6)). Its cur- rent prominence as a model organism stems from the work of Streisinger et al. (7), who pioneered its use to apply molecu- lar genetics to the study of vertebrate embryology, and Kimmel (8–10), who published detailed descriptions of cell differentia- tion and nervous system organisation (for review see (2)). The zebrafish was the subject of the first large-scale random mutagen- esis screens to be conducted in a vertebrate (11). These screens, conducted in 1996 in Boston (12) and Tübingen (13), gener- ated over 4,000 mutations and led to the identification of over 400 genes controlling vertebrate development. Since then there have been numerous technological advances (for review see (14– 22)), culminating in the zebrafish genome project, based at the Sanger Institute in Cambridge, which began in 2001 and will shortly be completed (http://www.sanger.ac.uk). The zebrafish is increasingly important in biomedical research (23–25), partic- ularly as a model of human disease (26, 27) and for the screening of therapeutic drugs (3, 28). Its strength as a model organism is that as a vertebrate it is more comparable to humans than inverte- brate model species such as Drosophila (29, 30), while being more tractable to genetic and embryological manipulation than mam- malian model species such as mice, in which such procedures are both more complicated and costly. Over 400 labs worldwide now routinely use the zebrafish in fundamental and applied research (http://www.zfin.org) and there is an increasing interest in its use as a model for understand- ing the genetic basis of behaviour (18, 31, 32). Figure 1.1 shows the number of papers on zebrafish behaviour published each decade since the 1970s, based on a search of Web of Science using “zebrafish” and “behaviour/behavior” as keywords. Despite this interest, it has attracted little attention from the behavioural ecol- ogy community, possibly because little is known about its natural ecology and few studies have been conducted on wild popula- tions. Most laboratory lines of zebrafish are the product of many generations in captivity, which is likely to have resulted in selec- tion for reproductive capacity, while relaxing selection for other traits, such as predator avoidance (33, 34). Thus, it is not clear in what respect and to what extent domesticated strains may dif- fer from wild fish, nor how much inter-population variation exists in nature. This chapter reviews the current state of knowledge of the ecology and behaviour of the zebrafish. The term behaviour is
- 23. Zebrafish Ecology and Behaviour 3 Fig. 1.1. The numbers of papers on zebrafish behaviour published since the 1970s, based on a keyword search in the Web of Science, up to mid 2009. used not in the sense of a simple reflexive response to stimuli but rather for complex patterns of behaviour such as those involved in social and reproductive behaviour. 2. Taxonomy 2.1. Taxonomic Status The zebrafish belongs to the family of freshwater fishes Cyprinidae, the most species-rich vertebrate family (35). There are currently approximately 44 danionin species (36), distributed throughout South and South East Asia. Their highest species diversity is in North East India, Bangladesh, and Myanmar (37). The name Danio derives from the Bengali name “dhani”, mean- ing “of the rice field” (38). Danios are included in the subfamily Rasborinae (39). They are characterised by small size (<120 mm total length), the presence of a “danionin notch”, in the ven- tromedial margin of the dentary, and a distinctive colour pattern based on alternating dark and light horizontal stripes, which may be broken up into blotches or bars. Danio rerio was first described by Francis Hamilton, a sur- geon with the British East India Company stationed principally in West Bengal at the beginning of the nineteenth century. He pub- lished An Account of the Fishes Found in the River Ganges and its Branches in 1822 that included 10 Danio species. D. rerio was later assigned to the subgenus Brachydanio, together with the other small Danio species with short dorsal fins and a reduced lat- eral line, Danio being reserved for the larger species of the group (40). Danio and Brachydanio were synonymised by Barman (37),
- 24. 4 Spence as there were no diagnostic characters that reliably separated the two groups. The first molecular phylogeny of the group was pro- duced by Meyer et al. (41, 42) based on 16S and 12S mitochon- drial DNA for nine species. This analysis showed that Danio was monophyletic with two subclades that were either deep bodied or slender bodied. Subsequent molecular studies (43, 44, 45, 46) supported this distinction, as did a combined molecular and mor- phological study by Sanger and McCune (47). Moreover, Parichy and Johnson (44) showed that hybrid viability and fertility among Danio species largely corresponded to the relationships inferred from molecular data. However, a more complete phylogeny, based on morpho- logical analysis and including 13 Danio species together with an additional eight closely related genera, proposed that Danio was paraphyletic, the deep- and slender-bodied clades forming sep- arate genera (48). The deep-bodied clade was thus assigned the distinct generic name of Devario, and includes most of the striped and barred danios (of which about 45 are considered valid), with Danio sensu stricto, (including D. rerio) restricted to nine species (48). A subsequent study using molecular data from a number of nuclear and mitochondrial genes and phylogenetic analysis confirmed this distinction, identifying Danio as monophyletic, being as closely related to Chela, Microrasbora and Inlecypris as to Devario (49). The closest relative of D. rerio is D. kyathit (49). The two genera (Devario and Danio) cannot be reliably dis- tinguished on the basis of proportional measurements alone, as there is considerable intra-species variation, mature females typ- ically being deeper bodied than males or juveniles. Although Devario tend to be larger, one of the large species, Danio dan- gila, is included in Danio (36, 44, 45, 46). However, the two genera are ecologically quite distinct, Devario spp. occurring in hill streams with clear running water, while Danio spp. are con- fined to lowland areas, typically inhabiting slow-flowing, turbid rivers and pools (36). 2.2. Appearance and Morphology Danio rerio rarely exceeds 40 mm body length (from the tip of the snout to the origin of the caudal fin (BL)). Its body shape is fusiform and laterally compressed, with a terminal oblique mouth directed upwards. The lower jaw protrudes further than the upper and the eyes are central and not visible from above. The diagnostic features for the species are an incomplete lateral line extending to the pelvic fin base, two pairs of barbels and five to seven dark blue longitudinal stripes extending from behind the operculum into the caudal fin (37). The anal fin is similarly striped, while the dorsal fin has a dark blue upper edge, bordered with white. The colour pattern comprises three types of pig- ment cell, dark blue melanophores, gold xanthophores and irides- cent iridophores (50, 51). Developmentally, two stripes first form
- 25. Zebrafish Ecology and Behaviour 5 centrally with subsequent stripes being added sequentially above and below (43). As with many teleosts, the melanophores can be concentrated or dispersed in response to stimuli, which appear to function for both camouflage (in response to light intensity (18, 52)) and signalling (since fish typically darken during aggres- sive display (31, 53)). Colour change appears to be under some degree of cognitive control; fish which were subjected to cyclical alternations of black and white backgrounds over 20 days showed an increase in the speed and degree of aggregation and dispersal of melanophores (52). Males and females are of similar coloura- tion, although males tend to have larger anal fins with more yel- low colouration (54, 55). The sex of juveniles cannot be reliably distinguished without dissection and while gravid females have a more rounded body shape, the most reliable diagnostic feature is the presence of a small genital papilla in front of the anal fin origin (54). 2.3. Domestic Aquarium Strains Zebrafish used for mutagenesis and screening are from lines bred in laboratories for many generations in order to maintain a sta- ble genetic background. They are also “cleaned up”; i.e. bred selectively to remove embryonic lethal mutations. The main cur- rently recognised wild-type lines from the Zebrafish International Resource Center are summarised in Table 1.1. For details of mutant lines see http://zfin.org The “Leopard” danio, which displays a spotted colour pat- tern instead of stripes, was originally thought to be a separate species, described as Brachydanio frankei (56). However, nei- ther molecular nor morphological analyses have differentiated between the two (41, 57) while hybrids were shown to produce fertile progeny (48). The Leopard danio is now known to be a spontaneous mutation of the wild-type D. rerio colour pattern (59), with homozygotes displaying a spotted pattern, while het- erozygotes have a disrupted stripe pattern (60). Leopard danio mutants are primarily bred for the aquarium trade but also occur in nature (R. Spence, pers. obs.). Another aquarium variant is the “longfin” D. rerio, which is a dominant mutation resulting in elongated fins (61). The commonly used wild-type strain, TL or Tübingen Long-fin displays both the “leopard” and “longfin” mutations (www.zfin.org). 2.4. Pigment Patterns in Danio spp. Comparison of pigment patterns among Danio species has pro- vided insights into their evolutionary relationships. Larval danios of different species exhibit an identical pigment pattern, which only differentiates into the adult pattern in about the third week of development (43). Interestingly, several D. rerio pigment pat- tern mutations resemble other Danio species (44). This remark- able concurrence in appearance raises the possibility that the alle- les expressed by zebrafish colour mutants are the same as those
- 26. 6 Spence Table 1.1 Wild-type zebrafish lines listed by the Zebrafish International Resource Center Name Description AB Derived from two lines purchased by George Streisinger from a pet shop in Albany, Oregon in the late 1970s. The currently used line ∗AB was derived from the original AB line in 1991–1992 by parthenogenesis AB/Tübingen An “official” line maintained as a cross but the term is also applied to crosses where the two parental lines are maintained separately C32 Derived from laboratory strains at Oregon. The current C32bc9 stock is a derivative of Steve Johnson’s inbred C32 Cologne Isolated at the Reugels/Campos-Ortega Lab, University of Cologne Darjeeling Collected in Darjeeling in 1987 and sent to Monte Westerfield at Oregon. A much faster swimmer than other wild-type strains. Used extensively for mapping as it contains many polymorphic markers Ekkwill (EKW) From Ekkwill breeders in Florida and maintained in Grunwald lab, University of Utah Hong Kong Stock obtained from a Hong Kong fish dealer HK/AB Hybrid of Hong Kong and AB wild-type lines HK/Sing Hybrid of Hong Kong and Singapore wild-type lines India Stock obtained from expedition to Darjeeling (wild isolate) Indonesia Stock obtained from Indonesian fish dealer Nadia Wild-caught about 40 miles east of Calcutta. The fish were collected from stagnant ponds and flood plains. Imported in 1999 by a wholesaler in Oregon. Established in the Oregon laboratory from an initial breeding of about 10 individuals Singapore Stock obtained from Singapore fish dealer SJA SJA is an inbred line of ∗AB isolated at the Stephen L. Johnson Lab, Washington University Medical School. Unlike ∗AB, which is bred to retain polymorphisms, this subline is bred to reduce polymorphism and is at least 85% monomorphic SJD Isolated at the Stephen L. Johnson Lab Tübingen Wild-type short fins. Strain used by Sanger for the zebrafish sequencing project. It was cleaned up to remove embryonic lethal mutations from the background before being used for mutagenesis and sequencing Tübingen long fin Homozygous for leot1, a recessive mutation causing spotting in adult fish, and lof dt2 a dominant homozygous viable mutation causing long fins. This is not the line used in the Sanger zebrafish sequencing project. It is genetically different from TU because it was bred differently and not “cleaned up”, and therefore retains a lot of polymorphisms WIK Derived from wild catch in India and used for genome mapping expressed in related Danio species. Consequently, these alleles may have played a role in colour pattern diversification among species (44). A spectacular array of adult pigment pattern mutants have been identified for zebrafish (59, 60). Many mutant colour
- 27. Zebrafish Ecology and Behaviour 7 patterns can be attributed to a single locus, and several pigment genes have been identified at the molecular level (63–65). In a study of colour pattern inheritance, Parichy and Johnson (44) showed that hybrids between zebrafish and four closely related Danio species all expressed pigment patterns resembling that of wild-type zebrafish. These findings imply that stripes may be ancestral in Danio spp. Thus the zebrafish may serve as a useful model for studying the genetic and developmental basis of colour pattern evolution as a mechanism for speciation (50, 51). 3. Ecology 3.1. Distribution and Habitat The natural range of the zebrafish is centred around the Ganges and Brahmaputra river basins in north-eastern India, Bangladesh and Nepal, although in the past specimens have also been col- lected in the Indus, Cauvery, Pennar, Godavari and Mahanadi river basins (Fig. 1.2, Table 1.2). In addition, it has been reported as occurring in the Krishna river basin (38) and in the Fig. 1.2. The natural distribution of the zebrafish. Major river systems indicated. Black dots indicate recorded occurrences.
- 29. Zebrafish Ecology and Behaviour 9 2006 India 21 87 Ganges Engeszer et al. (73) Tarania village, clarity <3 cm, no flow, silt substrate, submerged and flooded vegetation 2006 India 21 87 Ganges Engeszer et al. (73) Tarania village, clarity <3 cm, no flow, silt substrate, submerged and flooded vegetation 2005 Bangladesh 22 90 Ganges Spence et al. (66) Ditch on campus of Khulna University, 3 m wide, <1 m deep, Secchi depth 50 cm, no vegetation. Grassy bank. Some shade 2005 Bangladesh 22 90 Ganges Spence et al. (66) Isolated channel of R. Golamari, near Khulna. Approx. area 200 × 1,500 m, 50 cm deep, Secchi depth 19 cm, vegetation at margins, silt substrate. Grassy bank. No shade 2005 Bangladesh 24 90 Brahmaputra Spence et al. (66) Isolated pond, Sutiakali, near Mymensingh. Approx. area 30 × 50 m, 15 cm deep, silt substrate, vegetated. Grassy bank. No shade 2005 Bangladesh 24 90 Brahmaputra Spence et al. (66) Isolated pond, Sutiakali, near Mymensingh. Approx. area 10 × 12 m, 40 cm deep, Secchi depth 15 cm, silt substrate, no vegetation. Grassy bank. Some shade 2005 Bangladesh 24 90 Brahmaputra Spence et al. (66) Isolated pond, Sutiakali, near Mymensingh. Approx. area 10 × 10 m, 1 m deep, Secchi depth 30 cm, silt substrate, no vegetation. Grassy bank. Some shade 2005 Bangladesh 24 90 Brahmaputra Spence et al. (66) Large semi-natural pond at Bangladesh Agricultural University field station, Mymensingh. Approx. area 105 m 2 , 1 m deep, Secchi depth 30 cm, silt substrate, vegetation at margins. Vegetation on bank. No shade 2005 Bangladesh 24 90 Brahmaputra Spence et al. (66) Ditch connecting to paddy fields, Bangladesh Agricultural University field station, Mymensingh. 10 m wide, 50 cm deep, vegetation. Grassy bank. No shade (continued)
- 31. Zebrafish Ecology and Behaviour 11 1998 India 27 94 Brahmaputra Fang & Roos, Swedish Museum of Natural History Roadside ditch by the Sessa Tinali (Sessa crossing) on Dibrugarh – Jorhat road. About 15 m wide and 0.1–0.4 m deep with stagnant, brownish water. No shade. Plain with grass on land, no vegetation on bank, vegetation in water. Silt substrate 1998 India 27 95 Brahmaputra Fang & Roos, Swedish Museum of Natural History About 100 km SSE of Dibrugarh, small stream near R. Dilli. Stream about 2 m wide and 0.8 m deep with moderate current and yellow/brownish water. No shade. Plain with grass on land and bank, green algae in water. Silt substrate 1997 Bangladesh Brahmaputra Pritchard (67) Small shallow pools in a dry river bed and an adjacent spring-fed pond in a village of the Santal tribal group near to the India-Bangladesh border. Some vegetation 1997 Bangladesh Brahmaputra Pritchard (67) Artificial concrete channel at Northwest Fisheries, Saidpur, Bangladesh. Still, extremely turbid water. No vegetation 1997 Nepal Brahmaputra Pritchard (67) Pond 10 km N of Tangail, next to Tangail– Madhupur highway. Clear water, silt substrate and vegetation. 1.5 m deep 1996 Nepal Ganges Pritchard (67) Shallow ditches and pond on Ranpur campus of Royal Nepal Agricultural College, Chitwan. Clear, slow or still water, silt substrate, some vegetation 1996 Nepal 27 83 Ganges Edds, Kansas University Tribeni 1996 Nepal 28 80 Ganges Edds, Kansas University 3 km W of Pipariya, Shuklaa Phataa Wildlife Reserve 1996 Nepal 28 80 Ganges Edds, Kansas University Confluence of 3 rivers (Chaudhar, Bahuni, Gobraiya) at Royal Shuklaa Phantaa Wildlife Reserve (continued)
- 33. Zebrafish Ecology and Behaviour 13 1975 Nepal Ganges Roberts, California Academy of Sciences Chitawan Valley, low-lying mountain stream 1–2 miles S of Khoria Mohan in Someswar Hills (Hathimara Khola) 1975 Nepal Ganges Roberts, California Academy of Sciences Chitawan Valley, R. Reu near confluence with R. Rapti 1975 Nepal Ganges Roberts, California Academy of Sciences Farm pond 1–2 km east of Kalaiya or Khailaya 1972 India Mahanadi Rao, Zoological Survey of India Koraput District, Orissa 1961 India 19 81 Godavari Ross & Cavagnaro, California Academy of Sciences 9 miles north of Pharasgro, in pond among water plants 1961 India Mahanadi Lamba, Zoological Survey of India Balaghat District, Madhya Pradesh 1957 India Brahmaputra v. Maydell, Zoological Museum of Hamburg Raimona, R. Janali 1957 India 26 93 Brahmaputra v. Maydell, Zoological Museum of Hamburg Kaziranga, Mikir-Hills 1956 India 30 77 Brahmaputra v. Maydell, Zoological Museum of Hamburg Dharmawalla (Siwalik), R. Asan 1956 India 28 81 Brahmaputra v. Maydell, Zoological Museum of Hamburg Nishangara, Varei-Bach 1956 India 25 91 Brahmaputra v. Maydell, Zoological Museum of Hamburg Umsa, W. Assam, Khasi Hills 1956 India 25 91 Brahmaputra v. Maydell, Zoological Museum of Hamburg Garampani, Assam, R. Kopili 1955 India Sharavathi v. Maydell, Zoological Museum of Hamburg Jog-Falls 1949 India 24 85 Ganges Choata-Nagpur Survey, University of British Columbia R. Barakar near Tillya dam (continued)
- 35. Zebrafish Ecology and Behaviour 15 states of Rajasthan, Gujarat and Andhra Pradesh (river basins draining into the Arabian Sea) as well as northern Myanmar and Sri Lanka, although no location details are given (37). The relia- bility of some of the earlier records is questionable; either no spec- imens appear to have been collected (as in the case of records for Sri Lanka), or the specimen has been reclassified (as in the case of at least one species from Myanmar, now designated Danio kyathit (57)). Database records for this species should not be considered as complete. However, on the basis of confirmed occurrences, the zebrafish may be widely distributed over the Indian subcontinent; it may be overlooked in surveys on account of its small size and the fact that it has no value as a food fish, even to subsistence fishermen. The Indian subcontinent has a monsoon climate with wide seasonal variation in the extent of freshwater habitats. Some of the major river systems, such as the Ganges, run through low- lying areas that flood extensively during the monsoon months. The floodplains are characterised by oxbow lakes and blind chan- nels, which may have seasonal connections to the main river. In addition, these regions contain extensive areas of man-made lakes, ponds and irrigation channels constructed for fish and rice culti- vation. There is a wide range of temperatures within the natural range of zebrafish, from as low as 6◦C in winter to over 38◦C in summer. Zebrafish have typically been described as inhabiting slow- moving or standing water bodies, the edges of streams and ditches, particularly adjacent to rice fields (70, 38, 71). How- ever, they are also reported as inhabiting rivers and hill streams (72). This inconsistency in habitat preference probably results from the taxonomic confusion between Danio and Devario (36). Three surveys have systematically described their habitat prefer- ences; McClure et al. (68) captured zebrafish in three sites in the Ganges drainage in India, Spence et al. (66) captured them in nine sites in the Ganges and Brahmaputra drainages in Bangladesh, and Engeszer et al. (73) captured them in 14 sites in the Ganges and Brahmaputra drainages in India. In all three studies, zebrafish were found to occur in shallow water bodies with a visibility to a depth of ∼30 cm, frequently in unshaded locations with aquatic vegetation and a silty substrate. Zebrafish appear to be a floodplain rather than a true riverine species. They are most commonly encountered in shallow ponds and standing water bodies, often connected to rice cultivation. This association with rice cultivation may relate to the use of fer- tilisers that may promote the growth of zooplankton, a major component of the zebrafish diet (74). Rice paddies and shallow seasonal waters are also likely to be free from large predatory fish. Spence et al. (66) found no zebrafish in either rivers or temporary creeks that opened during the monsoon season. Where zebrafish
- 36. 16 Spence are found in streams and rivers, these typically have a low flow regime and zebrafish were most often encountered at the mar- gins (68, 73). Behavioural observations of their vertical distribu- tion indicated that they occupy the whole of the water column and occur as frequently in open water as among aquatic vegeta- tion (66). 3.2. Diet Zebrafish are omnivorous, their natural diet consists primarily of zooplankton and insects, although phytoplankton, filamen- tous algae and vascular plant material, spores and invertebrate eggs, fish scales, arachnids, detritus, sand and mud have also been reported from gut content analysis (68, 69, 74). The major- ity of insects identified in these studies were aquatic species, or aquatic larval forms of terrestrial species, particularly dipterans. It has been suggested that zebrafish may have some value in mosquito control (69). The high proportion of planktonic items in their diet indicates that zebrafish feed primarily in the water col- umn, however, terrestrial insects and arachnids are also consumed, suggesting surface feeding. The presence of inorganic elements and detritus suggests that zebrafish also feed from the substrate. In a study based on sampling over 12 months, dietary compo- sition appeared to differ significantly among months although no clear seasonal pattern was apparent (74). Additional data are required to determine the extent to which food items in the gut of zebrafish reflect selectivity on the part of the fish as opposed to seasonal availability of different prey. 3.3. Growth and Mortality Zebrafish growth is most rapid during the first 3 months fol- lowing hatching; afterwards the growth rate starts to decrease to approximately zero by about 18 months (74). Growth rates of domesticated strains in the laboratory have been reported as higher than those for wild fish. Eaton and Farley (75) reported an annual growth rate of 183 mm y–1 during the first 45 days of development, compared to 72 mm y–1 during the first 2 months in nature (74). This difference in growth rates could result from inadvertent selection for rapid growth or as a consequence of higher food intake in captivity. The latter explanation is more likely, as F2 offspring of wild-caught fish grow at an equiv- alent rate to domesticated strains under controlled conditions in the laboratory (C. Smith & R. Spence, unpublished data). Domesticated strains have also been reported to achieve a larger body size than some populations of wild fish (34). A length- frequency analysis based on sampling over 12 months from a lake population in Bangladesh showed the mean length of fish to be 25 mm after 1 year. The maximum BL observed was 35 mm (74), which is comparable to the typical range observed in laboratory strains. The size difference may be partly due to genetic factors (34, 76) with selection for fast growth and high fecundity among
- 37. Zebrafish Ecology and Behaviour 17 laboratory fish, but it may also reflect rearing conditions; in the laboratory, F1wild fish also achieve 35 mm BL after 18 months (R. Spence & C. Smith, unpublished data). Females tend to be larger than males both in domesticated and wild populations (74, 75, 77). The extent of variation in growth rates and body size among wild populations is unknown. The zebrafish appears to be primarily an annual species in nature, the spawning season commencing just before the onset of the monsoon (74). Length-frequency analysis showed two dis- tinct age classes during the summer months, representing repro- ductively mature 1+ year fish and a cohort of 0+ fish. Thus, the main period of rapid growth takes place during the monsoon months (June–September), a period of high temperatures (up to 34◦C) and food availability (78). Gerhard et al. (79) reported a mean life span of domesti- cated zebrafish of 42 months, with the oldest individual surviving for 66 months. However, instances of spinal curvature, a phe- notype caused by muscle degeneration and commonly associated with senescence (79, 80), become apparent in domesticated and wild zebrafish after their second year in captivity (R. Spence, pers. obs.). Spinal curvature was not observed in a wild population (74) and it is likely that fish die in natural populations before this con- dition develops. 3.4. Assemblage Where zebrafish are found, they tend to be among the most abun- dant species (66, 68, 73). Spence et al. (66) captured a total of 25 species from nine families that co-occurred with zebrafish over their range in Bangladesh, while Engeszer et al. (73) captured 36 species from 16 families. These were primarily small (<25 cm total length) indigenous species. Such species represent potential competitors of zebrafish. Zebrafish were often observed shoal- ing together with the flying barb Esomus danricus (Hamilton), another abundant cyprinid of similar size and appearance that is closely related to Danio (48). Other potential competitors are Puntius spp. and Aplocheilus panchax (Hamilton). The other danionin species found with zebrafish were Danio dangila (Hamilton), D. meghalayensis (Sen & Dey), Devario devario (Hamilton), Devario assamensis (Barman) and D. aequipinnatus (McClelland). McClure et al. (68) reported sig- nificant differences in the characteristic temperature, pH and cur- rent speed of the habitats in which different danionin species occurred; the Devario species typically inhabited faster flowing water whereas zebrafish were captured in the margins of streams and rivers. This corresponds with Fang’s (36) finding that the two genera occupy different microhabitats. 3.5. Predators The commonest predatory taxa captured with zebrafish were snakeheads, Channa spp., and the freshwater garfish, Xenentodon
- 38. 18 Spence cancila (Hamilton) (73, 64) although sampling protocols may have failed to capture other potential predators such as noctur- nal catfish. Engeszer et al. (73) additionally captured the cat- fish Mystus bleekeri (Day) and the knifefish, Notopterus notopterus (Pallas). Mastacembelids, which also co-occur with zebrafish, are oophagous and may be predators of zebrafish eggs and embryos, while odonate larvae may be predators of larval and juvenile zebrafish (73). Adult zebrafish are also predators of zebrafish eggs and larvae. Avian predators such as the Indian pond heron, Arde- ola grayii (Sykes), and the common kingfisher, Alcedo atthis L., are also ubiquitous in the floodplains of the Indian subcontinent and may feed on D. rerio. Laboratory studies have shown that zebrafish display fright reactions in response to both visual and olfactory cues associ- ated with predators. Dill (81, 82) used both living (largemouth bass, Micropterus salmoides (Lacepède)) and model predators to investigate zebrafish escape responses. The distance at which the response was elicited depended on the predator’s size and its approach velocity. Reactive distance did not differ significantly between living and model predators, although escape velocity was higher with living predators. Over repeated trials on successive days, zebrafish responded earlier and flight distance increased. No decline in response was detected when zebrafish were retested after a 10-day break. This effect may be an example of sec- ondary reinforcement; as the predator’s approach was associated with a negative experience, the fish began to respond before the initial threshold was reached. Bass and Gerlai (83) compared the responses of zebrafish to a sympatric predator (the leaf fish, Nandus nandus), an allopatric predator (the compressed cich- lid, Nimbochromis compressiceps), a sympatric harmless fish (the giant danio, Devario malabaricus) and an allopatric harmless fish (the swordtail, Xiphophorus helleri). The zebrafish, which were a domesticated line, showed an elevated fear response to the sympatric predator compared to the others. This would appear to indicate some kind of genetic involvement in anti-predator responses. In common with other ostariophysian fishes, zebrafish show alarm behaviours in response to a pheromone that is released as a result of injury to the epidermal cells (84, 85). The strength of the response is proportional to the concentration of alarm sub- stance in the water (86). Alarm behaviours include an increase in shoal cohesion and either agitated swimming or freezing on the substrate, a decrease in feeding rate and increase in aggres- sion. These behaviours have been interpreted as having an anti- predator function. Rehnberg and Smith (87) demonstrated that isolated zebrafish showed an alarm response to water containing alarm substance, so the response is independent of the presence of conspecifics.
- 39. Zebrafish Ecology and Behaviour 19 3.6. Parasites Little is known about the natural parasite fauna of zebrafish, or the role parasites play in their behaviour and ecology. In a pre- liminary study conducted in Bangladesh, based on an analysis of 120 specimens from seven sites, infection by 20 species of meta- zoan parasites and three protozoans was observed (R. Spence & C. Smith, unpublished data). The majority of parasites were larval stage digeneans, cestodes and acanthocephalans, while ectoparasite infection was rare. Infection by Acanthostomum sp., Centrocestus sp. and one diplostomoid species was observed in all the locations sampled, with 100% prevalence being observed for the metacercariae of Acanthostomum sp. in one site and Centro- cestus sp. in two sites. In laboratory stocks, infection by the microsporidian Pseudoloma neurophilia is common (88). It infects the central nervous system, cranial and spinal nerves, and skeletal muscle of zebrafish, causing emaciation, ataxia and spinal malformations. It is not clear whether vertical transmission of this parasite can occur in zebrafish. Captive zebrafish have also been subject to infec- tion by the nematode Pseudocapillaria tomentosa, which infects the gut; symptoms include inflammation, emaciation and intesti- nal carcinomas (89). P. tomentosa can be transmitted directly and infects entire laboratory colonies. There are many possible explanations for this phenomenon but the finding that nematode infection appears to be rare in nature may indicate that zebrafish have not evolved natural immunity to the effects of parasitism by nematodes. 4. Reproductive Behaviour 4.1. Spawning Cycle Much of the scientific literature on zebrafish reproduction has been concerned with how best to maximise the supply of eggs for research (reviewed by Laale (54)) and, until recently, almost noth- ing was known about the reproductive ecology of wild zebrafish. In zebrafish, all gonads initially develop as ovaries, which in males start to differentiate at approximately 5–7 weeks post hatching (10–15 mm TL) through an intersexual stage, finally developing into normal testes by approximately the third month of develop- ment (12–17 mm TL), depending on strain and rearing condi- tions (90, 91). The genetic mechanism of sex determination in zebrafish is unknown. However, there is evidence that food sup- ply or growth rate affects sex determination, with faster growing individuals developing as females and slower growing individuals as males (92). Based on samples collected from a population in Bangladesh, sex ratios in nature appear to be 1:1 (74).
- 40. 20 Spence In the laboratory, domesticated zebrafish strains breed all year round whereas in nature spawning is more seasonal. However, larger females collected in January (outside the main spawning season) have been found to contain mature ova, indicating that reproduction may not be cued by season, but may instead be dependent on food availability, which is likely to co-vary with season (66). Furthermore, reproductive maturity appears to be related to size rather than age; wild and domesticated zebrafish appear to reach reproductive maturity at similar sizes, despite hav- ing different growth rates. Eaton and Farley (75) showed that domesticated zebrafish reared at 25.5◦C reached maturity after 75 days, when females were 24.9 mm BL and males 23.1 mm. In laboratory conditions, F1 wild zebrafish also reach reproductive maturity at approximately 23 mm BL (R. Spence, pers. obs.). Pairs of zebrafish left together continuously spawn at fre- quent but irregular intervals (77) and a single female may produce clutches of several hundred eggs in a single spawning. In a study by Spence and Smith (93) inter-spawning intervals ranged from 1 to 6 days, with a mean of 1.5 days, producing clutches ranging from 1 to over 700 eggs, with a mean of 185 (± SD 149). Clutch size correlated positively with both female body size and inter- spawning interval. Eaton and Farley (77) reported that inter- spawning interval increased with age, from a mean of 1.9 days in 12-month-old fish to 2.7 days 3 months later. Clutch size also increased over this period from a mean of 158–195. No equiva- lent data are available for wild zebrafish, but inter-spawning inter- vals tend to be greater and clutch sizes smaller than domesticated strains (R. Spence, pers. obs.). Ovulation is dependent on female exposure to male gonadal pheromones; male holding water, testis homogenates and testis fractions containing steroid glucuronides will induce ovulation but fail to do so in females rendered anosmic by cauterising the nasal epithelium (94, 95). Eaton and Farley (77) showed that exposure to a male for 7 h in the afternoon was sufficient to enable eggs to be stripped from females the following morning. However, eggs were never obtained from isolated females more than once in any 5-day period after exposure to a male. Thus it appears that all mature ova are released in a single spawning bout (77, 96). The presence of a male is essential for females to spawn eggs. Females kept in isolation or older females can become “eggbound” (Fig. 1.3a, b) which can be lethal in severe cases. Dissections of eggbound females showed a 3 × 3 mm plug con- sisting of necrotic clumped eggs clogging the oviduct, preventing any further successful spawning (Gerlach unpublished results). Regular exposure to males and spawning dishes can prevent this development. Interestingly, despite the fact that egg production is non-continuous, females exposed to male pheromones for several
- 41. Zebrafish Ecology and Behaviour 21 Fig. 1.3. Female zebrafish a before and b after being housed alone for 3 weeks. The belly of the females increased, on average, by 69 ± 24% (n = 10). Grid = 0.5 cm2. (Reproduced by kind permission of Gabi Gerlach). days prior to spawning produce more eggs of higher quality than females isolated for several days (97). This effect could be a con- sequence of the concentration of pheromones to which they are exposed. Bloom and Perlmutter (98) showed that both sexes pro- duce pheromones that function as inter- and intra-sexual attrac- tants, and have different effects at different concentrations. For both sexes, the intra-sexual response was elicited at a lower con- centration than the inter-sexual response. Eggs are non-adhesive and demersal, with a diameter of approximately 0.7 mm. They are released directly over the sub- strate with no preparation of the substrate by either sex and there is no parental care. Eggs become activated on contact with water and even in the absence of sperm, undergo a series of pro- grammed developmental steps. Unfertilised eggs develop a perivi- tilline space but fail to develop beyond the first few cleavages (99). Hatching takes place between 48 and 72 h at 28.5◦C, depending on the thickness of the chorion and the muscular activity of the embryo inside, both of which can vary within a group of embryos (5). Immediately after hatching, the larvae (measuring ∼3 mm) attach to hard surfaces by means of small secretory cells in the epidermis of the head (54). Attachment at progressively higher
- 42. 22 Spence levels enables them to reach the surface to which they need to gain access in order to inflate their swim bladders (100). This process occurs from about 72 h post-fertilisation, whereupon swimming, feeding and active avoidance behaviours commence (5). 4.2. Mating Behaviour It is well known that spawning in domesticated zebrafish is influ- enced by photoperiod (101). Zebrafish show a distinct diurnal activity pattern, synchronised with the light/dark and feeding cycles. The first activity peak occurs immediately after illumination with two further peaks in the early afternoon and the last hour of light (61, 102). Spawning activity coincides with the first activity peak and usually commences within the first minute of exposure to light following darkness, continuing for about an hour (103). Field observations have shown that spawning in zebrafish under natural conditions is also largely limited to a short period at dawn (104). Notably, wild-caught zebrafish held in captivity are more likely than domesticated strains to spawn at times other than first light (R. Spence, pers. obs.). Extended day length may be a con- tributory factor in the seasonal onset of spawning in nature. It was noted by Breder and Rosen (101) that adding a dash of cold water to aquaria could encourage spawning in zebrafish. Thus, it may be that a drop in water temperature or an increase in water level may be additional cues used by zebrafish. In nature, zebrafish spawn during periods of heavy rain (R. Spence, pers. obs.). Courtship behaviour in zebrafish consists of a male chasing the female rapidly, often nudging her flanks with his snout and attempting to lead her to a spawning site (see below), swimming around or in front of her in a tight circle, or figure of eight, with his fins raised. If she does not follow, he may alternate between circling the female and swimming back and forth between the female and the spawning site. Once over a spawning site he swims closely alongside the female, spreading his dorsal and caudal fins around her so that their genital pores are aligned, and may oscil- late his body at high frequency and low amplitude. This behaviour triggers oviposition in the female and sperm is released simulta- neously. This sequence of behaviours is repeated throughout the spawning period, females releasing between 5 and 20 eggs at a time. Male courtship behaviour is most active in the first 30 min and although it continues for about an hour, few females extrude eggs after the first 30 min (103). Wild zebrafish display similar courtship and territorial behaviours during spawning as have been described in domesticated strains (104). Under more natural con- ditions, courtship involves males actively pursuing females, who utilize the whole water column, alternately swimming towards the surface and then diving steeply down to the substrate to spawn. Small groups of 3–7 fish usually take part in these chases. Courtship behaviour in the male is triggered by female pheromones. In a study by van den Hurk and Lambert (94)
- 43. Zebrafish Ecology and Behaviour 23 males, but not females, were attracted to ovarian extracts injected into the aquarium. Anosmic males failed to court females while control males only courted females that had ovulated. Further, anosmic males were extremely aggressive, suggesting that ovarian pheromones also inhibit aggression that is common in both sexes during foraging. Zebrafish typify a basic mating pattern common to many cyprinid fishes; they are group spawners and egg scatterers (101). Females will spawn directly onto a bare substrate, but when pro- vided with an artificial spawning site, such as a plastic box filled with marbles, will preferentially use it for oviposition (105). Some male zebrafish are territorial during mating (105). Both territo- rial and non-territorial males show the same courtship behaviour but whereas non-territorial males pursue females, territorial males confine their activities to within a few body lengths of a spawn- ing site and chase other males away when they try to approach. A study by Spence and Smith (105) examined the effects of manipulating density and sex ratio on the behaviour of these territorial males. Aggression rates increased at higher densities. However, while courtship behaviour increased with density under a female-biased sex ratio, when the sex ratio was male-biased courtship rate decreased relative to that observed at low densi- ties. A subsequent microsatellite parentage analysis showed that the reproductive success of territorial males was also density dependent (106). At low densities territorial males sired signif- icantly more offspring than non-territorial males. However, at higher densities territorial males were no more successful than non-territorials. Thus male zebrafish display two distinct mating tactics, territorial defence and active pursuit of females, the adop- tion of which is flexible and may be density dependent. Another study (107) used a higher density level and found that terri- toriality broke down completely and aggression was reduced in consequence. Thus it is likely that aggression will be highest at intermediate densities, depending on the availability of defend- able territories. Density can also affect female reproductive success, mean per capita egg production decreasing at higher densities (105, 107). A parentage analysis indicated that this effect was due to females spawning smaller clutches, rather than some females being excluded from spawning (106). There are several possible expla- nations for reduced female egg production at high densities; increased male-male aggression may interfere with female ovipo- sition attempts and/or competition may arise among females for access to spawning sites. Alternatively, reduced female egg pro- duction may arise through pheromonally mediated reproductive suppression. Females exposed to the pheromones of other females for several days prior to spawning have been shown to be sig- nificantly less likely to spawn compared to isolated females (97).
- 44. 24 Spence Further, dominant females produce more eggs than subordinates (97). In a study on female territoriality conducted in a large 2 × 2 m aquarium, Delaney et al. (108) showed that females avoid the presence and, therefore, also the direct exposure to pheromones of other females. Females have a significant preference to stay with one or several males over other females. Tested in a T-maze, an increasing concentration of chemical cues from female zebrafish elicited avoidance behaviour in other females (109). Thus, com- petition among both males and females may play a role in the zebrafish mating system. 4.3. Mate Choice The existence and nature of female mating preferences can be difficult to demonstrate in species where male competition plays a significant part in the mating system; matings are likely to be determined by the dominant male excluding other males rather than females actively choosing mates. There is some evidence that female zebrafish prefer larger males (110), and body size tends to correlate with dominance in teleost fishes (111). When female egg production is used as a measure of preference, female zebrafish do appear to prefer some males over others (93). However, while these preferences do not correlate with male dominance, neither do females correspond in their choice of males (93). In view of the role played by pheromones in the reproductive behaviour of both sexes, it is possible that mating preferences may be based on olfactory cues. For instance, female zebrafish prefer the odour of unrelated males to unfamiliar brothers (112). In the zebrafish mating system, the two mechanisms of sexual selection, male- male competition and female preference, may operate in oppo- sition. If females do not prefer dominant males, their preferences may undermine the ability of dominant males to monopolise mat- ings. Further, competition among males for mating opportuni- ties may be balanced by similar competition among females (97). Indeed, variance in reproductive success among females is equiv- alent to that among males, and consequently the opportunity for sexual selection is weak in zebrafish (106), borne out by the fact that they do not display striking sexual dimorphism. 4.4. Ovipostion Choice Females are selective with respect to sites for oviposition. In choice tests conducted both with domesticated fish in the lab- oratory and with wild fish in a field-based mesocosm, females preferred a gravel substrate to silt (104). Territorial males were also observed to defend gravel-substrate spawning sites in prefer- ence to silt. This preference appears to relate to spawning site quality; egg survival is enhanced by incubation in a substrate that allows oxygenated water to circulate while protecting them from disturbance and cannibalism. In the laboratory, a prefer- ence for vegetation was also observed, although vegetation did not affect survival. Vegetation is thought to be important in the
- 45. Zebrafish Ecology and Behaviour 25 survival of larval zebrafish; they possess attachment organs that may assist them in reaching the surface to inflate their swim bladders (54). Sessa et al. (113) studied oviposition preference in relation to a depth gradient (0–4 cm) and found that females spawned preferentially in very shallow water. In the types of habitat where zebrafish are common, such as floodplain ponds, the substrate is often silty and zebrafish are thought to spawn in shallow vegetated areas that offer protection from predators (73, 104). Therefore, there may be competition for access to sites that afford better water circulation as well as protection for eggs and larvae. Choice of oviposition site is one of the few ways in which oviparous species with no parental care can maximize offspring survival. Thus, if females actively choose oviposition sites, males may increase their reproductive success by guarding such sites. 5. Social Behaviour 5.1. Shoaling Preferences Zebrafish are a shoaling species; shoaling behaviour commences soon after hatching and increases with age (114) although shoal- ing preferences do not develop until fish reach the juvenile stage, c. 10 mm BL (115). Miller and Gerlai (116) showed that the average inter-individual distance between individual zebrafish in a shoal remained constant over multiple days. Group cohesion represents a balance between predator avoidance and competi- tion for food. During feeding (when food was evenly dispersed) inter-individual distance increased; when presented with a model aerial predator, while in a bare tank with no hiding places, the shoal responded by scattering and quickly reassembling into a tight group (116). Shoaling behaviour appears to be innate; fish reared in isola- tion quickly form shoals when placed together (117). McCann and Matthews (118) showed that zebrafish reared in isolation did not discriminate between shoals of conspecifics, pearl dan- ios, Danio albolineatus (Blyth), or guppies, Poecilia reticulata (Peters), suggesting that species identification is learned. McCann and Carlson (119) tested this by cross-rearing zebrafish with the closely related unstriped pearl danio. Cross-reared individu- als showed a reduced preference for associating with conspecifics. Engeszer et al. (120) showed that preferences for different intra- specific phenotypes are also learned. Wild-type zebrafish cross- reared with the stripeless pigment mutant nacre preferred the colour pattern of those with which they had been raised, irrespec- tive of their own appearance. Based on a comparison of shoaling preferences among five different danio phenotypes, stripes appear
- 46. 26 Spence to be a key shoaling cue (121). These studies suggest that species recognition in the zebrafish is mediated by a process of phenotype matching against a template based on early experience. Engeszer et al. (115) found that the visual preference of juvenile wild- type zebrafish for like phenotype remained even when their social environment was manipulated by placing individuals in groups of nacre shoalmates for 30 days. However, McCann and Carlson (119) found that the visual preference of cross-reared subjects was eroded after similar manipulation. These observations together suggest that template formation involves both genetic and learned components. Zebrafish have also been shown to use olfactory cues in both species and kin recognition (112). In a series of odour flume choice tests, juvenile zebrafish preferred conspecifics to het- erospecifics, unfamiliar kin to non-kin, and familiar to unfamiliar kin. Gerlach et al. (122) showed that kin recognition is based on olfactory imprinting, with a very specific 24-h developmen- tal window requiring exposure to kin on day 6 post-fertilisation. There was no evidence of self-matching; larvae reared in isolation did not imprint on their own chemical cues. Exposure to non-kin at the critical stage did not result in imprinting which suggests some genetic involvement in the process. Thus, social preferences in zebrafish may be based on individual recognition as well as phenotype matching. Individual recognition may play a role in zebrafish since this species is known to establish dominance hier- archies (93, 97, 123). The mechanism underlying this olfactory recognition is not yet known. Shoaling decisions in zebrafish are also influenced by shoal size and activity level. In a test of shoaling preferences, Pritchard et al. (124) showed that individuals generally preferred larger shoals. However, when shoal activity level was manipulated by changing the water temperature, fish preferred the more active shoal, regardless of size. Preferences also appear to differ between the sexes (125). Male zebrafish preferred to associate with female shoals compared to males but had no preference for shoal size. However, females preferred to associate with the larger shoal, regardless of whether it was composed of males or females. Zebrafish appear to be able to assess the nutritional state of con- specifics; food-deprived individuals preferred to shoal with well- fed conspecifics, and had increased foraging success than when shoaling with other food-deprived individuals (126). Tests of shoaling preference based on visual cues have been conducted between wild-type zebrafish and various aquarium variants: leopard danios (127), longfin (128) and the transgenic GlofishTM, which are genetically engineered to express red flu- orescent proteins (129). No significant preference was detected in any of these tests. However, Engeszer et al. (130) com- pared shoaling preferences among 17 different pigment pattern
- 47. Zebrafish Ecology and Behaviour 27 mutants or closely related species and showed that, while wild and laboratory zebrafish exhibited similar preferences, there was a marked difference between the sexes. Male preferences were based on species and stripe patterning but female preferences did not correlate with a priori identifiable traits. While most tests of shoaling preference are based on dichotomous choice tests, Saverino and Gerlai (131) analysed video footage of shoals of test and stimulus fish swimming together, to determine inter- individual distances, and found that zebrafish shoaled more closely with conspecifics. They also presented fish with computer animated images of zebrafish, modifying their colour, location, pattern and body shape and found a preference for yellow and avoidance of elongated images. 5.2. Aggression and Dominance Zebrafish of both sexes can establish dominance hierarchies. Aggressive interactions involve chasing and in some cases biting. Display behaviour involves pairs of fish orienting head to tail with their fins splayed and slowly circling one another while ascend- ing (R. Spence, personal observation). This behaviour operates within and between the sexes; its function is not clear but it may be a means of individual recognition that reinforces dominance ranks. Once dominance relationships become established, aggres- sion becomes less intense (53). When fish are housed in pairs, the dominant individual often appears darker and utilises the entire aquarium, while subordinates are pale and occupy a smaller area (53). Dominance relationships appear to be relatively stable over time, at least over the duration of 5-day experiments (105, 123). Moreover, males separated for 4 days have been shown to re- establish identical dominance ranks once reunited (G. Gerlach, unpublished data). The sex of an individual does not appear to be an important factor in determining its dominance rank (123). The relation- ship between body size and dominance is unclear, partly because studies often control for size (93, 105, 123). However, in stud- ies using fish of different sizes, Hamilton and Dill (132) found that size correlated positively with rank, while Basquill and Grant (133) found that it was not. Dominance has been demonstrated both during mating behaviour, where males establish territories around spawning sites (105) and foraging, where dominant indi- viduals attempt to monopolise a food source (123, 132, 133). It is not known whether males that are territorial during spawning are also dominant during foraging. In a study of zebrafish foraging behaviour, Gillis and Kramer (134) manipulated fish density and food patch profitability. Zebrafish formed shoals but aggressive interactions took place near feeding sites. The distribution of fish was affected by patch profitability, with more fish being concentrated around the most profitable food patch. However, the variability in the distribution
- 48. 28 Spence between the three patches was greater when fish density was lower. At high densities, there were more fish in the least prof- itable patch and fewer in the most profitable patch than would be predicted by an ideal free distribution model (135). Aggressive interference did not fully explain the density-related reduction in foraging efficiency; aggressive interactions increased with patch profitability but decreased at high population densities. Thus, foraging distributions may also be influenced by non-aggressive interactions, while aggressive interactions are ameliorated at high densities. Aggression and food monopolisation are also influenced by habitat structure. Basquill and Grant (133) compared lev- els of aggression in a vegetated versus a non-vegetated habitat. Aggression and food monopolisation by the dominant fish were lower in the vegetated habitat. This effect could be because the presence of vegetation makes the environment more difficult to defend. An alternative explanation is that a vegetated environ- ment is perceived as safer; dominant fish may be more willing to forage in open habitats where predation risk is higher, while to subordinate fish the perceived benefit of shoaling in a risky habitat may outweigh the cost of reduced foraging efficiency. In order to test these two hypotheses, Hamilton and Dill (132) com- pared aggression and resource monopolisation among three habi- tats, open, vegetated and unvegetated with overhead cover. When allowed to choose, fish preferred to forage in the covered habitat and there was no effect of vegetation. There was no difference in aggression among habitats, but resource monopolisation was greater in the open “risky” habitat. Rearing environment may also influence aggression and dom- inance. Marks et al. (136) found that fish raised in an hypoxic environment were less aggressive and spent more time in refugia than those reared in a normoxic environment. This result suggests that zebrafish offer a potential model for exploring phenotypic plasticity in behaviour, particularly developmental plasticity. 5.3. Exploratory Behaviour Shoaling behaviour can increase the probability of an individual fish detecting and avoiding predators (137). A related behaviour is predator inspection, whereby individual fish leave a shoal briefly to approach a predator. These two traits are known to be at least partly genetically determined in zebrafish. Wright et al. (138) showed differences in “boldness” (defined as the propensity to approach a novel object, in the shape of a black cylinder sus- pended in an experimental aquarium) among laboratory raised wild (F2) zebrafish from four different populations. An intra- population study indicated a genetic component to shoaling ten- dency (the time an individual fish spent associating with a stimulus shoal), although there was no equivalent inter-population differ- ence. In a further study, Wright et al. (34) compared boldness
- 49. Zebrafish Ecology and Behaviour 29 and shoaling tendency between wild (F2) and laboratory zebrafish (AB line). The AB fish showed reduced shoaling tendency and increased boldness compared to wild fish, presumably as a result of relaxed selection for anti-predator behaviours. Robison and Rowland (33) similarly compared the Nadia wild (F5) strain with a transgenic line TMI, which contains a green fluorescent protein transgene, allowing them to be visually distinguished from other strains in a mixed aquarium. They found that Nadia were less sur- face orientated, were more likely to freeze on the bottom of the aquarium when presented with a novel object, and were less likely to inspect novel objects compared to TMI fish. Hybrids between the two strains showed intermediate responses and inter-strain dif- ferences were still apparent among strains reared in mixed tanks, suggesting that the behaviour was not learned. It is also possible that the results of both these studies reflect pre-existing strain differences and are not related to domesti- cation. A further study using Nadia, TMI and an additional domesticated strain (SH) revealed significant inter-strain differ- ences across five behavioural measures, although the observed relationships within strains were relatively weak and occasion- ally inconsistent (139, 140). These observations, together with the inter-population differences among wild fish identified by Wright et al. (138) indicate the need for caution in interpreting behaviours as indicative of particular behavioural patterns such as domestication. 6. Cognitive Behaviour Learning mediates many aspects of animal behaviour, includ- ing social interactions, foraging, navigation and predator avoid- ance. In zebrafish, the preference for associating with other fish is innate, while the preference for particular colour patterns is based on learned behaviour. Individuals raised in isolation do not dis- play colour pattern preferences whereas cross-reared individuals prefer to associate with the colour pattern with which they were raised (120, 127). The preference effect of cross-rearing does not persist once fish are housed in groups of the same colour pat- tern, so the early learned preference can be modified by later experience (119). However, zebrafish reared with others of the same colour pattern retain the preference even when subsequently housed with an alternative colour pattern, indicating that there may be some genetic involvement in colour pattern preference (115). Learned preferences are mediated by olfactory as well as visual cues; zebrafish can differentiate between familiar and unfa- miliar conspecifics on the basis of odour, and thus, appear capable of individual recognition (112, 122).
- 50. 30 Spence The response shown by zebrafish to alarm substance (see Section 3.5) is also innate but appears to function as a means of learned predator recognition (86). Alarm substance can ini- tiate a conditioned response to an innocuous odour, such as morpholine, when the two are presented simultaneously (141). Hall and Suboski (142) further elicited a learned response to a visual cue by pairing alarm substance with a red light as well as with morpholine. Thus, conditioning can operate across dif- ferent sensory modalities. Hall and Suboski (143) also demon- strated second order conditioning whereby fish conditioned with alarm substance to respond to either light or morpholine, then learned to react to the second neutral stimulus when presented in combination with the first conditioned stimulus in the absence of alarm substance. The mechanism for communicating learned predator recognition appears to be classical conditioning, pair- ing of an unconditioned stimulus (alarm substance) with a condi- tioned stimulus (light or morpholine) to produce a conditioned response (alarm reaction). Conditioned responses can develop after a single trial, and a response can be obtained even when there is a time delay of several minutes between presentation of the unconditioned and conditioned stimuli (144). Furthermore, conditioned responses can be passed on to naïve fish, a process known as social facilitation. Naïve fish exposed to morpholine when in the company of morpholine-sensitized fish subsequently display an alarm reaction to morpholine. The naïve fish retain this learned response when solitary or in the company of a new group of naïve fish (141). An alternative approach to studying learning is to use an oper- ant conditioning paradigm, whereby fish are trained to swim in a specific direction for a food reward paired with a visual cue. This approach has been used to study spatial memory, landmark use and orientation in other species (145), and the few studies available indicate that zebrafish are potentially a useful model for research in this area. In a study to investigate spatial learn- ing and memory, Williams et al. (146) trained adult zebrafish to swim alternately to one or other side of a divided aquarium to receive a food reward. Once trained, the fish could remember the task after a 10-day period during which they were fed ad libitum in another aquarium. Zebrafish were also able to learn to swim into one of three compartments when the one containing the reward was cued by a white light (147). A three-choice design provides better evidence of learning than a two-choice design, as the level of a chance response is reduced to a third. Williams et al. (146) reported that fish learned the task in approximately 14 trials, although Bilotta et al. (147) reported wide individual vari- ability in speed of learning. When food rewards were withheld, the training effect was quickly lost (146, 148). Given the strong shoaling instinct of zebrafish, an alternative reward shown to be
- 51. Zebrafish Ecology and Behaviour 31 effective in associative learning is the sight of other fish, or even of computer-generated images of fish (149, 150). Little is known about the development of learning capacity. Williams et al. (146) found that age affected acquisition of con- ditioned responses in zebrafish. Juveniles of 6–8 weeks learned the task as well or better than adults, whereas those of 3–4 weeks were not able to do so. It was not clear whether this was a result of limited cognitive capacity or because the task presented to the fish was too physically demanding. A related question, which has not been investigated in zebrafish, is the extent to which habi- tat complexity during rearing influences cognitive development. Research with other fishes and comparisons among populations suggests that learning in fish may be related to the demands of their environment (151). The majority of studies of learning involve testing individ- ual fish (152). However, in a shoaling species like zebrafish, fish may perform better in groups; the stress of being isolated may inhibit learning ability in isolated individuals. Moreover, fish are known to be able to learn by watching others (153). However, Gleason et al. (154) found that while zebrafish learned an avoid- ance response to an electric shock fastest in groups of five or more, single fish learned faster than pairs. Thus the relationship between learning and group size may not be straightforward. Steele et al. (155) obtained similar results in exploratory feeding behaviour in response to alanine, a ubiquitous amino acid in the aquatic environment that functions as a chemical attractant and is the primary constituent of many prey odours. They found that the fastest response was elicited in groups of four fish, but single fish responded faster than groups of two, six or eight. Group size has not been studied in relation to spatial learning in zebrafish. Miklósi and Andrew (156) used beads of different colours and patterns to study the effects of habituation to stimuli. Based on video footage of zebrafish biting responses, they concluded that habituation is mediated by cerebral lateralisation of function; responses are controlled by different cerebral hemispheres under different circumstances. In trials, fish initially approached the bead with the right eye but in subsequent trials, once the object was familiar, used their left eye. Miklósi and Andrew concluded that right hemisphere control (i.e. left eye) mediates escape/attack responses (automatic behaviour), whereas left hemisphere (right eye) control is used in assessing novel stimuli and involves the inhibition of Mauthner cell discharge. Many studies of learning are based on the use of neutral stim- uli. However, in many species, innate receiver biases have evolved that cause them to respond more strongly to certain stimuli, and thus affect learning outcomes. Biases can exist at any level along the signal reception and processing continuum, from stimula- tion of a primary sensory receptor to synthesis at higher levels
- 52. 32 Spence of integration, including learning, memory and decision making (157). Both learned preferences and innate receiver biases operate in the context of foraging. Spence and Smith (158) raised groups of fish on diets consisting solely of one colour: red, blue, green or white. When fish were subsequently tested for their colour prefer- ences in a foraging context, each group responded most strongly to red, irrespective of the colour of food with which they had been conditioned. However, there was also a significant effect of condi- tioning. The observed sensory bias towards red may have evolved as a function of the nature of the transmission environment that zebrafish inhabit, in combination with an adaptive preference for carotenoid compounds in their diet (158). Different tasks have been shown to elicit different prefer- ences. Colwill et al. (148) used a T-maze with different coloured arms (green versus purple or red versus blue) to assess visual dis- crimination learning in zebrafish. They found that while fish could be trained to swim down whichever coloured arm was associated with a food reward, they learned faster and retained the response longer when the colour associated with the reward was purple or blue than when it was green or red. Thus, not only were the stim- uli not perceived as equal, but the colour preferences shown in this context differed from those in the foraging study by Spence and Smith (158). Similarly, two studies reached different con- clusions about whether zebrafish prefer a dark or light environ- ment. Serra et al. (159) found that zebrafish spent more time in a black chamber than a white one and concluded that they have an innate preference for dark environments. In contrast, Gerlai et al. (160) concluded that zebrafish did not prefer a dark envi- ronment; fish initially avoided a dark chamber and on habituation spent equal amounts of time in illuminated and dark chambers. Clearly, the existence of innate preferences needs to be under- stood when designing behavioural protocols for learning studies in zebrafish. 7. Genetic Basis of Behaviour The relationship between genes and complex behaviours is not straightforward (161). Behavioural syndromes comprise numer- ous individual components, involving multiple, interacting genes whose expression is influenced by the environment. The start- ing point in such research is to identify behavioural syndromes that can be quantified, with simple, reliable protocols that allow high-throughput screening, either for mutagenesis or nat- urally occurring behavioural variation. Much of the pioneering behavioural genetics research has utilised Drosophila as a model;
- 53. Zebrafish Ecology and Behaviour 33 genes have been identified that control complex syndromes, such as learning and memory, mating behaviour and circadian rhythms (161, 162). The advent of functional genomics has enabled research to be extended to other species (163, 164). In comparison, there is a paucity of studies on complex behaviour in zebrafish, although it is recognised as having great potential as a model for understanding the genetic basis of human behavioural disorders (18, 19). One area of interest has been the effect of drugs of abuse on behaviour. Darland and Dowling (165) conducted a behavioural screen for cocaine addiction using the conditioned place preference paradigm (CPP), whereby the drug is paired with a neutral stimulus in one compartment of the aquarium and the amount of time the fish spends in each compartment is measured before and after administration of the drug. Three out of 18 families of mutagenised fish showed abnormal responses in the CPP and were subjected to further behavioural screens, testing spatial cognition in a T-maze, swim- ming behaviour, and sensitivity to light. Each family had differ- ent behavioural profiles, which were shown to be heritable, each supposed as representing a different single gene mutation that affected addiction (165). Lau et al. (166) used CPP to demon- strate a preference by wild-type zebrafish for both food and mor- phine as rewards. In contrast, the too few mutants, in which the basal forebrain DA and 5HT neurons are selectively reduced, lacked the morphine preference, while still displaying a prefer- ence for food. This result, whereby a single gene mutation can dissociate the preference for a natural reward and an addictive drug, indicates that the two preferences are controlled by differ- ent pathways. Gerlai et al. (160) designed a series of simple, easily quan- tifiable tests to examine the effects of alcohol administered at different concentrations on locomotion, aggression, shoaling ten- dency, alarm response, light/dark preference and pigmentation. These tests could be used to identify individuals with abnormal responses to alcohol. Echevarria et al. (167) similarly used a bat- tery of tests to examine the effects of NMDA and dopaminergic manipulation (using MK-801 and SKF 38393) on activity level and shoaling tendency. Several studies have also compared the effects of acute and chronic alcohol administration among differ- ent zebrafish strains. Inter-strain differences were detected in star- tle response, predator avoidance, aggression and shoal cohesion, suggesting that there is a genetic basis to both initial sensitivity and the development of tolerance to alcohol (168–170). Zebrafish may also be a suitable model for studying the genetic basis of social behaviour. Larson et al. (53) showed that there are clear differences between dominant and subordi- nate fish in the expression of arginine vasotocin, a neurohor- mone known to mediate social behaviour such as aggression,
- 54. 34 Spence courtship and parental behaviour in vertebrates, although the system varies among taxa. Dominance relationships are not fixed and must, therefore, involve differential expression of different genetic pathways. Tropepe and Sive (171) suggested that a forward genetics screening approach might be employed to model the behavioural deficits involved in autism using zebrafish. As deficits in social behaviour are strongly characteristic of autism, behaviours such as courtship and shoaling may represent a suitable paradigm for sociability. In mice, tests of exploratory behaviour have been used as a paradigm for anxiety and fear, exploratory behaviour tend- ing to be negatively correlated with anxiety (172). Using a similar approach, Wright et al. (34) utilised the pronounced differences between wild and laboratory strains of zebrafish in willingness to approach an unfamiliar object (boldness) and attempted to iden- tify quantitative trait loci associated with these phenotypes (see Chapter 5 in this book). Other complex behaviours that offer potential for genetic analysis are learning and memory. Protocols where fish are trained to swim in a particular direction for a food reward can be used to assess speed of learning and retention time between different strains of fish, fish reared under different conditions, or known behavioural mutants. These protocols have also been used to assess the effects of drugs of abuse on learning and memory (173, 174). Yu et al. (175) studied cognitive aging in zebrafish, comparing 1, 2 and 3-year-old fish. They found that the younger fish performed better in both temporal and spatial learning and that CPP could be established more quickly. In addition, cogni- tive aging was accelerated in mutant and gamma-irradiated fish. Genetic analysis of cerebral lateralisation of function may offer insights into the molecular basis of habituation. For instance, the mutant frequent situs inversus (fsi), which shows reversal of asym- metry in many cerebral and visceral organs, showed reversal of behavioural asymmetry in some tests but not others, suggesting that at least two different mechanisms are involved in lateralisa- tion of function (176). Zebrafish have also been used to investigate the effects of anthropogenic disturbance on fish behaviour. Larsen et al. (177) studied the effects of endocrine disrupting chemicals on zebrafish sexual development and courtship behaviour. Exposure to environmentally realistic concentrations of 17α-ethinyloestradiol (EE2) from egg until sexual maturity resulted in a female-biased sex ratio, while males displayed female secondary sexual charac- teristics such as the development of urogenital papillae, rounder body shape and smaller, less distinctly patterned anal fins. Male courtship behaviour proved more resistant to the effects of EE2 and only a few biological males at the highest concentration treat- ment were unable to induce spawning. Another study investigated
- 55. Exploring the Variety of Random Documents with Different Content
- 56. hennes ojämna, nervösa springande och fröjdade sig vid tanken på att få taga henne trött i sina armar och lyfta henne upp i vagnen och fråga henne, om hon nu lärt sig, att det bästa hon hade att göra vore att uppge all egen vilja och blindt följa honom. Men så såg han henne stanna och tveka, då hon kom till vägen, som ledde till den andra byn. Och så, med ett raskt och energiskt beslut, kastade hon sig in på denna väg och försvann snart bakom husen. Han förstod hennes mening. Å, hon tänkte, att han skulle springa efter henne dit! Hon tänkte att han skulle komma förskräckt och ångerfull och tigga henne om att komma tillbaka med honom! En stygg tanke kom öfver honom. Han satte sig upp i vagnen och sade till bonden att köra vidare. Vi ska då inte vända om efter signoran? frågade denne. Nej, det behöfs icke. Alie väntade med spänning i sitt lilla rum på hotellet hela kvällen. Naturligtvis skulle han gifva sig ut att söka henne, då han icke såg henne komma. Och då låg det så nära till hands att gissa, att hon tagit sin tillflykt hit, att det ej kunde slå felt, att han ju komme hit och frågade efter henne. Men det blef natt, och han kom icke. Detta var han således i stånd till! Större var icke hans kärlek, än att han kunde veta henne ensam ute i mörkret utan att ens taga reda på, hvad som blifvit af henne. Men om han trodde, att hon nu skulle vara den, som först kom till honom, så misstog han sig. Hon ville vänta tåligt på honom, en dag, två dagar — och kom han icke då — ja, så måste hon ju tro, att han grep denna anledning för att bli fri — och så hade hon ingenting annat att göra än taga en biljett på postvagnen och försvinna. Följande dag väntade Andrea på henne hela dagen. Han var säker om, att hon skulle komma, men han kände sig ändå nästan förbittrad på henne för att hon kunnat lämna honom på detta sätt. Han gick hela dagen af och an utanför huset och rökte cigaretter, den ena efter den andra, kastade bort dem halfbrända och rullade nya, i det han spejade utåt vägen. En underlig kyla smög sig mer och mer öfver hans sinne. Å, var det icke mer bevändt med hennes hängifvenhet än så! Svek den för ett så litet prof? Ja, då hade han ju misstagit sig helt och hållet på henne, det var en illusion när han
- 57. trott sig ha vunnit henne så helt, att hon ej mer skulle kunna frigöra sig. Ja, hvad tjänade då hela leken till. Å, han borde väl vetat det, att något verkligt kunde det icke bli. När hade han någonsin haft ett helt förhållande i sitt lif. Och han hade ju alltid vetat, att detta måste sluta en dag, lika väl som alla andra. Han hade bara inte trott, att det skulle komma så snart. Men det var bäst, som det var, det var sannerligen hög tid att nu blifva fri, han hade ju varit på väg att haka sig fast vid denna flicka på det mest lumpna, banala sätt. I morgon skulle han taga diligensen och gifva sig af därifrån. Nu visste han då åtminstone, att hon skulle trösta sig, han, som så många gånger fruktat för, att han en dag skulle drifvas till att göra henne en obotlig sorg. Gudskelof, det var då ingen fara. Han gick ännu och rökte och spejade utåt vägen, när mörkret föll på. Och han hade blifvit så nervös och retlig, att han höll på att piska upp en pojke, som kom springande mot honom, som om han haft något att säga honom, för att sedan blott begära en soldo. Han hejdade flera bönder, som kommo körande, med den meningslösa frågan, om de ej sett en dam på vägen, och när värdinnan på hotellet frågade honom, om ej signoran skulle komma igen i dag heller, bad han henne ursinnigt, att hon icke skulle blanda sig i, hvad som icke angick henne. Alie hade under tiden med en viss förtviflans kallblodighet öfverlagt, hvad hon hade att göra. Hur kunde hon resa med blott den lilla kassa, hon hade i fickan och utan några reseffekter? Nej, hon ville vänta ännu en dag och sedan ville hon sända ett bud till honom med en biljett, däri hon bad honom att tillställa henne hennes tillhörigheter. Hon hade skrifvit och kasserat och skrifvit om igen denna biljett många gånger under dagens lopp, hade mildrat dess ursprungliga bitterhet, tills den slutligen blifvit nästan öm. Vi ha funnit det tillfälle, du sökt för att skiljas, tänkte hon sig till slut skrifva. Låt oss minnas hvarandra utan bitterhet — — — hvad mig beträffar — skall jag alltid välsigna de outsägligt lyckliga dagar, du skänkt mig. Detta kom under en ström af tårar. Allt som dagen led blef hon mer och mer vekt stämd. En våldsam, oresonlig längtan började att få makt med henne. Ej återse honom
- 58. mer! Det var ju icke möjligt. Slut för alltid! Och hon skulle nu anträda den långa, oändliga återresan till Sverge ensam, hon skulle sitta dag och natt i kupén med denna dödande sorg i hjärtat — för att slutligen komma fram — till hvad? Till ett lif af evig saknad, till dagar och nätter och veckor och år, som ej skulle ha annat innehåll för henne än ett ständigt rufvande öfver hvad hon ägt och mistat. Nej, det var att bli tokig åt, och hon skulle bli det, om hon kom åter hem. Hvad var då att göra? Resa härifrån, det måste hon — hon måste låta honom veta, att hon rest — men sedan? Då hon for i diligensen, om natten, öfver det vilda bergspasset! Hon ville taga platsen på taket, denna förtjusande dubbla fåtölj, i hvilken de båda suttit, när de kommit, och hvarifrån bråddjupen på sidorna syntes så svindlande — och så — om natten — med ett språng! Ja, det var det enda, som återstod henne. Mot aftonen greps hon af en outhärdlig ångest. Tänk, om han redan förverkligat, hvad hon tänkte på. Om han tagit afton- diligensen och rest! Nej, hvad var klockan? Diligensen gick kl. 9. Det var ännu tid. Och förtviflad, besinningslös, glömmande allt annat för den enda tanken att hindra honom att resa, började hon att springa vägen fram åt byn. Han stod ännu utanför hotellet och rökte. Det var redan mörkt, och han väntade henne icke mer. Men han hade vägrat att gå in till bordet, där de andra gästerna samlats till aftonmåltiden. Då värdinnan envisades att han dock skulle äta något, hade han dundrat ett: lämna mig i fred! så energiskt, att ingen mer tordes närma sig honom. Han såg den stora, tunga postvagnen med sex hästar komma långsamt släpande upp emot stationen, och han stod just och funderade på, om han skulle gifva sig af redan i kväll, då han hörde små, ifriga steg och såg en liten, kvinnlig gestalt komma springande emot sig. Han kastade bort cigaretten, tog ett par steg emot henne, öfvertygade sig om att det var hon och hans ögon lyste upp i jubel, det brusade för hans öron och surrade i hans tinningar, som vore han färdig att förlora medvetandet, han öppnade sina armar och drog henne, utan ett ord, med sig in i deras rum. Hon föll
- 59. storgråtande till hans bröst, och de voro båda som från besinningen, gräto och skrattade om hvartannat, samt kväfde hvarandra med sina omfamningar, utan att på länge komma till tals. Och då hon slutligen ville börja tala, tystade han henne. Låt mig bara känna, att jag har dig igen, sade han och blef sittande med henne tätt sluten intill sig långt inpå natten, utan att tillåta henne att röra sig. Vet du hvad Runeberg säger, vågade Alie blott en gång sakta hviska: »I himlen af deras kärlek föddes ej ordens skyar mera.» Efteråt återkom han ofta till detta. Det är inte värdt du talar om att lämna mig, ty nu har du ju sett, att du inte kan det, sade han. Om jag också skulle önska att bli fri från dig, ska det inte lyckas mer — du ska nog hålla mig fast. Hon försökte bestrida det. Det var för hans skull, hon gjorde det, och inte blott för sin egen. Hon visste, att hon skulle gjort honom olycklig, om hon lämnat honom på detta sätt. Men om hon en gång blefve öfvertygad om, att det vore bättre för honom — Inte ens då, afbröt han. Du ska hålla mig fast ändå — men hvarför försvarar du dig, som om det vore en anklagelse? Förstår du inte, att detta är min lycka. Men du själf — du skulle verkligen lämnat mig, om jag inte kommit tillbaka? Ja, det är säkert. Hvad du är underlig! Du vill alltid, att jag ska göra allt — och själf vill du inte räcka ut en hand för att hålla mig. Ja, ser du — det är därför att vi ä af så olika ålder. När man är ung, som du, har man tillräcklig entusiasm för att tro det lönar sig att strida för något. När man däremot är gammal och lifserfaren — Vi ä ju alldeles jämnåriga. Personligen, ja, men inte som ras. Du är af ett ungt folk, du. Kom i håg, hvad Giusti säger i dikten till Gino Capponi på tal om barbarerna:
- 60. Ma il libro di natura Ha l’entrata e l’uscita: Tocca a loro la vita E a noi la sepoltura. E poi, se lo domandi, Assai siamo campati. Gino, eravamo grandi, E là non eran nati. (Men naturens bok har inkomster och utgifter: Dem tillkommer lifvet, oss förgängelsen. Och om du mig spörjer, länge nog ha vi lefvat. Gino, vi voro stora, då de ej voro födda.) Ser du, fortfor han i samma lekfulla ton. Vår kärlekshistoria är i smått barbarernas intagande af det gamla Roma. Kan du tänka dig saken omvändt — att romarne skulle tågat ut till barbarerna? Ja, det gjorde ni väl också mången gång. Ja, på sin tid naturligtvis. När vi ännu voro tillräckligt unga för att vara ett eröfrarfolk. Men när vi en gång hade nått själfva höjden af världsmakt och kultur — då fanns det ingenting som skulle förmått oss att anstränga oss mer. Vi behöfde det nya blodet, vi läto oss i det hela ganska gärna styras af främmande eröfrare, emedan det var bekvämare — men om någon skulle sagt oss, att i barbarernas land funnes själfva den eviga ungdomens lifskälla, tror du att vi då skulle tågat ut för att finna den? Nej, vi skulle sagt: — förutsatt att vi trott på hela historien — att vara evigt ung kan visst vara bra, men kostar det så mycket ansträngningar, får det hellre vara. Fy, det är afskyvärdt! Du gör mig helt förtviflad, när du talar så. Jag däremot finner mig mycket bra i fångenskapen hos min urfriska lilla barbarkvinna. — Hvad tänker du på? Hvilken allvarsam min? Jag tänker på, att jag så förfärligt gärna ville sätta dig på prof en gång — ville se, om du verkligen, verkligen inte skulle kunna kämpa en dust för att vinna mig.
- 61. Det är en dålig tanke, som du bör afstå ifrån. Allt hvad som heter strid och kamp är så oskönt — jag hatar det så mycket, att jag icke skulle hålla ut många dagar förrän jag frågade mig själf: är nu också verkligen målet priset värdt? Och om jag fullföljde ändå — hvilket jag troligen inte skulle göra — så skulle jag i alla fall inte ha någon glädje af det, ty de obehag, som varit, skulle kasta sin skugga också öfver framtiden. Tänk, hvad du är olik oss andra — Rikard t. ex. Rikard, ja! Han är ju en äkta, typisk barbar — som tycker om att strida för stridens egen skull. Alie kunde ej låta bli att önska inom sig, att Andrea ägt något af denna stridslust, som hon förut klandrat hos Rikard. Hon skulle varit tryggare för deras framtid då. Men hon tänkte på hans svaghet i detta fall som en mor tänker på de brister hos sitt barn, som göra det illa rustadt för lifvet, utan en skugga af klander, blott med ett oändligt behof att få stödja och styrka.
- 62. S XIII. ommaren led mot sitt slut, dagarna begynte bli korta och det föll snö och blåste bitande kallt här uppe bland bergen. Hvarje dag hade diligensen med sig ett par extra vagnar, som ändå ej räckte till för den massa resande, hvilka strömmade bort till varmare trakter eller tillbaka till sina hembygder. Men där voro två, som icke visste, hvart de skulle taga vägen, som med oro sågo denna upplösning omkring sig, emedan den för dem betydde slutet på en period af fullkomlig lycka, på hvilken ingen fortsättning tycktes kunna följa. De hade alltjämt uppskjutit att afgöra något om framtiden, och nu stodo de där inför nödvändigheten att fatta ett beslut. Men hvilket? De vågade ej ställa den frågan till hvarandra, de sågo med växande ångest de för hvarje dag bortdragande främlingsskarorna, som om en bit af deras lycka dragit bort med dem. Se på alla dessa belåtna borgare, som nu återvänder till sina hem, sade Andrea. De har varit ute och förstrött sig, har hållit öppen börs en tid och gjort extravaganser — men nu gäller det att begynna spara och slita igen, nu kommer hvardagslifvets sträf ofvanpå feriedagarna — skulle du ha lust att byta öde med någon af dem? Neeej. Du drar på det. Men hur kan ett hvardagslif följa ofvanpå sådana feriedagar som våra? Det är omöjligt. Om vi skulle göra en alpbestigning — men en riktigt svår och utan förare — vi förirra oss, mörkret och snöstormen öfverfaller oss — vi gör ett falskt steg — det är så lätt — en lina bunden om lifvet för att göra oss oskiljaktiga — och så: buona notte! Det vore en lösning så god som en annan — bättre kanske än någon annan. Det hade ej på länge kommit någon ny gäst till hotellet, då en afton ett ungt par steg ur diligensen och begärde ett rum för natten.
- 63. De sade sig ämna fortsätta resan följande dag och medförde intet bagage. De fingo sitt rum vägg i vägg med Alies och med en dörr emellan, hvilken var tillstängd med en tung, gammal soffa, där Alie och Andrea brukade tillbringa sina aftnar tillsammans. De hade med ett visst intresse iakttagit de nykomna vid bordet. Hon var påfallande vacker med stolta, energiska drag, stora, djupblå, lidelsefulla ögon och en yppig, vällustig mun. Han hade ett mycket fint, ädelt formadt hufvud och en smärt, elegant gestalt. Men det var något trött och slappt öfver hela hans personlighet, oaktadt han ännu var helt ung, ögonen voro urgräfda med blåa skuggor, hufvudet nästan kalt, uttrycket prägladt af lifsleda och ett slags dof förtviflan. De drucko mycket vin vid bordet, och hon talade hela tiden lifligt. Efter middagen drogo de sig strax tillbaka till sitt rum, och Andrea och Alie hörde genom dörren, att de efter ett kort sysslande genast lade sig till sängs. Själfva sutto de däremot ännu uppe och läste, då de, ungefär en timme efter sedan allt blifvit tyst där inne, hörde ett skott smälla af, ett rop och strax därefter ännu två andra skott. Alla människor i hotellet störtade till, man bröt upp dörren, som var läst, och fann dem båda ligga döda i hvarandras armar. Alie kunde ej hämta sig från detta intryck. Om kvällarna, när de sutto i sin soffa, lyssnade hon ofta inåt det andra rummet, som lämnats tomt med öppna dörrar och fönster, sedan de båda döda burits ut därifrån. Och oupphörligt, natt och dag kommo de tillbaka i hennes tankar, dessa båda unga, som valt att dö tillsammans, hellre än att låta lifvet skilja dem åt, och de utöfvade som en egendomligt dragande makt på hennes fantasi. På Andrea hade däremot denna händelse haft en motsatt verkan. Nå, där har de nu stulit min goda idé, sade han med sitt vanliga begär att vända allt i skämt. Två par på samma ställe — det vore ju nästan komiskt, i synnerhet hvad det andra paret beträffar. Därtill ä vi dock för goda, du och jag, att gå och apa efter den första bästa. Vi måste hitta på en originellare lösning. Alie log med en smärtsam dragning i mungiporna. För mig vore den tillräckligt originel ändå, min ambition går inte längre, sade hon.
- 64. De sutto en afton efter måltiden inne i Alies rum, där det var så kallt, att de, för att hålla sig någorlunda varma, krupit upp i soffan, tätt tillsammans med en pläd öfver fötterna. Ett enda ljus stod bredvid dem på bordet och de läste vid dess sken tillsammans, som de alltid brukade, Ariostos Orlando Furioso. Detta stora verk i sex band hade varit hela deras sommarläsning, och de voro nu nära slutet. Aldrig hade en af dem under hela deras samvaro öppnat en bok för att läsa ensam. Endast gemensamt kunde de njuta af hvad det vara måtte; med armarna om hvarandra, med bådas ögon i boken, omväxlande läsande högt — så hade de tillbragt alla aftnarna här uppe, och det lilla bergshotellets bristande komfort, rummets nakenhet och kyla försvann inför den värme och stämning, som genomströmmade dem där de sutto hopkrupna tillsammans, helt uppfyllda af sin läsning och af hvarandras närhet. När han läste, lyssnade hon med spänd uppmärksamhet till hvarje tonfall, ifrig icke blott att förstå hvarje ord af innehållet, utan också att låta sina öron så mättas med själfva språkets ljud, så att hon sedan, i sin ordning, skulle kunna läsa väl. Och hon hade på detta sätt förvärfvat sig ett nästan fulländadt uttal af italienskan, så att han med oblandad njutning kunde höra henne föredra sina älsklingsskalder. Endast någon gång, på de allra vackraste ställena, afbröt han henne och sprang upp samt föredrog ur minnet. Alie hade med lefvande medkänsla följt Bradamantes och Ruggieros kärlekshistoria med dess så mänskliga, psykologiskt sanna konflikter, och de voro just vid den intressanta punkten, då den stolta amazonen förklarat, att ingen annan man skulle äga henne än den, som visste att öfvervinna henne i tvekampen, då det knackade på dörren. Fördjupade som de voro i sin läsning och öfvertygade om att det ej kunde vara någon annan än uppasserskan, som kom för att göra i ordning bäddarna till natten, ropade de: kom in! utan att ändra ställning och utan att ens se upp från boken. Det hände ju aldrig, att någon besökande kom till dem. Dörren öppnades och någon kom in, men först efter ett par minuter blefvo de uppmärksamma på, att denne ej rörde sig ur stället. De sågo båda upp på en gång och upptäckte en manlig gestalt, som stod orörlig vid dörren. I ett ögonblick hade Alie kastat
- 65. undan filten, hvari de suttit insvepta ända upp till hakan, och med ett litet utrop stod hon på golfvet, ansikte mot ansikte med — Rikard. Det dröjde ännu flera minuter, innan någon talade. Slutligen utbrast Andrea: Hur har ni burit er åt för att få rätt på oss? Det må jag säga var en mästerkupp. Det var inte så svårt som det kan tyckas, svarade Rikard, talande med ansträngning och i upprörd ton, samt undvikande att möta Alies blick. Andrea inbjöd honom med handen att stiga fram. Han kastade liksom en skygg och förlägen blick kring rummet, som bar alla spår af deras förtroliga samlif, rynkade lite på ögonbrynen samt satte sig slutligen långt ifrån dem. En af mina bekanta i Stockholm, fortfor han, var nyss här på genomresa. Han såg Alie ute i förbifarten, gjorde efterfrågningar om henne på hotellet och fick reda på allt. Min mor blef alldeles öfverväldigad af sorg och förtviflan — men äfven utan hennes önskan skulle jag inte tvekat ett ögonblick att resa strax och göra allt hvad som kunde stå i min förmåga att rädda Alie från — en så förnedrande ställning. Alie, som hela tiden stått orörlig framför honom med upplyftadt, trotsigt tillbakakastadt hufvud, men bortvänd blick och två djupa flammor på kinderna, gjorde här en rörelse, som för att afbryta honom, men han hejdade henne. Jag vet, hvad du vill säga, sade han. Jag vet, att du inte tror mig äga tillräcklig opartiskhet för att blanda mig i denna sak. Jag kände detta också själf förra året och det var det, som förmådde mig att lämna dig som jag gjorde. Tror du, att någon hänsyn skulle kunna förmått mig att annars — tror du, att jag annars skulle lämnat något medel oförsökt att återföra dig till sans och besinning. Men jag var inte opartisk då, och jag fruktade för att låta min känsla inverka på mitt omdöme. Men nu har jag arbetat ärligt hela detta år på att öfvervinna denna svaga punkt hos mig själf — hela min sträfvan har varit att uppfostra mig själf till att bli för dig det enda jag ännu kunde bli — en god och trofast och fullkomligt osjälfvisk bror. Jag kände att den stund skulle komma, då du allt för väl behöfde en sådan — här är jag nu och du kan lugnt räcka mig handen — du
- 66. behöfver heller inte vara så rädd att se mig i ögonen — han ansträngde sig för att själf stadigt möta hennes blick, som dock ännu vek undan — du må tro, att den kamp, jag haft, inte varit den lättaste — jag kan väl säga, att jag aldrig i mitt lif förr till den grad behöft all den viljekraft, hvaraf jag är i besittning — men det har lyckats, och det är nog. Det blef åter en stunds tystnad. Alie kände som en varm blodström kring hjärtat — ja, denna osjälfviska tillgifvenhet var som en utsträckt hand till en drunknande — men hon kunde ej tala ännu. Hvad är således nu er afsikt? frågade slutligen Andrea torrt, med ett litet satiriskt, sårande leende. Min afsikt är helt enkelt den, att erbjuda Alie en brors stöd, om hon behöfver det — och om hon inte känner sig behöfva det, att använda allt det inflytande, jag möjligen kan ha — eller rättare — inte jag, ty jag gör inte anspråk på att ha något inflytande öfver henne — men det inflytande, som förnuftsskäl och en varm tillgifvenhet kan utöfva — för att förmå henne att rycka sig lös, innan det ännu är för sent — från ett förhållande, som m å s t e sluta illa. Och om Alie nu vill återvända med mig, så lofvar jag henne, att hon inte bara ska finna ett kärleksfullt hem med den ömmaste mor och syskon och syskonbarn, utan också — hon ska inte bara finna en oförminskad tillgifvenhet hos alla dem, hon med allt skäl kan räkna för de sina — utan hon ska också alltid aktas och äras såsom den, hon alltid varit — inte skuggan af ett klander eller en misstro ska möta henne — mina barn ska uppfostras att i henne se den kvinna, som jag för min del alltid ska sätta högst bland alla jag känt — vid sidan af min mor och — min hustru. Andrea såg spörjande och pröfvande på Alie, som stod där blek och liflös som en bildstod. Alie ensam har att afgöra här, sade han. Jag kan inte lofva henne detsamma som ni — en lugn familjelycka. Hvad jag har att bjuda henne är alltid ett kamplif under svåra förhållanden — vare sig hon blir min hustru eller ej — må hon alltså fritt välja! Nu lyfte Alie för första gången blicken och såg på honom med ett uttryck af så djup smärta, sugande ömhet och förtviflans ångest att han skulle velat falla till hennes fötter och bedja henne förlåta sig att
- 67. han ej kunde annat än sönderslita henne på detta sätt. Han k u n d e ej annat. Om det hade gällt både hans och hennes lif kunde han ej i denna stund lagt ett ord i vågskålen för att påverka hennes beslut. Jag begär naturligtvis inte, att du ska bestämma dig i kväll, afbröt slutligen Rikard den pinsamma tystnaden. Jag är beredd att vänta — så länge du vill — jag har tagit in på det andra hotellet — jag ska inte besvära dig — när du vill kalla mig till dig är jag beredd, det är allt — du kan fullständigt förfoga öfver mig. Därmed afskedade han sig och Andrea följde honom till dörren, utan att Alie ännu hade växlat en blick med honom eller uttalat ett enda ord under hela tiden han varit inne i rummet. Då de åter blefvo ensamma och dörren var låst, löste sig hennes krampaktiga stelhet, hon kastade sig på golfvet framför Andrea med hufvudet i hans knä och utbröt med en ström af tårar: Andrea! Andrea! bed mig stanna. Bed mig! Han sköt henne bort, häftigt, nästan våldsamt, och sprang upp. Jag kan inte! ropade han. Hon kröp efter honom på golfvet, hon hängde sig fast vid hans knän och upprepade blott under strömmande tårar: bed mig! bed mig! Säg bara ett ord, att du vill det, säg bara, att utan mig ska du bli olycklig, ska du gå under. Säg, att du, liksom jag, hellre vill bära allt, än skiljas! Bed mig, Andrea! Jag kan inte — jag kan inte! upprepade han som utom sig, i det han sprang undan. Han fick fatt i sin hatt, störtade sig på dörren, ryckte upp den och sprang ut i natten. Då dörren stängdes om honom, kom det som en isande kyla öfver henne. Hon reste sig upp, stirrade i spegeln på sitt uppsvullna ansikte, ordnade sitt hår och begynte därefter att packa sin koffert. Hon skilde på sina och hans saker, hvilka i öm oreda lågo om hvarandra, hon räknade kallsinnigt sina egna näsdukar ifrån hans, läste namnet i de böcker, som voro hans och lade dem åt sidan, alltsammans under ett slags drömlikt tillstånd, som ginge hon i sömnen eller i yrsel. Hon hade ej något klart medvetande om hvad som förestod, blott en känsla af stingande, nästan olidlig smärta, någonstans, hon visste icke hvar, samt en andnöd, så att hon emellanåt tyckte sig skola kväfvas. Hon tog de sex i röda band
- 68. inbundna volymerna af Ariosto, som han gifvit henne, och ställde sig att läsa den sonett, han skrifvit till henne på första sidan. Hon läste den flera gånger och tyckte, att den var så underligt tom och meningslös. Därefter satte hon sig att skrifva en biljett till Rikard, däri hon förklarade sitt beslut att afresa med honom följande morgon med den postvagn, som gick kl. 7. Hon ringde och tillsade, att biljetten genast skulle bäras dit, samt att hon ville bli väckt kl. 6 och ha räkningen för sista veckan. Hon hade alltid fordrat att få betala sina egna utgifter. Sedan allt detta var omställdt, kofferten packad och stängd, handkappsäcken öppen för att hon på morgonen kunde lägga ned de sista effekterna, lade hon sig uttröttad och med en sådan känsla af absolut tomhet i hjärnan, att hon ej ens förmådde reflektera öfver det underliga i att Andrea dröjde så länge ute. Hon somnade genast i en tung, död sömn, med ljuset ännu brinnande på bordet, men vaknade efter ett par timmar och kom strax till fullt medvetande, samt for upp i bädden med stark hjärtklappning. Hon såg sig om i rummet och märkte, att Andrea varit inne och gått igen. Han hade tagit sin ytterrock, som förut låg på en stol, samt sitt cigarrettfodral, som hon ännu under packningen hade lagt framme på toalettbordet. Hon visste, att det var hans vanliga tillflykt, när han var upprörd — att spatsera och röka. Natten var kall, men månljus — hon såg det genom fönstren, som hon försummat att stänga för. Hon såg på klockan. Ännu blott 1. Först om sex timmar skulle hon kunna resa — detta tycktes henne nu en oändlighet. Hon kände nu samma otåliga längtan efter det, hon så länge fruktat värre än döden, som Dante så djupt träffande skildrar hos de fördömde vid deras första inträde i helvetet, då de skola stiga ned i Karons färja för att låta sig föras mot oerhörda kval: Chè la divina giustizia gli sprona Sì che la tema si volge in disio. (Ty den gudomliga rättvisan sargar dem så, att fruktan förvandlas i åstundan.)
- 69. Ja, hon längtade efter morgonen, hon längtade efter att sitta i vagnen och se för sista gången dessa drag, denna gestalt, som hon älskat så våldsamt, så vanvettigt, utan att dock kunna vinna det, på hvilket hon samlat all sin själs energi med en sådan intensitet, att det syntes henne som blödde hon däraf ur tusen sår — att vinna honom helt och för alltid. Han hade ej kunnat gifva henne det sista prof, som hon satt såsom villkor, som hon måste sätta som villkor för att knyta sig varaktigt till honom — han hade ej kunnat uttala det enda lilla ord, som skulle varit nog för att få henne att underkasta sig allt. Hon hade gifvit efter punkt för punkt af sina fordringar, hade vikit steg för steg — men i detta sista kunde hon ej vika. Han måste en gång vilja, klart och bestämdt vilja äga henne för alltid — hon hade förgäfves väntat därpå hela tiden, och när han ej heller nu kunde taga ett sådant beslut, så hade hon intet val. Han kom och gick ännu ett par gånger under natten; hvilade sig stundtals i soffan, men gick snart ut igen. Hon låg med slutna ögon och bultande pulsar, räknande klockans knäppningar tills hon somnade däraf, alltid för att åter vakna med en ångestfull känsla under bröstet, som skulle hon svimma eller kväfvas. Han hade sett den stängda kofferten, sina egna saker ordnade i en rad på en stol och förstått allt. Och en dof förbittring jäste inom honom mot henne, så stark, att han ej ens kunde förmå sig att närma sig henne. Å, hur hade hon kunnat bedraga honom så! Han hade trott, ja, han hade slutat med att tro, att han här skulle finna denna lifvets helhet och fullhet, som var hans sinnes djupaste kraf — trott på en kärlek, höjd öfver hvarje prof — och där var hon nu färdig att svika honom så gränslöst. Han gick omkring med sjudande hämndtankar i sitt sinne. Denne Rikard, denne tarflige moralist och kälkborgare med sina äckliga, banala fraser — han skulle velat gå upp på rummet, där han sof, och gifva honom ett knytnäfsslag i ansiktet och sedan slåss med honom på lif och död. Och hon, som låg där och sof helt lugnt, sedan hon med en sådan ordning och metod räknat hans näsdukar och tagit vara på sina egna — ja, han skulle vilja döda äfven henne. Hvarför inte. Inte göra som det där andra paret — bara helt enkelt döda henne.
- 70. Han stannade nu slutligen bredvid hennes säng och såg på henne. Hon slumrade lätt men oroligt samt andades flämtande. Han hade många gånger förr gladt sig åt, hur vacker hon var, när hon sof. Äfven nu lade sömnen ett visst skimmer af rodnad öfver hennes ansikte. Hon låg på sidan, med ena handen under kinden, den andra utbredd på täcket. Håret bucklade sig mjukt kring tinningarna och utefter ryggen, läpparna voro halföppna och hade ännu i sömnen en sakta skälfning som i smärta — äfven ögonlocken ryckte något — men den profil, som aftecknade sig mot kudden, var så beundransvärdt ren och fin, så själfullt känslig och mjuk, att Andrea öfverväldigades af rörelse och kastade sig öfver henne med en omfamning, som nästan var en kväfning. Hon for upp och stirrade honom i ansiktet med den yrvaknas förskräckta blick. Han grep med båda händerna hårdt om hennes hals och höll henne så. Hon kom till besinning, läste som ett begynnande vansinne i hans ansikte, kastade sig tillbaka mot kuddarna, slöt ögonen och sade med svag, flämtande stämma, men ett lyckligt leende: Ja, ja! döda mig! Gör det! Jag ber dig — döda mig! Hans händer sjönko långsamt tillbaka och den onaturliga spänningen i hans drag gaf vika, i det tårar strömmade till hans ögon. Då kastade hon armarna om hans hals och hängde sig fast vid honom med hufvudet tillbakakastadt, kramade sitt bröst intill hans och bad ifrigt, inträngande, med fullt medvetande: Döda mig! Eller låt oss dö tillsammans — som de andra! Så mycket älskar du mig? frågade han, och det började sprida sig ett skimmer af lycka öfver hans ansikte. Men hvarför vill du då lämna mig? Jag vill ju inte lämna dig — jag kan det inte. Därför vill jag dö. Och hvem tvingar dig att lämna mig? Du. Jag! som bara väntat, bara hoppats på denna stund — som kände att hela min tro på lifvet, den enda möjligheten att bli botad från en skepticism, som dödade mig, var om du kunde ge mig detta yttersta prof — nu vet jag det säkert, du ska alltid hålla mig fast. Nu kan vi
- 71. gärna trotsa allt och gifta oss — du har gifvit mig den kraft, som fattades mig, nu kan jag strida, nu kan jag arbeta för dig. Du ska bara fordra det af mig, så går det. Hon kramade armarna hårdt omkring honom och snyftade ännu länge mot hans hals. Men det var inte längre tårar af förtviflan, utan af en lycka, som hon kände vara allt för stor för att kunna vara varaktig. Hon visste väl, att hon i denna stund invigde sig till ett lif af oafbruten kamp, och det var ångest och bäfvan i den sällhet, hvarmed hon gick framtiden till mötes. Hon visste, att den fullkomliga lyckan endast existerar i ett moment och att den alltid köpes dyrt.
- 73. Transcriber’s note: Källa: Anne-Charlotte Leffler: Ur Lifvet. Femte Samlingen., Z. Hæggströms Förlags-Expedition, Stockholm 1890, pp. 1-232. Originalets stavning och interpunktion har bibehållits. Titelns »kvinlighet» blev i andra delen stavat »kvinnlighet». Båda delar har därför registrerats under samma titel, »Kvinnlighet och erotik», men respektive originalets stavning har bibehållits i respektive individuell text. Uppenbarliga fel har rättats (innan/efter): ... rum och den sistnämde ordnade soffan åt henne samt ... ... rum och den sistnämnde ordnade soffan åt henne samt ... ... nu också Agot — å, hvad de voro löjliga! Men hon ... ... nu också Aagot — å, hvad de voro löjliga! Men hon ... ... armarna på Agot. ... ... armarna på Aagot. ... ... öppna balkongdörrarna utåt stjärhimmeln, som framskymtade ... ... öppna balkongdörrarna utåt stjärnhimmeln, som framskymtade ... ... kom, som meddelade henne att kan ej kunde komma ... ... kom, som meddelade henne att han ej kunde komma ... ... lämna dörren öppen? Nej, det var bättre att läsa ... ... lämna dörren öppen? Nej, det var bättre att låsa ... ... här resan, som kunde vara bätte att inte ha tänkt ... ... här resan, som kunde vara bättre att inte ha tänkt ... ... mörket, så hade hon här ingen annan utväg än att ... ... mörkret, så hade hon här ingen annan utväg än att ... ... Stigen fortsatte ju där långs strömmen, ... ... Stigen fortsatte ju där längs strömmen, ... ... Då de åter blefvo ensamma och dörren var läst, ... ... Då de åter blefvo ensamma och dörren var låst, ...
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