characterization and comparison of the arginine vasotocin...
TRANSCRIPT
Universidade de Lisboa
Faculdade de Ciências
Departamento de Biologia Animal
Characterization and comparison of the arginine
vasotocin (AVT) neurons in the brain of
phylogenetically close wrasses, with different levels of
cooperative behaviour.
Ana Rute Martins de Mendonça
Mestrado em Ecologia Marinha
2012
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Universidade de Lisboa
Faculdade de Ciências
Departamento de Biologia Animal
Characterization and comparison of the arginine
vasotocin (AVT) neurons in the brain of
phylogenetically close wrasses, with different levels of
cooperative behaviour.
Dissertação orientada por: Doutora Marta C. Soares (ISPA) e
Professor Doutor Paulo Fonseca (FCUL)
Ana Rute Martins de Mendonça
Mestrado em Ecologia Marinha
2012
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Acknowledgements
To those who, somehow, helped throughout the production of this thesis, I would like to
show my gratitude:
Dr. Marta Soares for accepting me as her student, supervising my work, giving such
valuable comments and trusting me;
Professor Dr. Rui Oliveira, for all the insight, vital help and attention given when I
mostly needed, and for trusting me and my work;
Professor Dr. Paulo Fonseca for accepting to be my co-supervisor and for all the support
given during the process;
Professor Dr. Eduardo Barata for giving me the opportunity of working with him in the
past and for being a source of inspiration since then;
Dr. Matthew Grober for kindly providing the anti-AVT antibody and patiently helping
during the ICC protocol troubleshooting;
Dr. Jim Goodson, for the productive talk we had once and the advises that made the
difference for an optimal staining with the ICC protocol;
All the IBBG team, specially Ana Isa Fialho, Ana Isabel Faustino, Catarina Bacelar, and
Sara Cardoso, for helping me keep my head up and with whom I had the opportunity to
develop lovely friendships; Ana Sofia for always having a word of encouragement;
Gonçalo Oliveira, for the tips and great help with the statistics; Magda Teles for the
initial push and all those that followed (not enough words for all the help, a thousand
thanks); Miguel Simões for the knowledge, help and laughing moments; Olinda
Almeida my AVT partner, for all the help throughout the last many months; Silvia
Costa, for all the encouragements;
The Cleaner Wrasse Team, which I had the pleasure to meet at Lizard Island: Albert
Ros, Alizée Derendinger, Ana Pinto, Nichola Raihani, Philippe Vullioud, Sharon
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Wismer and Tim Lyons, for the new friendships, making me grow and all the fun.
Special thanks to Philippe, my buddy, for the immeasurable help, road trips, nice
scientific discussions, comments on my writing, support, laughs, and so on...;
All the staff from LIRS for all the patience, help and kindness: Anne Hoggett and Lyle
Vail, Tania and Bob Lamb and Marianne and Lance Pearse;
All the other friends that are part of this thesis, specially Ana Leitão, for being my
forever co-worker with who I will always learn something more and final comments on
my writing; Carolina Martins, for being around since ever (for what I remember) and
João Cardoso for the support and understanding;
Finally, I don’t even have enough words to thank my family and, as they are the ones
who have highly supported me, I would like to dedicate them all the effort spent on this
work (Mãe e Pai, mil obrigados por tudo o que me têm proporcionado, pela paciência
com os meus maus humores e principalmente pela família linda que criaram; Avó
Guida, a tua bondade e capacidade de ajuda são e serão sempre inspiradoras para mim,
obrigada!; Raquel e Maria, não me imagino sem vocês, são as minhas luzinhas,
obrigada por cafés e chocolates, risadas, birras e amuos… e tudo e tudo e tudo! Família,
gosto-vos por tudo o que são!).
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Resumo
Cooperação é um dos mais intrigantes tipos de interações sociais presentes na natureza,
pois sai fora do contexto de contante competição e típico comportamento egoísta entre
indivíduos, proposto por Charles Darwin na sua teoria da Seleção Natural. A evolução e
manutenção de cooperação têm sido considerados autênticos puzzles evolucionários
devido à inerente dificuldade em explicar por que razão indivíduos agem em benefício
de outros.
O termo cooperação é usado para definir interações entre dois ou mais indivíduos cujos
benefícios da interação suplantam os custos (investimentos). Este tipo de interações são
denominadas por “mutualismos”, quando ocorrem entre indivíduos de espécies
diferentes. Os mutualismos de limpeza marinhos estão entre os mais estudados, e a
associação entre o peixe limpador obrigatório Labroides dimidiatus e os seus peixe-
clientes é um exemplo típico. Estes sistemas sociais de natureza interespecífica
implicam a existência de pelo menos um indivíduo limpador (normalmente de tamanho
mais pequeno) que inspeciona o corpo, boca e cavidades branquiais de outro indivíduo
(denominado cliente) em busca de ectoparasitas, muco, tecido morto e escamas (dos
quais se alimenta).
Embora muito se saiba sobre os mecanismos comportamentais que descrevem e testam
a existência e manutenção de interações cooperativas e mutualísticas, pouco trabalho
tem sido feito para compreender os mecanismos fisiológicos subjacentes a estas
relações sociais. Recentemente foi proposto que o comportamento social é controlado
por processos endócrinos, segundo os quais os esteroides sexuais (testosterona,
progesterona e estradiol), esteroides do stress (como o cortisol) e alguns candidatos
peptídicos, tais como os neuropéptidos da família da vasopressina (AVP)/oxitocina
(OT), desempenham papéis cruciais.
O sistema nervoso central é um importante coordenador das relações entre o animal, o
ambiente que o rodeia e o comportamento que expressa numa dada situação. Foi
recentemente proposta a existência de uma rede neural subjacente à expressão de
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comportamentos sociais (“social behaviour network”; SBN). Esta é uma rede de áreas
cerebrais importante na regulação da expressão de comportamentos sociais. Os
neuropéptidos (péptidos produzidos no cérebro com capacidade de influenciar outros
neurónios) são capazes de alterar a resposta desta rede neuronal e consequentemente a
expressão comportamental. Assim, estas moléculas são potenciais agentes moduladores
dos comportamentos expressos no decorrer de interações mutualísticas, através de ações
na SBN. O neuropéptido arginina vasotocina (AVT) e o seu homólogo arginina
vasopressina (AVP; nos mamíferos) têm sido implicados numa grande variedade de
comportamentos sociais em vertebrados, incluindo peixes, anfíbios, répteis, aves e
mamíferos.
Em teleósteos, a AVT é produzida em neurónios neurosecretores da área pré-óptica
(POA), que se divide em três grupos neuronais: parvocelular (pPOA), magnocelular
(mPOA) e gigantocelular (gPOA). Os neurónios da pPOA projetam maioritariamente
para a pituitária (onde os neuropéptidos são armazenados antes de serem transportados
para a periferia através do sistema circulatório), enquanto os neurónios da gPOA
projetam sobretudo para outras áreas do cérebro (como por exemplo as incluídas na
SBN). Os neurónios da mPOA projetam para a pituitária e também para outras áreas
cerebrais.
A maioria dos estudos sobre os efeitos da AVT em comportamentos sociais tem-se
focado em interações sociais intraespecíficas. Recentemente, foi sugerido que este
neuropéptido tem também um efeito importante na modulação de comportamentos
sociais a nível interespecífico, no sistema mutualista que inclui o peixe limpador L.
dimidiatus. De facto, um trabalho recente mostrou que injeções de AVT diminuem a
probabilidade destes peixes iniciarem interações mutualísticas, enquanto injeções de um
antagonista tem efeitos inversos.
No seguimento do estudo acima mencionado, o presente trabalho tem como principal
objetivo caracterizar e comparar o fenótipo neuronal da AVT em duas espécies
filogeneticamente próximas, que divergem na expressão de comportamentos
mutualísticos: um limpador obrigatório (L. dimidiatus) e um não-limpador (Labrichthys
unilineatus). As duas espécies vivem em sistema de harém, sendo o macho o maior
indivíduo do grupo social, que possui o território. Os limpadores L. dimidiatus
dependem das interações mutualísticas para sobreviver, pois obtêm alimento
exclusivamente a limpar os seus clientes. O não-limpador L. unilineatus, não apresenta
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qualquer comportamento de limpeza durante todo o seu ciclo de vida, alimentando-se
exclusivamente do muco produzido pelos pólipos dos corais presentes no seu território.
Adicionalmente, pretende-se identificar possíveis diferenças relacionadas com o sexo
dos indivíduos.
Para tal, foi usada uma técnica de imunocitoquímica no sentido de detetar
imunorreatividade à AVT em secções de tecido cerebral. As células imunorreativas
foram contadas e medidas em cada um dos grupos neuronais (pPOA, mPOA e gPOA) e
foram feitas comparações entre espécies e entre sexos para cada espécie.
As duas espécies analisadas apresentaram neurónios AVT-imunorreativos (-ir)
limitados à área pré-óptica, o que está de acordo com outros estudos que mostram que
este é o principal local de produção de AVT, em teleósteos. Os neurónios
parvocelulares, que começam a surgir acima do quiasma óptico, são os mais anteriores e
ventrais, e são constituídos por células pequenas e redondas, geralmente monopolares
ou sem neurites óbvias. Este grupo celular estende-se para uma posição dorso-caudal,
até à região onde começam a aparecer os primeiros neurónios magnocelulares. Estes
ocupam uma localização medial na POA, têm aproximadamente o dobro da área dos
neurónios parvocelulares e geralmente apresentam um axónio evidente que se estende
em direção ao trato pré-óptico-hipofisial. Os neurónios gigantocelulares começam a
aparecer numa posição dorsal relativamente aos magnocelulares e estendem-se
caudalmente. Estas são as maiores células, apresentam geralmente várias neurites
evidentes. Este padrão de distribuição está de acordo com a proposta conservação
evolucionária das características neuroanatómicas do sistema AVT em teleósteos.
Os resultados quantitativos mostram que a espécie limpadora tem menos e menores
células AVT-ir relativamente à não-limpadora, e que estas diferenças são apenas
significativas no grupo celular gPOA. Muitos outros estudos têm encontrado diferenças
quantitativas a nível intra- e interespecífico neste grupo celular. As células AVT-ir do
grupo gPOA têm sido particularmente associadas à expressão de comportamentos
agressivos, sendo que espécies com maiores ou mais células, ou com maiores níveis de
produção de mRNA nesta área, tendem a ser mais agressivas. Este efeito é
provavelmente mediado pelas projeções destas células para zonas do cérebro onde
ocorre a modulação dos comportamentos sociais. Adicionalmente, foi também
demonstrado que injeções de AVT na POA induzem o afastamento social.
Possivelmente, a evolução favoreceu neurónios gigantocelulares AVT-ir mais pequenos
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e em menor quantidade, consequentemente diminuindo a expressão de potenciais
comportamentos agressivos e afastamento social interespecíficos, favorecendo assim o
desenvolvimento de interações mutualísticas na espécie limpadora.
A espécie não limpadora apresentou ainda uma maior densidade de fibras
imunorreativas em volta dos neurónios gPOA, relativamente à espécie limpadora, que
embora apresente algumas fibras estas ocorrem em menor densidade. Possivelmente, a
elevada densidade de fibras observada na espécie não limpadora está relacionada com
uma maior necessidade destas células comunicarem entre si (através de comunicação
dendrítica), de forma a sincronizarem a libertação axonal de péptidos noutras áreas do
cérebro. Isto pode ser particularmente importante em L. unilineatus para modular a
expressão de comportamentos agressivos. No entanto, a partir das secções de tecido
usadas neste trabalho, não é possível determinar se as fibras encontradas são extensões
dendríticas dos corpos celulares dos neurónios gPOA. Assim, esta sugestão deve ser
tomada com alguma precaução e trabalhos futuros deverão usar secções de tecido mais
finas para verificar esta situação.
O número e tamanho dos neurónios nos outros dois grupos celulares não foi
significativamente diferente entre as duas espécies. Os neurónios pPOA e mPOA
projetam para a pituitária, onde têm a capacidade de estimular a secreção de ACTH e
consequentemente modular a reatividade do eixo hipotálamo-pituitária-interrenal (HPI).
Assim, a falta de diferenças nestes grupos celulares entre as duas espécies pode sugerir
que: a) os animais não se encontravam sob stress, e a falta de diferenças pode refletir
processos fisiológicos semelhantes às duas espécies; b) caso os animais estivessem sob
stress, as respostas fisiológicas não foram diferentes entre as duas espécies; c) o
confinamento a que os animais estiveram sujeitos durante o transporte representou um
stress crónico, causando habituação do sistema AVT nas duas espécies. As duas últimas
hipóteses apresentadas são as mais prováveis pois o confinamento espacial é uma fonte
de stress fisiológico.
Adicionalmente às projeções para a pituitária, o grupo neuronal mPOA também projeta
para outras áreas cerebrais capazes de modular comportamentos sociais. A falta de
diferenças entre as duas espécies neste grupo celular vem enfatizar a ideia de que a
expressão de comportamentos mutualísticos poderá estar associada às projeções centrais
das células AVT-ir da área gPOA.
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Ao contrário do esperado, não foram encontradas diferenças no número ou tamanho dos
neurónios AVT-ir entre sexos, em ambas as espécies. Diferenças entre sexos eram
esperadas com base em diferenças comportamentais entre machos e fêmeas. Em L.
dimidiatus sabe-se que os machos apresentam maiores níveis de agressividade
intraespecífica, que é dirigida às fêmeas quando estas são desonestas com os seus
clientes (i.e. quando elas ingerem muco em vez de ectoparasitas). Em L. unilineatus, os
machos patrulham e defendem o seu território contra possíveis machos competidores.
Muitos trabalhos têm associado diferenças entre sexos nas características das células
AVT-ir a esteroides sexuais, principalmente androgénios. No entanto, existem outros
estudos que também não encontraram diferenças significativas entre sexos. A falta de
diferenças entre machos e fêmeas pode refletir uma falta de influência dos androgénios
no sistema AVT, nas duas espécies usadas no presente estudo. No entanto o presente
estudo não foi desenhado para responder a questões sobre a influência de outras
hormonas no sistema AVT, pelo que experiências futuras devem testar a hipótese
proposta de uma forma mais fidedigna.
Em conclusão, este estudo mostra, pela primeira vez, diferenças no fenótipo neuronal da
AVT entre duas espécies filogeneticamente próximas que diferem na expressão de
comportamentos mutualísticos. A espécie mutualista Labroides dimidiatus apresenta
menos e menores neurónios gPOA, em relação à espécie não mutualista. Neste trabalho
eu sugiro que os mais baixos níveis de AVT na espécie limpadora (inferidos pelo menor
tamanho e número de neurónios AVT-ir) desempenham um papel importante na
expressão de comportamentos mutualísticos, possivelmente modulando a predisposição
para aproximação, interação e cooperação com parceiros sociais interespecíficos.
Adicionalmente, o grupo neuronal gPOA e as suas projeções extra-hipotalâmicas
aparentam ser responsáveis pela expressão dos elevados níveis de pro-socialidade
interespecífica típicos do peixe limpador. Embora a maioria dos estudos sobre a relação
do sistema AVT e comportamentos sociais se concentrem em relações intraespecíficas,
o presente estudo fornece evidências de que este sistema desempenha também um papel
importante na modulação de comportamentos sociais entre espécies diferentes, e
concretamente comportamentos mutualísticos.
Palavras-chave: Mutualismos de limpeza; Cooperação; Arginina vasotocina; Área pré-
óptica; Labroides dimidiatus; Labrichthys unilineatus.
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Summary
Mutualistic interactions are particular social interactions where individuals of different
species cooperate in order to gain benefits. A textbook example of mutualism is
provided by the bluestreak cleaner wrasse (Labroides dimidiatus), which feeds on
clients’ ectoparasites, mucus, scales and dead or infected tissue. Although much is
known about the ultimate functions of cooperative behaviour, studies on their
underlying proximate mechanisms are scarce. The nonapeptides arginine vasotocin
(AVT) and its mammalian homologue, are well known for their function in the
modulation of several conspecific social behaviours. Recently, AVT was also shown to
play an important role in the modulation of interspecific cooperative behaviours in the
cleaner wrasse, as AVT injections decreased the cleaners’ willingness to engage in
cleaning interactions. In this study, I aimed to characterize and compare the AVT
neuronal phenotypes of two closely related species that live in similar environments, but
diverge in the expression of interspecific cooperative behaviours: an obligate cleaner (L.
dimidiatus) and a non-cleaner, corallivore labrid (Labrichthys unilineatus).
Additionally, I aimed to identify sex differences in AVT neuronal phenotypes, as they
might be responsible for the expression of sex-related social behaviours. Species
differences were restricted to the gPOA (known to have extrahypothalamic projections).
Cleaners had smaller and less numerous AVT immunoreactive (-ir) neurons, compared
to the non-cleaners. I propose that this neuronal preoptic group plays an important role
in the modulation on interspecific cooperative behaviours. Also, I suggest that smaller
and less numerous neurons projecting to extrahypothalamic brain areas, might have
been selected to lessen the expression of potential interspecific aggressive behaviours
and interspecific social withdrawal, thus facilitating the evolution and maintenance of
mutualistic interactions. No intersexual differences were found for both species which
might reflect a lack of androgen interaction with the AVT system. Alternatively, the
lack of sex differences might reflect that both males and females engage in interspecific
cooperative relationships, and the expressed mutualistic behaviours do not differ
between sexes.
Keywords: Cleaning mutualism; Cooperation; Arginine vasotocin; Preoptic area;
Labroides dimidiatus; Labrichthys unilineatus.
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Table of contents
Acknowledgements 4
Resumo 6
Summary 12
List of Abbreviations 16
1. Introduction 18
1.1. Cooperation and mutualisms as complex social interactions 18
1.2. Marine cleaning mutualisms 19
1.3. Proximate mechanisms underlying mutualistic interactions 20
1.4. Arginine vasotocin/vasopressin system and physiological roles 21
1.5. Behavioural roles of AVT/AVP 22
1.5.1 Aggressive behaviour and social dominance 22
1.5.2. Sexual behaviour 23
1.6. Is there a role of AVT in the modulation of cleaning mutualisms? 24
1.7. The model species: a comparative approach 25
1.8. Objectives and predictions 26
2. Materials and Methods 28
2.1. Subjects 28
2.2. Tissue Preparation 29
2.3. Immunocytochemistry 30
2.4. Quantification 31
2.5. Statistical analysis 33
3. Results 36
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3.1. Qualitative characterization and distribution of preoptic AVT-ir cells in L.
dimidiatus and L. unilineatus 36
3.2. Quantitative comparisons of preoptic AVT-ir cell size 38
3.3. Quantitative differences of preoptic AVT-ir cell number 39
4. Discussion 42
4.1. The distribution of AVT-ir neurons 42
4.2. Species differences in AVT-ir neurons 42
4.3. Sex differences in AVT-ir neurons 46
4.4. Final comments 47
5. References 50
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List of Abbreviations
ACTH – adrenocorticotropin hormone
AH – hypothalamus
AVP – arginine vasopressin
AVT – arginine vasotocin
BNST - bed nucleus of stria terminalis
BNSTm – medial amygdala and the
media bed nucleus of stria terminalis
DAB – diaminobenzidine
gPOA – gigantocellular preoptic area
HPI – hypothalamus-pituitary-interrenal
ir - immunoreactive
IT - isotocin
LS – lateral septum
mAMY- medial amygdala
mPOA – magnocellular preoptic area
OT – oxytocin
PB - phosphate buffer
PBS - phosphate buffer saline
PFA - paraformaldehyde
PHT – preopticohypophyseal tract
POA – preoptic area
pPOA – parvocellular preoptic area
PVN – periventricular nucleus of the
hypothalamus
SBN – social behaviour network
SCN – suprachiasmatic nucleus
SON – supraoptic nucleus
VMH – ventromedial hypothalamus
Vs - supracommisural nucleus of the
ventral telencephalon
Vv – ventral nucleus of the ventral
telencephalon
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1. Introduction
Beyond the necessity of interacting with the environment, animals must interact with
others at some point of their lives. Social interactions, have been intensively studied as
they represent important ecological traits, and imply the communication between
individuals, with fitness consequences for all involved parties (West et al., 2007b).
1.1. Cooperation and mutualisms as complex social interactions
Cooperation is one of the most intriguing and interesting types of social interactions, as
it falls out of the context of constant competition, fight and ‘struggle for life’ between
individuals, suggested by Charles Darwin (Noë, 2006). The existence of cooperation
remained for a long time puzzling from an evolutionary point of view, as it was difficult
to explain why animals should behave to benefit of others (Sachs et al., 2004). The term
cooperation is largely used to define interactions between two or more individuals with
positive outcome (inclusive fitness) for all of them, i.e. the benefits of interacting
outweigh its costs (investments) (Bshary & Bergmüller, 2008). When this type of
interactions occur amongst individuals of different species, they are often termed
mutualistic interactions (Bshary & Bergmüller, 2008). Both cooperative and mutualistic
relationships can be maintained stable as long as all the partners involved can profit
(Komdeur, 2006).
Many behavioural mechanisms have been studied to explain how cooperation and
mutualisms evolved and are maintained in natural populations. Such mechanisms can be
divided into two categories: those that bring indirect fitness benefits (gained from aiding
related individuals) and those that bring direct fitness benefits to the actor (gained by
producig offspring; West et al., 2007a). The most interesting mechanisms are those
trying to explain the evolution of cooperation between non-related individuals, such as
reciprocation. Reciprocity can be direct (an individual A reciprocates benefits back to a
previous investor B) or indirect (an individual A invests on an individual B, and
receives benefits from an individual C, based on A’s reputation).
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Many other mechanisms responsible for the evolution of cooperative interactions have
been proposed, each based in different initial assumptions, characteristic of the wide
variety of biological systems expressing cooperation (for reviews on this and other
behavioural mechanisms see Nowak, 2006; Sachs et al., 2004; West et al., 2007a; West
et al., 2007b).
1.2. Marine cleaning mutualisms
Mutualistic relationships can be found all over the world in all habitats and a variety of
life forms can be involved, including bacteria, fungi, algae, plants and animals (Sachs et
al., 2004). Marine mutualisms, such as the association between the Indo-pacific blue-
streak cleaner wrasse (Labroides dimidiatus) and its fish clients, is a textbook example
of mutualistic cooperation (e.g. Trivers, 1971).
Cleaners are usually small animals (such as shrimps or small fish) that inspect the body
surface, mouth and gills of other larger fish (the clients) searching for ectoparasites and
dead tissue (Cote, 2000; Grutter, 1999). Their existence can therefore affect fish
communities by improving their growth and survival (Waldie et al., 2011). Clients can
visit cleaning stations (i.e. the cleaners’ established territories) several times per day,
and cleaners can have over two thousand interactions in a day, eating up to 1200
parasites per day (Grutter, 1995).
At first hand, it looks like a fair system where both parties win: cleaners gain an easy
meal and clients get rid of ectoparasites. Nonetheless, this type of system can be
affected by exploitation. In the L. dimidiatus system, cleaners are dishonest (cheat) by
ingesting mucus and scales (which they prefer) from its clients, that are costly for the
clients to replace (Grutter & Bshary, 2003; Grutter & Bshary, 2004). Therefore, clients
need to control dishonest behaviour by punishing the cleaner (usually with aggressive
chases) (Bshary & Grutter, 2002). It’s also frequent to see cleaner males punishing
females after a dishonest behaviour of the later (Raihani et al., 2010). Such aggressive
events keep the females cooperative and less likely to be dishonest in the following
interactions (Raihani et al., 2012).
Although much is known about the ecology and behaviour of cooperative/mutualistic
interactions, and particularly about the L. dimidiatus system, little work has been
20
conducted in order to unravel the proximate mechanisms responsible for cooperative
behaviours.
1.3. Proximate mechanisms underlying mutualistic interactions
Recently, the physiological aspects underpinning mutualistic interactions have started to
receive attention, specially the health benefits for clients in the form of stress reduction.
For example, Bshary and colleagues (2007) have first found an association between the
presence of cleaner organisms (cleaner shrimps or cleaner wrasses) and reduced stress
in client fish, as measured by cortisol levels. Additionally, RU486 (a glucocorticoid
receptor antagonist) decreases clients’ motivation to seek out cleaning interactions,
without affecting conspecific interactions (Ros et al., 2012). This authors then suggest
that higher levels of cortisol, as a consequence of ectoparasite infection (Fast et al.,
2006), increases the clients’ propensity to interact with cleaners in order to lessen
parasite load. Therefore, stress might be a proximate mechanism that promotes
cooperative interactions (Ros et al., 2012).
Additionally, in a recent review, Soares and colleagues (2010) proposed a
neuroendocrine control of cooperative/mutualistic behaviours, where stress steroids
(cortisol), sex steroids (testosterone, oestradiol and progesterone) and the neuropeptides
from the vasopressin (AVP)/oxytocin (OT) family might play important roles.
The central nervous system is a key link and coordinator of the interactions between the
animal, the surrounding environment and the behaviour expressed in a given situation
(Meek & Nieuwenhuys, 1998). In fact, it has been proposed a brain network capable of
regulating the expression of social behaviours: the brain social behaviour network -
SBN (Newman, 1999). This network, first proposed for mammals by Newman (1999),
comprises six major nodes that have been shown to control several social behaviours
and have bidirectional connections: the extended medial amygdala (medial amygdale,
mAMY, and the medial bed nucleus of stria terminalis, BNSTm), the lateral septum
(LS), the preoptic area (POA), the anterior hypothalamus (AH), the ventromedial
hypothalamus (VMH) and the midbrain (Newman, 1999). There is now growing
evidence from studies on fish and birds, suggesting that the SBN must have arisen
earlier in the vertebrate evolution (Goodson & Bass, 2002; Goodson, 2005). In bony
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fish six main brain nodes have been identified to be homologues of those of mammals
and to have large implications in the regulation of social behaviours: the
supracommisural nucleus of the ventral telencephalon (Vs; amygdala homologue);
POA; AH; ventral nucleus of the ventral telencephalon (Vv, septum homologue);
anterior tuberal nucleus (at least part of this nucleus is homologous to the VMH) and
midbrain (Goodson, 2005).
Neuropeptides, such as those belonging to the AVP/OT family, are capable of altering
this network response and consequently the behavioural output (Goodson, 2005),
making them great candidates to modulate the behaviours expressed during
cooperative/mutualistic interactions through actions in the SBN.
1.4. Arginine vasotocin/vasopressin system and physiological roles
The term neuropeptide refers to peptides produced in brain neurons that have the
capability to influence other neurons (Strand, 1999). The neuropeptide arginine
vasotocin (AVT) and its mammalian homologue AVP, belong to the nine amino-acid
peptide family (nonapeptides), where isotocin (IT) and its mammalian homologue OT
are also included (Strand, 1999).
Arginine vasotocin/vasopressin are mainly produced in the brain and have been
implicated in a variety of physiological and behavioural functions in fish, amphibians,
reptiles, birds and mammals (e.g. Almeida et al., 2012; Bradshaw et al., 2007; Moore &
Lowry, 1998; Rose et al., 1995; Srivastava et al., 2010).
In teleosts, AVT is produced in neurosecretory neurons of the preoptic area - anterior
hypothalamus, that are known to project both to the posterior pituitary (where it is
stored before delivery to the periphery, via bloodstream) and other brain regions (Butler
& Hodos, 2005; Holmgvist & Ekström, 1995). The teleost preoptic area (POA) is
commonly divided in three neuronal groups: parvocellular (pPOA), magnocellular
(mPOA) and gigantocellular (gPOA).
Vertebrate evolution seems to have been accompanied by an expansion of this system,
resulting in additional cell groups responsible to produce these neuropeptides in higher
vertebrates (Balment et al., 1993; Goodson & Bass, 2001; Moore & Lowry, 1998). In
22
mammals, the major production of AVP comes from the supraoptic nucleus (SON;
homologue of teleost mPOA and gPOA) and periventricular nucleus of the
hypothalamus (PVN; homologue of pPOA of teleosts). Other production sites of the
mammalian brain include the suprachiasmatic nucleus (SCN), mAMY and BNST
(Carter et al., 2009).
Physiological processes where AVT/AVP have been largely implicated include
cardiovascular reflexes, metabolism and osmorregulation (Balment et al., 2006; Strand,
1999; Warne & Balment, 1997). The regulation of the osmotic balance is probably the
best studied physiological function of this neuropeptide (Balment et al., 1993; see
Balment et al., 2006 for review) and in teleosts this is extremely important for species
exposed to salinity variations. Indeed, AVT secretion from the pituitary has an
important role in fish osmotic balance through actions in the kidney to regulate urine
production and water retention (Balment et al., 1993; Perrott et al., 1991).
1.5. Behavioural roles of AVT/AVP
Insel and Young (2000) refer to neuropeptide systems as particularly suitable for the
regulation of behaviour due to their neuromodulatory functions, discrete location in
neural pathways and plasticity. The AVT/AVP systems have been deeply studied in
relation to its influence in the modulation of social behaviours, and the majority of the
studies has been carried out in a sexual/reproductive context (e.g. Goodson & Bass,
2001; but see Perrone et al., 2010; and also Thompson & Walton, 2004). In teleosts, the
main focus has been the role of AVT on aggression, social status (dominance vs.
subordinance) and sexual behaviour (see Godwin & Thompson, 2012 for review).
1.5.1 Aggressive behaviour and social dominance
The relationship between AVT/AVP and aggressive behaviours has been referred to be
species-specific (Goodson, 2008). Although, some recent studies suggest that these
differences may also be related to each species’ social system (e.g. Dewan et al., 2008;
Dewan et al., 2011). In territorial species AVT seems to cause a decrease in aggression
levels whereas in the colonial species the effects appear to be contrary, increasing the
23
expression of aggressive behaviours (e.g. Backström & Winberg, 2009; Lema & Nevitt,
2004a; Lema, 2006).
Nevertheless, a study carried out in a wild population of the territorial damselfish
(Stegastes leucosticus) contradicted the results described above, as intramuscular
injections of AVT were able to produce an increase in the levels of aggression
(Santangelo & Bass, 2006). Recently, it has been proposed that the assumed species-
specific effects of AVT in aggression may be a result of social context and might be
specific of each social phenotype, as different patterns of AVT release from the various
cell groups can be responsible for distinct behavioural patterns within and between
species (Goodson et al., 2009). Indeed, Goodson and colleagues (2009) showed that
AVT can produce opposite effects in the territorial estrildid finch Uraeginthus
granatina, depending on the social context and the social phenotype or status of the
individuals used.
Social dominance and aggressive behaviours are tightly linked, and thus some studies
have also associated AVT with individual social status. In zebrafish (Danio rerio) the
neuronal phenotype is markedly different between dominant and subordinate males
(Larson et al., 2006). Dominant individuals only show cells in the magnocellular
component of the preoptic area (in this study includes both mPOA and gPOA). On the
other hand, subordinate individuals only have pPOA cells (Larson et al., 2006).
In fact, cellular characteristics of mPOA and gPOA neuronal groups have been linked to
the expression of aggressive behaviours, through their projections to areas belonging to
the SBN (Dewan et al., 2011; Dewan & Tricas, 2011). On the contrary, pPOA neurons
have been suggested to play an important role both on subordinate behaviour and stress
modulation (Backström et al., 2011; Gilchriest et al., 2000; Pavlidis et al., 2011),
through stimulation of adrenocorticotropin hormone (ACTH) release and subsequent
activation of the hypothalamus-pituitary-interrenal (HPI) axis (Balment et al., 2006).
1.5.2. Sexual behaviour
AVT is also known for its effects on the expression of sexual behaviour. For instance, in
the peacock blenny Salaria pavo, nest holder males are courted by females and sneaker
males, which attempt to gain access to nests and sneak fertilizations (Almada, 1995). In
24
this species AVT production levels are correlated with the production of courtship
behaviour as both females and sneakers have higher expressions of AVT mRNA in the
POA (Grober et al., 2002). In the social system of the tropical bluehead wrasse
Thalassoma bifasciatum there are two types of brightly coloured terminal phase (TP)
males: territorial males (T-TP) own a spawning site, court females, display high levels
of aggression toward other males and do not feed; and the non-territorial males (NT-TP)
that do not own a spawning site, show low levels of aggressive and courtship behaviour
and feed on corals (Semsar et al., 2001). When injected with AVT, NT-TP males
increase their display of both courtship and aggressive behaviours, to similar levels of
territorial males. On the other hand, T-TP when injected with a AVT V1 receptor
antagonist (Manning compound) showed a decrease in courtship and aggression,
abandoning their spawning sites to feed (Semsar et al., 2001). In addition, females and
initial phase males (which are very similar to females in appearance and don’t court or
defend territories) did not show any response to AVT treatments (Semsar & Godwin,
2004).
These experiments show that, in fact, AVT is of major importance in the expression of
relevant social behaviours.
1.6. Is there a role of AVT in the modulation of cleaning mutualisms?
So far, most studies linking the AVT/AVP system to social behaviour has been done in
a purely intraspecific and reproductive context. However, little is known about the
physiological mechanisms underpinning cooperative interspecific and conspecific
interactions. Recent evidence shows that the AVT pathways might play an important
role in the regulation of interspecific cooperative behaviours (cleaning behaviour) and
conspecific social behaviours among cleaner wrasse females L. dimidiatus (Soares et al.,
in review). In comparison to saline intramuscular injections, AVT significantly
decreased both the cleaners’ likelihood of engaging in a mutualistic interaction and the
quality of the service provided (measured as the duration of the inspection) (Soares et
al., in review). Cleaners treated with manning compound (an AVT antagonist) were
more prone to interact with the clients, but the quality of the service decreased [as
measured by an increase in client jolts - a measure of dishonest behaviour (Soares et al.,
2008)]. Moreover, conspecific social behaviours increased in fish injected with AVT,
25
LabrichthynesLabrichthys unilineatus
Diproctacanthus xanthurus
Labropsis australis
Labroides dimidiatus
Labroides bicolor
Labroides rubrolabiatus
Obligatory cleanerFacultative cleanerNon cleaner
…
suggesting that the mechanisms already in place for the regulation of conspecific social
behaviours, might have been used for the modulation of interspecific behaviours
(Soares et al., in review).
In order to further inspect this suggestion, a comparison of the AVT system between
related species, with and without the expression of interspecific cooperative behaviours,
is of great importance. Such comparisons allow to disclose important relationships
between brain and behaviour (Insel & Young, 2000).
1.7. The model species: a comparative approach
Two closely related labrid species (also called wrasses) were used in the present study:
the Indo-pacific blue-streak cleaner wrasse Labroides dimidiatus and the Tubelip
wrasse Labrichthys unilineatus.
L. dimidiatus and L. unilineatus belong to the Labrichthynes lineage and diverged from
a common ancestor in the early Miocene (~20 Ma) (Cowman et al., 2009). L. dimidiatus
evolved as an obligatory cleaner, while L. unilineatus evolved as a corallivore, targeting
to coral mucus (Cole et al., 2010) (Figure 1).
L. dimidiatus is a territorial protogynous species and lives in harems, where the sole
male is always the larger fish (Nakashima et al., 2000). This species is well known for
its interspecific social system, where cleaners feed exclusively on what they remove
from clients (Bshary, 2001). Thus, from the cleaners’ perspective this is an obligatory
mutualism, as they completely depend on their clients to feed and hence to survive.
Figure 1 – Cladogram representing the evolution of the Labrichthynes lineage. Adapted from (Cowman et al.,
2009)
26
L. unilineatus is a territorial, haremic and protogynous species with only one male per
territory (Colin & Bell, 1991). This species is a generalist obligate corallivore (Cole et
al., 2010; McIlwain & Jones, 1997), and the mucus produced by the coral polyps is the
main target (Cole et al., 2009; Cole et al., 2010). Male tubelip wrasses have large
feeding territories which they defend from other male competitors, whereas females are
more associated to patches (McIlwain & Jones, 1997). Additionally, this species does
not show mutualistic behaviours at any time of its life cycle (Cowman et al., 2009),
therefore referred to as non-cleaner hereafter.
1.8. Objectives and predictions
As a follow up of the field and laboratory study about AVT’s influence on mutualism
(Soares et al., in review; see section 1.7), the present study’s major goal is to
characterize and compare the AVT neuronal phenotypes of two closely related species
that live in similar environments, but diverge in the expression of mutualistic
behaviours: an obligate cleaner (L. dimidiatus) and a non-cleaner, corallivore labrid (L.
unilineatus).
An additional aim of this study is to identify sex differences in AVT neuronal
phenotypes, as they might be responsible for the expression of sex-specific social
behaviours.
I used an immunocytochemical technique to detect AVT immunoreactivity (-ir) on brain
tissue sections. Cells present on the three components of the POA (pPOA, mPOA and
gPOA) were measured and counted. Comparisons were taken to test the following
predictions:
i) Species should diverge in terms of neuronal phenotypes, explaining the strong
differences in behavioural output (i.e. the expression or not of mutualistic behaviours).
As suggested by a previous study, increasing the endogenous AVT level results in a
decrease of the expression of interspecific cooperative behaviours in L. dimidiatus
(Soares et al., in review). Therefore, I expect that cleaners have smaller and/or less
numerous AVT-ir neurons. More precisely, these differences should be evident in the
neuronal groups that project to extrahypothalamic areas (mPOA and gPOA) and are
therefore capable of modulating the behaviours expressed;
27
ii) Males and females should diverge in their AVT neuronal phenotypes, as a mirror of
higher aggressive levels in males than females. L. dimidiatus aggressive behaviours are
usually directed towards females (Raihani et al., 2012), whereas L. unilineatus also can
express aggressiveness towards other conspecific males (McIlwain & Jones, 1997).
Therefore, I expect that males have larger and/or more numerous AVT-ir cells than
females, especially in those neuronal groups that project to extrahypothalamic brain
centres.
28
2. Materials and Methods
2.1. Subjects
All the subjects tested in this study were purchased from a local distributor of tropical
marine fishes (T.M.C.; Tropical Marine Centre, Lisbon, Portugal). L. dimidiatus
specimens were collected in Maldives, and L. unilineatus were collected in Indonesia.
Fish were housed in plastic bags with seawater and oxygen and freighted to Portugal by
air. All the animals arrived to Portugal between July 2011 and January 2012 and were
kept in quarantine in the T.M.C. facility tanks for a maximum of one week, and then
transported to the laboratory at ISPA (Lisbon, Portugal).
A total of 9 L. dimidiatus [4 males, standard length (SL, mean ± SEM)= 6.275 ± 0.253
cm, and 5 females SL (mean ± SEM)= 6.080 ± 0.252 cm] and 8 L. unilineatus [3 males,
SL (mean ± SEM)= 11.333 ± 1.225 cm, and 5 females, SL (mean ± SEM)= 8.620 ±
0.907 cm] were used in this study. There were no differences between genders in SL for
either species (one-way ANOVA, L. dimidiatus: F1,7 = 0.29, p = 0.607; L. unilineatus:
F1,6 = 3.251, p = 0.121). L. unilineatus were significantly larger than L. dimidiatus (one
way ANOVA, F1,15 = 18.487, p < 0.001). All data presented is corrected for SL as
described in the statistical analysis section below.
The sex of the individuals was confirmed in both species by direct inspection of the
gonads with the help of an aceto-carmine protocol (Wassermann & Afonso, 2002)
whenever needed. According to this protocol female gonads were identified by the
presence of oocytes with a well defined nucleoli (Figure 2 A, B) whereas male gonads
were identified by the presence of cysts (Figure 2 C, D) (Wassermann & Afonso, 2002).
29
2.2. Tissue Preparation
Fish were deeply anesthetised with tricaine methanesulfonate (MS-222, PHARMAQ),
measured for SL, and perfused transcardially with 0.9% heparinised 0.1 M phosphate
buffer saline solution (PBS, pH 7.4) for 1 to 2 minutes, followed by 4%
paraformaldehyde (PFA) in 0.1 M PBS for 30 min. This protocol allowed the fixative’s
administration through the circulatory system of the deeply anesthetised animal,
Figure 2 - Labrichtys unilineatus gonadal tissue was stained with aceto-carmine to better visualize female and male
typical cells. A – Female gonad (100X); B – Female gonad (1000X). Individual cells are easily distinguished from
each other and a defined nucleoli is present in each cell; C- Male gonad (100X); D – Male gonad (1000X). Typical
cell arrangement of a male gonad with small cysts present throughout the gonadal tissue.
30
ensuring a quick and efficient tissue fixation. Brains were then removed, post-fixed in
4% PFA for 1 to 2 hours and transferred to 0.1 M PBS until further processing, which
never took longer than 5 days. One day prior to sectioning, brains were cryoprotected in
30% sucrose in 0.1 M PBS and kept at 4 ºC overnight. All solutions were made fresh
every day to avoid bacterial or fungal growth.
2.3. Immunocytochemistry
Cryoprotected brains were embedded in Tissue-Tek® (optimal cutting temperature
compound), frozen at -80 ºC and then placed in the cryostat chamber at -25 ºC for 1h,
allowing the tissue to reach the optimal cutting temperature. Brains were sectioned with
the cryostat at 25 µm in the coronal plane. Alternate brain sections were collected onto
two series of Silane-prep glass slides (Sigma) and stored at -80 ºC until reacted. All
brains were completely processed within six days following the perfusion.
One of the alternate series was immunoreacted following a protocol adapted from
Grobber and Bass (1991). At day 1, slides were defrosted at room temperature and the
tissue sections were surrounded with a PAP pen for immunostaining (Sigma), which
functioned as a hydrophobic barrier. Slides were covered with 0.1 M Phosphate Buffer
solution (PB; pH 7.4) for 7 minutes, drained and washed again for another 7 minutes,
and blocked with 0.4% Triton® X-100 SigmaUltra (Sigma) in 0.1 M PB with 2.8%
Goat Serum (Sigma) for 20 minutes. Slides were then drained and covered with 3%
H2O2 in 0.1 M PB for 20 minutes, for endogenous peroxidise blockage. After two
additional washes (7 minutes each) with 0.1 M PB, the primary AVT antibody was
applied in a final dilution of 1:7500 and slides were incubated overnight in a sealed
humidified chamber at 4 ºC. The antibody used was generously donated by Dr. Matthew
Grober (Georgia State University, USA) and has been successfully used in similar
studies done with different teleost species (e.g. Dewan et al., 2008; Maruska et al.,
2007; Maruska, 2009). Day 2 started with two 7 minutes washes (0.1 M PB) followed
by incubation with the biotinylated goat anti-rabbit secondary antibody (KPL) for 30
minutes, two additional washes (7 minutes each) and incubation with peroxidase
labelled streptavidin (KPL) for another 30 minutes. After two additional 0.1 M PB
washes of 7 minutes the slides were reacted with diaminobenzidine (DAB) chromogen
peroxidase substrate kit (Vector Laboratories, Inc.) according to the manufacturer’s
31
instructions, for 3-6 minutes or until golden-brown coloration was achieved. Slides were
then immersed in distilled water to stop the reaction, dehydrated in an ethanol series
(70%, 85%, 95%, 3 x 100%; 1 minute each bath, with the exception of the last bath of 2
minutes), cleared in xylol (Sigma) (two baths, one of 1 minute and the second of 5
minutes) and coversliped with Cytoseal 60 mounting media (Richard Allen Scientific).
Several controls have been conducted to assure specificity of this antibody in other
species (Dewan et al., 2008; Maruska et al., 2007; Maruska, 2009). Nonetheless, I have
also controlled for a possible non-specific labelling of the neurons by pre-adsorbing the
antibody with 8 µM AVT peptide (Sigma) overnight at 4 ºC. The next day the
immunocytochemistry protocol was conducted in two alternate series of the same brain.
The first series received the diluted antibody whereas the second series received the
diluted antibody pre-adsorbed with the peptide as described above. All the other steps
were maintained. The first series showed normal immunostaining, while the control
series did not show any stained neuron, thus demonstrating the specificity of the
antibody used.
2.4. Quantification
Slides were coded so that the specimen sex or species were not known during the
quantification process.
The complete brain series were inspected for localization of AVT-immunoreactivity in
the brain. AVT-ir neuron cell bodies were only detected in the POA and therefore all
quantifications for cell number and soma size were done in this region. Each AVT-ir
cell was assigned to either the parvocellular (pPOA), magnocellular (mPOA) or
gigantocellular (gPOA) cell group based on descriptions of cells’ neuroanatomical
location, size and morphology made for the teleost brain by Bradford and Northcutt
(1983). This nomenclature has been widely used in similar studies (Lema, 2006;
Maruska, 2009; Semsar & Godwin, 2003).
Cell size was determined from digital images captured at a magnification of 400X with
a digital camera (Olympus C-2020 Z) attached to a microscope (Olympus BX50). After
calibration for magnification the cross-sectional area of four to twenty randomly
32
selected cells from each cell group was calculated using ImageJ software, by tracing the
cell profile. The cell profile area was automatically calculated by the software.
Cell number was determined by visually counting the cells belonging to each cell group
with the microscope. Whenever an accurate attribution of a cell to a cell group was not
possible, the cell diameter was measured (using the microscope ruler) and recorded as
“unidentified cell”. A correlation line between cell area (measured with ImageJ) and
cell diameter (measured with the microscope ruler) was used in order to correctly
classify each unidentified cells a posteriori (Figure 3). This took into account the
individual distribution of cell area in the three cell groups (pPOA, mPOA and gPOA).
Figure 3 - Regression line between cell profile area (µm2) and cell diameter (µm). Coefficient of
determination (R2) is a measure of how strong the association between the two variables is. The
regression equation allowed calculating a predictable cell profile area for any observed cell diameter.
Only cells presenting a discernible perimeter from the background and at least one
neurite were measured and counted. For pPOA cells only the discernible perimeter
criterion was used as neurites were not always visible.
The larger cell measured had a profile area of 747.66 µm2, which according to the
regression line described above corresponds to a cell diameter of 38.20 µm. As the
distance between two alternate tissue sections equals 50 µm and given that the antibody
y = 0.0373x + 10.311
R² = 0.8924
0
10
20
30
40
50
60
0 200 400 600 800 1000 1200
Cel
l d
iam
eter
(µ
m)
Cell profile area (µm2)
33
is only likely to penetrate a few micrometers of the tissue sections (Foran & Bass,
1998), it is not expected that the same cell was counted twice in alternate brain sections.
2.5. Statistical analysis
All descriptive statistics given in the text are the mean ± standard error of the mean
(SEM), unless stated differently. In order to determine the effect of body size on cell
number and cell size, linear regressions tests were used. Several significant relationships
between cell characteristics and fish SL were found (Table 1) for L. unilineatus.
Therefore, and because this study aims to make comparisons between the two species,
all the data were corrected for fish body size by dividing cell size and cell number by
fish SL (cm).
Differences between species and sexes for AVT-ir cell profile area and number were
assessed using two-way repeated measures ANOVA with species (L. dimidiatus vs. L.
unilineatus) and sex (male vs. female) as independent factors and cell group (pPOA vs.
mPOA vs. gPOA) as a within-subjects factor. Repeated measures ANOVA were
followed by planned comparisons for least square means, in order to compare species
and sexes in each cell group.
34
Table 1 – Linear regression tests for AVT-ir cell profile areas and numbers versus body length. Results are presented
for both species together and separately, showing that the regressions are only kept significant for Labrichtys
unilineatus.
r2 p
Cell size
pPOA ,588 0,001
mPOA ,719 < 0.001
gPOA ,793 < 0.001
Cell number
pPOA ,263 0,051
mPOA ,266 0,049
gPOA ,859 < 0.001
L. dimidiatus
Cell size
pPOA ,170 0,311
mPOA ,005 0,871
gPOA ,095 0,421
Cell number
pPOA ,099 0,448
mPOA ,041 0,631
gPOA ,318 0,114
L. unilineatus
Cell size
pPOA ,586 0,027
mPOA ,772 0,004
gPOA ,570 0,03
Cell number
pPOA ,148 0,395
mPOA ,190 0,328
gPOA ,730 0,014
35
36
3. Results
3.1. Qualitative characterization and distribution of preoptic AVT-ir cells in L.
dimidiatus and L. unilineatus
All AVT-ir cells were found within the preoptic area and no qualitative differences in
the pattern of cell distribution were found between the two species. The parvocellular
neuronal group (pPOA) is the most rostral and ventral consisting of small (L.
dimidiatus: 45.4 ± 8.4 µm2; L. unilineatus: 69.7 ± 5.3 µm
2) round cells usually
monopolar or without obvious neurites (Figure 4 A, B). This cell group starts above the
optic chiasm in the ventral surface of the forebrain and extends dorso-caudally until the
region where magnocellular neurons start to appear. The pPOA cells extend more
laterally than any of the other cell groups. The magnocellular neurons (mPOA) appear
dorsally to the last pPOA cells and occupy a medial location in the preoptic area lying
against the third ventricle. They have approximately twice the size of parvocellular
neurons (L. dimidiatus: 94.6 ± 6.7 µm2; L. unilineatus: 141.6 ± 8.4 µm
2) and usually
have one prominent axon that extends towards the preopticohypophyseal tract (PHT)
(Figure 4 C, D). The gigantocelular (gPOA) neuronal group begins dorsal to the mPOA
cells and extends caudally, presenting fewer cells per tissue section. These are the
largest AVT-ir cells (L. dimidiatus: 128 ± 5.7 µm2; L. unilineatus: 539.1 ± 64.3 µm
2;
Figure 4 E, F).
37
Qualitative differences in gPOA cell characteristics were found between the two
species. Gigantocellular neurons in L. unilineatus are multipolar, usually having many
fibres surrounding them and extending in/from different directions (Figure 5 B). In L.
Figure 4 – Photomicrographs of AVT-immunoreactive (-ir) neurons in the brain of Labroides dimidiatus on the left
(A, C, E) and Labrichthys unilineatus on the right (B, D, F). Cross-sections through the preoptic area illustrate the
relative position and appearance of the three cell types: parvocellular (A, B), magnocellular (C, D) and
gigantocellular (E, F). Magnification is 200X.
38
dimidiatus gigantocelular neurons were typically solitary cells, bipolar or multipolar but
with much less processes surrounding the cell body (Figure 5 A).
3.2. Quantitative comparisons of preoptic AVT-ir cell size
There was a main effect of species (L. unilineatus > L. dimidiatus, F1,12 = 79.41, p <
0.001) and cell type (parvocellular neurons < magnocellular neurons < gigantocelular
neurons, F2,24 = 107.71, p < 0.001, all planned comparisons p < 0.001) in the size of
AVT-ir neurons.
Sex had no effect in the size of AVT-ir cells (F1,12 = 1.23, p = 0.289).
There was a significant interaction between cell type and species (F2,24 = 40.60, p <
0.001), which was due to differences in cell size between the two species being
restricted to gPOA cells (F1,12 = 40.27, p < 0.001; all other planned comparisons p >
0.05; Figure 6).
Figure 5 – Photomicrographs of AVT-ir neurons in the gPOA of Labroides dimidiatus (A) and Labrichthys
unilineatus (B), showing qualitative differences between the species in the amount of AVT-ir fibers surrounding this
type of cells (section 3.1). It is also visible the difference in cell size between species (section 3.2). Magnification is
400X.
39
0
10
20
30
40
50
60
70
pPOA mPOA gPOA
Cel
l P
rofi
le A
rea
(µm
2)
/ cm
SL
L. dimidiatus
L. unilineatus
***
3.3. Quantitative differences of preoptic AVT-ir cell number
There was a main effect of cell type on the number of AVT-ir cells (F 2, 24= 3.96, p =
0.033). Planned comparisons showed that gigantocellular were significantly more
numerous than parvocellular neurons (F 1, 12 = 5.90, p = 0.032), and that there was a
marginally non-significant trend for mPOA cells to be more numerous than pPOA (F
1,12 = 4.51, p = 0.055), whereas no significant differences were observed between the
number of mPOA and gPOA cells (F 1,12 = 0.17, p = 0.691).
Species and sex had no effect on the number of AVT-ir cells (Species: F1,12 = 1.95, p =
0.187; Sex: F1,12 = 0.50, p = 0.491).
There was a significant interaction between cell type and species (F 2,24 = 4.23, p =
0.027). Planned comparisons between the two species for each cell group showed that
species differences were restricted to the number of gPOA cells (F1,12 = 22.21, p <
0.001; Figure 7).
Figure 6 – Cell area for AVT-ir neurons corrected for fish standard length (cm) within the
preoptic area in Labroides dimidiatus (light bars) and Labrichthys unilineatus (dark bars). Data
are presented as mean ± SEM. Asterisks indicate significant differences between species (planned
comparisons; ***p < 0.001).
40
0
2
4
6
8
10
12
14
16
18
20
pPOA mPOA gPOA
Cel
l N
um
ber
/ c
m S
L
L. dimidiatus
L. unilineatus ***
Figure 7 - AVT- -ir neuron number corrected for fish standard length (cm) within the preoptic
area in Labroides dimidiatus (light bars) and Labrichthys unilineatus (dark bars). Data are
presented as mean ± SEM. Asterisks indicate significant differences between species (planned
comparisons; ***p < 0.001).
41
42
4. Discussion
This study shows evidence for interspecific differences in arginine vasotocin (AVT)
neuronal phenotypes between two phylogenetically close labrid species that differ in
their social system, one species being an obligate cleaner (L. dimidiatus), thus engaging
in repeated interspecific mutualistic interactions (Trivers, 1971), whereas the other (the
non cleaner, coral feeder L. unilineatus) does not engage in such interactions (Cole et
al., 2010).
4.1. The distribution of AVT-ir neurons
The two species examined herein had AVT-ir neurons limited to the three cellular
groups of the preoptic area, which is in accordance with what has been reported for
other teleost species (e.g. Dewan et al., 2008; Grober et al., 2002; Lema & Nevitt,
2004b; Maruska, 2009; Miranda et al., 2003; Santangelo & Bass, 2010). Preoptic AVT
cells are well known for their physiological, endocrine and behavioural roles, and there
is a general consensus that the POA represents the main (and usually the only) site of
production of this neuropeptide in fish (Bradford Jr. & Northcutt, 1983; but see Foran &
Bass, 1998). Thus, the distribution pattern of AVT-ir neurons in the two species used in
this study is in line with the suggested conserved evolution of the neuroanatomical
features of the teleost AVT system (Goodson & Bass, 2001).
4.2. Species differences in AVT-ir neurons
Quantitative results of this study demonstrate that the non-cleaner L. unilineatus had
larger and more numerous AVT-ir cells than the cleaner L. dimidiatus, and that these
differences were restricted to the gPOA cell group.
Several other studies have found intra- and interspecific differences in gigantocellular
neurons, which have been associated to differences in social systems (Dewan et al.,
2011) aggression (Dewan & Tricas, 2011), dominance status (Larson et al., 2006)
43
and/or breeding season (Maruska, 2009). In a study involving two related butterflyfish
species that differ in their social system, the territorial aggressive Chaetodon
multicinctus has larger AVT-ir neurons in relation to C. miliaris, a shoaling less
aggressive species (Dewan et al., 2008). This species differences in cell size were
restricted to the mPOA and gPOA cell groups and were hypothesized to reflect the
differences in the social systems and consequent social behaviours displayed.
Additionally, in a recent experiment focusing that same territorial butterflyfish, Dewan
and Tricas (2011) showed a positive relationship between gPOA cell numbers and
individual aggression level towards a mirror, and suggested that this effect might be due
to neuronal projections to areas where behaviour modulation occurs.
In fact it has been showed that gPOA cells mainly project to extrahypothalamic areas
such as the telencephalon and thalamus (Holmgvist & Ekström, 1995; Saito et al.,
2004), where behavioural modulation occurs (Goodson, 2005), suggesting that this cells
have important roles in the modulation of social behaviours. This modulation likely
occurs by means of neuronal projections to other brain centres, such as those belonging
to the SBN (Goodson, 2005). Indeed, in teleosts, the density of AVT-ir varicosities
(putative locations of peptide release that likely originate in the POA) in the ventral
nucleus of the ventral telencephalon (Vv) is a strong predictive of aggressive/territorial
species (Dewan et al., 2011). Also, in a comparative study with rodents of the genus
Peromyscus, higher AVP immunoreactivity in the lateral septum (homologous of the
teleost Vv) was also shown to be a good predictor of more aggressive species (Bester-
Meredith et al., 1999).
Arginine vasotocin is also linked with pro-sociality levels in the highly social male
goldfish Carassius auratus. Infusions of this peptide in the POA inhibited social
approach behaviours towards a conspecific, possibly through behavioural modulation in
the telencephalon (Thompson & Walton, 2004). This suggests that AVT also plays a
role in social approach and social withdrawal. In a recent study involving the cleaner
fish L. dimidiatus, Soares and colleagues (in review) showed that higher levels of AVT
result in a decrease of interspecific social approach and in the expression of mutualistic
behaviours.
Together with these previous findings, the present results suggest that the smaller size
and number of gPOA neurons of the cleaner fish, in comparison to the non-cleaner,
44
might have facilitated the evolution and maintenance of cleaning behaviours and the
ability to engage in mutualistic interactions. Possibly, evolution favoured smaller and
less numerous neurons projecting to extrahypothalamic brain areas in order to lessen the
expression of potential interspecific aggressive behaviours and interspecific social
withdrawal, thus enhancing mutualistic interactions.
It is not yet known if size and number of immunoreactive cells are precise descriptors of
the amount of peptide being used by the brain, and further studies should address this
issue. Nonetheless, taking size and number of gPOA neurons as a proxy for the
potential amount of AVT being transported and released in brain areas responsible for
the modulation of aggressive behaviours, then cleaners might have lower levels of AVT
in this system. Such lower levels can possibly be responsible for the expression of
mutualistic behaviours.
The higher fibre densities surrounding the gPOA neurons of the non-cleaner fish might
be related to a higher requirement of cell communication, in comparison to the cleaner
fish. Such communication is important to synchronize peptide release in distant brain
areas and increase the expression of territorial and aggressive behaviours in L.
unilineatus. Indeed, AVT neurons are able to communicate with each other (Saito et al.,
2004) through their dendrites. Additionally, dendritic release of nonapeptides is
partially independent of distant axonal release, suggesting that dendrites have an
autoregulatory activity that modulates distant peptide releases (Ludwig, 1998).
Nevertheless, the hypothesis proposed above should be taken cautiously as it is not
possible to determine that the reported fibres are dendrites. This issue should be
addressed, for example by using thinner tissue sections in further studies. Alternatively,
the higher fibre density in L. unilineatus might only result from the larger and more
numerous cells in the gPOA. Therefore, quantitative measures of fibre density and
correlations with cell number and size in this neuronal group, for both species, could
provide a better understanding about this question.
It is also important to note that AVT might not be the solely responsible for the
suggested hypothesis as other hormones might interact with the AVT system (Delville
et al., 1996; Ferris, 1992) or even exert a direct effect on mutualistic (Soares et al.,
2010) and aggressive behaviours (Nelson & Trainor, 2007). For instance, serotonin is
known to reduce the AVP-facilitated aggressive behaviours in golden hamsters
45
(Delville et al., 1996). Also in the teleost Thalassoma bifasciatum, males treated with
fluoxetin (a serotonin reuptake inhibitor) exhibited lower aggression levels compared to
saline controls (Perreault et al., 2003). Such behavioural modulation is due, at least in
part, to serotonin interactions with the AVT system, as AVT mRNA production was
twofold lower in the three preoptic regions (Semsar et al., 2004).
No differences between the two species in number or size of pPOA and mPOA neurons
were detected in this study. Both pPOA and mPOA neurons project to the pituitary
(Bradford Jr. & Northcutt, 1983; Strand, 1999) and are capable of regulating other
peripheral mechanisms such as osmorregulation and stress response (Balment et al.,
1993; Balment et al., 2006).
Osmorregulation is not a primary variable in this study as both species were kept at the
supplier (T.M.C.) facility tanks, with stable temperature and salinity. Accordingly,
differences between species that could be related with different osmotic pressures were
not found in this study.
Parvocellular AVT neurons in fish also play an important role in stress response. In
rainbow trout (Oncorhynchus mykiss) acute confinement stress events increase AVT
mRNA expression in parvocellular neurons of the preoptic area, without affecting
magno- or gigantocellular neurons (Gilchriest et al., 2000). Those results support the
idea that the pPOA neurons innervating the pituitary are involved in the stimulation of
ACTH release in response to acute stressors and therefore are able to modulate the HPI
axis (Balment et al., 2006).
In the present study it is possible that physiological stress was induced to the animals
used, due to confinement during transportation. The lack of differences in pPOA and
mPOA AVT-ir neurons between the two species might support one of the three
hypotheses: a) the animals used in this study were not stressed and the lack of
differences might reflect similar physiological processes. However, this hypothesis
unlikely as confinement is a major source of physiological stress, as described above; b)
if the animals were stressed, the stress response was not significantly different between
the two species; c) unnatural confinement was applied for a long period of time
(approximately 1 week between catch and arrival to the laboratory) and represented a
chronic stressor, causing an habituation of the AVT system. In contrast to acute stress
responses, pPOA neurons in rainbow trout did not respond to chronic stress, despite the
46
elevated concentrations of plasmatic cortisol (Gilchriest et al., 2000). The hypothesis b)
and c) are then possible candidates to explain the lack of species differences in pPOA
and mPOA neural groups. However, future studies are needed in order to show that in
L. dimidiatus and L. unilineatus, chronic stress has similar effects to the ones described
for trout.
Additionally to pituitary projections, magnocellular AVT neurons have also been
reported to be involved in the expression of behaviours that are dependent of social
phenotype, as they can also project to brain centres responsible for behavioural
modulation (Holmgvist & Ekström, 1995). For example, in the tropical bluehead wrasse
(Thalassoma bifasciatum), different levels of AVT production in this cell group are
associated with different sexual phenotypes, and consequent differences on the display
of sexual and aggressive behaviours (Godwin et al., 2000). Cleaners and non-cleaners
did not differ in number and size of mPOA cells, which further suggests that mutualistic
behaviours are more likely to be modulated by central projections of the gPOA AVT
neurons rather than those from the mPOA group.
4.3. Sex differences in AVT-ir neurons
Contrary to the initial predictions, no sex differences in any AVT-ir cellular group were
found in the cleaner fish L. dimidiatus or in the non-cleaner L. unilineatus. However,
many studies have found sex differences in AVT cell characteristics, often associated
with gonadal sex hormones (Goodson & Bass, 2001) and behavioural differences
between males and females (e.g. Grober et al., 2002; Lema & Nevitt, 2004b).
The possibility that this lack of differences mirrors a lack of behavioural differences
between sexes is unlikely. The expected differences for the cleaner fish were based on
the fact that males show higher levels of intraspecific aggression, directed towards
females in response to their dishonest behaviours towards the clients (Raihani et al.,
2010; Raihani et al., 2012). Concerning the non-cleaner fish, males own a territory
(Colin & Bell, 1991) that they actively patrol and defend from other competitor males
(McIlwain & Jones, 1997). As females don’t show such territorial behaviour (McIlwain
& Jones, 1997), intersexual differences in the neuronal AVT phenotype were also
expected for this species.
47
Other studies did not find any differences between sexes in the AVT system. For
example, in the goldfish no differences in neuronal AVT phenotypes were found
between sexes (Parhar et al., 2001). Moreover, sex steroid treatments (Estradiol,
Testosterone and 11-ketotestosteron) did not produce any effect on preoptic neurons in
that species (Parhar et al., 2001). The lack of sex differences in AVT-ir neurons in
cleaners and non-cleaners might reflect a lack of androgen modulation on the AVT
system in both species. However, androgens might still modulate intraspecific
aggression through direct actions on aggressive behaviours. Nonetheless, the present
study was not designed to test other hormonal systems or their interactions with AVT,
and further experiments should test that hypothesis in a more direct way. For example,
the effect of androgen implants/injections and castration might give a better view of
those interactions.
Alternatively, the lack of sex differences in the cleaner fish might merely reflect the fact
that both males and females engage in cleaning interactions and the expressed
mutualistic behaviours do not differ between sexes (Bshary, 2003; Tebbich et al., 2002).
The lack of sex differences in the non-cleaner wrasse should be taken cautiously. In this
study it was only possible to use three males of this species and the absence of sex
differences might be due to the small sample size used. Although the p values are high
for intersexual differences in cell size and number, increasing the sample size (for
example matching the females’ sample size) would provide more reliable results.
4.4. Final comments
This is the first study to show differences in AVT-ir neuronal phenotype between two
closely related teleost species that differ in the expression of mutualistic behaviours.
The cooperative cleaner wrasse L. dimidiatus showed less and smaller gPOA neurons,
in comparison to the non-cleaner tubelip wrasse L. unilineatus. In light of a previous
study showing the effects of AVT on mutualistic behaviour, here I suggest that the
lower levels of AVT in the cleaner wrasse (inferred by smaller and less numerous AVT-
ir neurons) might have a role in the predisposition to approach, interact and cooperate
with interspecific social partners. Additionally, the gPOA cellular group and its
extrahypothalamic projections appear to be responsible for such levels of interspecific
48
pro-sociality. Although most studies focusing on the AVT system centre their attention
on conspecific social behaviours, the present findings provide further evidence that
AVT might also play an important role in the modulation of interspecific social
behaviours. This possibly occurs through the same AVT action mechanisms that
modulate intraspecific social behaviours in other species. Moreover, and as suggested in
our previous study (Soares et al., in review), the AVT system already in place for th
modulation of intraspecific social behaviours, might have been recruited for the
modulation of mutualistic behaviours. Further studies are also needed to test possible
interactions with other hormonal systems and thus, fully understand the contribution of
the AVT system for the cleaner wrasse social system and other mutualistic/cooperative
systems.
49
50
5. References
Almada, V.C., 1995. Courting females: ecological constraints affect sex roles in a natural population of the blenniid fish Salaria pavo. Anim Behav. 49, 1125-1127.
Almeida, O., Gozdowska, M., Kulczykowska, E., Oliveira, R.F., 2012. Brain levels of arginine vasotocin and isotocin in dominant and subordinate males of a cichlid fish. Hormones and Behavior.
Backström, T., Winberg, S., 2009. Arginine-vasotocin influence on aggressive behavior and dominance in rainbow trout. Physiology & Behavior. 96, 470-475.
Backström, T., Schjolden, J., Øverli, Ø., Thörnqvist, P.-O., Winberg, S., 2011. Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness. Hormones and Behavior. 59, 180-186.
Balment, R.J., Warne, J.M., Tierney, M., Hazon, N., 1993. Arginine vasotocin and fish osmoregulation. Fish Physiology and Biochemistry. 11, 189-194.
Balment, R.J., Lu, W., Weybourne, E., Warne, J.M., 2006. Arginine vasotocin a key hormone in fish physiology and behaviour: A review with insights from mammalian models. General and Comparative Endocrinology. 147, 9-16.
Bester-Meredith, J.K., Young, L.J., Marler, C.A., 1999. Species differences in paternal behavior and aggression in peromyscus and their qassociations with vasopressin immunoreactivity and receptors. Hormones and Behavior. 36, 25-38.
Bradford Jr., M.R., Northcutt, R.G., 1983. Organization of the diencephalon an pretectum of the ray-finned fishes. In: Fish Neurobiology. Vol. 2, R.E. Davis, R.G. Northcutt, ed.^eds. University of Michigan Press, Ann Arbor, pp. 117-164.
Bradshaw, D., Ladyman, M., Stewart, T., 2007. Effect of hypernatraemia and the neurohypophysial peptide, arginine vasotocin (AVT) on behavioural thermoregulation in the agamid lizard, Ctenophorus ornatus. General and Comparative Endocrinology. 150, 34-40.
Bshary, R., 2001. The cleaner fish market. In: Economics in nature. Vol., R. Noë, J.A.R.A.M. van Hoof, P. Hammerstein, ed.^eds. Cambrige University Press, Cambrige, pp. 146-172.
Bshary, R., Grutter, A.S., 2002. Asymmetric cheating opportunities and partner control in a cleaner fish mutualism. Animal Behaviour. 63, 547-555.
Bshary, R., 2003. The cleaner wrasse, Labroides dimidiatus, is a key organism for reef fish diversity at Ras Mohammed National Park, Egypt. Journal of Animal Ecology. 72, 169-176.
Bshary, R., Bergmüller, R., 2008. Distinguishing four fundamental approaches to the evolution of helping. Journal of Evolutionary Biology. 21, 405-420.
Butler, A.B., Hodos, W., 2005. Overview of the forebrain. In: Comparative Vertebrate Neuroanatomy. Vol., ed.^eds. John Wiley & Sons, Inc., pp. 341-372.
Carter, C.S., Keverne, E.B., Editors-in-Chief: Donald, W.P., Arthur, P.A., Susan, E.F., Anne, M.E., Robert T. RubinA2 - Editors-in-Chief: Donald W. Pfaff, A.P.A.S.E.F.A.M.E., Robert, T.R., 2009. The neurobiology of social affiliation and pair bonding. In: Hormones, Brain and Behavior (Second Edition). Vol., ed.^eds. Academic Press, San Diego, pp. 137-166.
Cole, A.J., Pratchett, M.S., Jones, G.P., 2009. Effects of coral bleaching on the feeding response of two species of coral-feeding fish. Journal of Experimental Marine Biology and Ecology. 373, 11-15.
51
Cole, A.J., Pratchett, M.S., Jones, G.P., 2010. Corallivory in tubelip wrasses: diet, feeding and trophic importance. Journal of Fish Biology. 76, 818-835.
Colin, P.L., Bell, L.J., 1991. Aspects of the spawning of labrid and scarid fishes (Pisces: Labroidei) at Enewetak Atoll, Marshall Islands with notes on other families. Environmental Biology of Fishes. 31, 229-260.
Cote, I.M., 2000. Evolution and ecology of cleaning symbioses in the sea. Oceanography and Marine Biology, Vol 38. 38, 311-355.
Cowman, P.F., Bellwood, D.R., van Herwerden, L., 2009. Dating the evolutionary origins of wrasse lineages (Labridae) and the rise of trophic novelty on coral reefs. Molecular Phylogenetics and Evolution. 52, 621-631.
Delville, Y., Mansour, K.M., Ferris, C.F., 1996. Serotonin blocks vasopressin-facilitated offensive aggression: Interactions within the ventrolateral hypothalamus of golden hamsters. Physiology & Behavior. 59, 813-816.
Dewan, A.K., Maruska, K.P., Tricas, T.C., 2008. Arginine vasotocin neuronal phenotypes among congeneric territorial and shoaling reef butterflyfishes: species, sex and reproductive season comparisons. Journal of Neuroendocrinology. 20, 1382-1394.
Dewan, A.K., Ramey, M.L., Tricas, T.C., 2011. Arginine vasotocin neuronal phenotypes, telencephalic fiber varicosities, and social behavior in butterflyfishes (Chaetodontidae): Potential similarities to birds and mammals. Hormones and Behavior. 59, 56-66.
Dewan, A.K., Tricas, T.C., 2011. Arginine vasotocin neuronal phenotypes and their relationship to aggressive behavior in the territorial monogamous multiband butterflyfish, Chaetodon multicinctus. Brain Research. 1401, 74-84.
Fast, M.D., Muise, D.M., Easy, R.E., Ross, N.W., Johnson, S.C., 2006. The effects of Lepeophtheirus salmonis infections on the stress response and immunological status of Atlantic salmon (Salmo salar). Fish & Shellfish Immunology. 21, 228-241.
Ferris, C., 1992. Role of vasopressin in aggressive and dominant/subordinate behaviors. Annals of the New York Academy of Sciences. 652, 212-226.
Foran, C.M., Bass, A.H., 1998. Preoptic AVT immunoreactive neurons of a teleost fish with alternative reproductive tactics. General and Comparative Endocrinology. 111, 271-282.
Gilchriest, B.J., Tipping, D.R., Hake, L., Levy, A., Baker, B.I., 2000. The effects of acute and chronic stresses on vasotocin gene transcripts in the brain of the rainbow trout (Oncorhynchus mykiss). Journal of Neuroendocrinology. 12, 795-801.
Godwin, J., Sawby, R., Warner, R.R., Crews, D., Grober, M.S., 2000. Hypothalamic arginine vasotocin mRNA abundance variation across sexes and with sex change in a coral reef fish. Brain Behavior and Evolution. 55, 77-84.
Godwin, J., Thompson, R., 2012. Nonapeptides and Social Behavior in Fishes. Hormones and Behavior.
Goodson, J.L., Bass, A.H., 2001. Social behavior functions and related anatomical characteristics of vasotocin/vasopressin systems in vertebrates. Brain Research Reviews. 35, 246-265.
Goodson, J.L., Bass, A.H., 2002. Vocal–acoustic circuitry and descending vocal pathways in teleost fish: Convergence with terrestrial vertebrates reveals conserved traits. The Journal of Comparative Neurology. 448, 298-322.
Goodson, J.L., 2005. The vertebrate social behavior network: Evolutionary themes and variations. Hormones and Behavior. 48, 11-22.
Goodson, J.L., 2008. Nonapeptides and the evolutionary patterning of sociality. In: Advances in Vasopressin and Oxytocin: From Genes to Behaviour to Disease. Progress in Brain Research, Vol. 170, I.D.L.R. Neumann, ed.^eds., pp. 3-15.
Goodson, J.L., Kabelik, D., Schrock, S.E., 2009. Dynamic neuromodulation of aggression by vasotocin: influence of social context and social phenotype in territorial songbirds. Biology Letters. 5, 554-556.
52
Grober, M.S., George, A.A., Watkins, K.K., Carneiro, L.A., Oliveira, R.F., 2002. Forebrain AVT and courtship in a fish with male alternative reproductive tactics. Brain Research Bulletin. 57, 423-425.
Grutter, A.S., 1995. Relationship between cleaning rates and ectoparasite loads in coral-reef fishes. Marine Ecology-Progress Series. 118, 51-58.
Grutter, A.S., 1999. Infestation dynamics of gnathiid isopod juveniles parasitic on the coral-reef fish Hemigymnus melapterus (Labridae). Marine Biology. 135, 545-552.
Grutter, A.S., Bshary, R., 2003. Cleaner wrasse prefer client mucus: support for partner control mechanisms in cleaning interactions. Proceedings of the Royal Society of London. Series B: Biological Sciences. 270, S242-S244.
Grutter, A.S., Bshary, R., 2004. Cleaner fish, Labroides dimidiatus, diet preferences for different types of mucus and parasitic gnathiid isopods. Animal Behaviour. 68, 583-588.
Holmgvist, B.I., Ekström, P., 1995. Hypophysiotrophic systems in the brain of the Atlantic salmon. Neuronal innervation of the pituitary and the origin of pituitary dopamine and nonapeptides identified by means of combined carbocyanine tract tracing and immunocytochemistry. Journal of Chemical Neuroanatomy. 8, 125-145.
Insel, T.R., Young, L.J., 2000. Neuropeptides and the evolution of social behavior. Current Opinion in Neurobiology. 10, 784-789.
Komdeur, J., 2006. Variation in individual investment strategies among social animals. Ethology. 112, 729-747.
Larson, E.T., O'Malley, D.M., Melloni Jr, R.H., 2006. Aggression and vasotocin are associated with dominant-subordinate relationships in zebrafish. Behavioural Brain Research. 167, 94-102.
Lema, S.C., Nevitt, G.A., 2004a. Exogenous vasotocin alters aggression during agonistic exchanges in male Amargosa River pupfish (Cyprinodon nevadensis amargosae). Hormones and Behavior. 46, 628-637.
Lema, S.C., Nevitt, G.A., 2004b. Variation in vasotocin immunoreactivity in the brain of recently isolated populations of a death valley pupfish, Cyprinodon nevadensis. General and Comparative Endocrinology. 135, 300-309.
Lema, S.C., 2006. Population divergence in plasticity of the AVT system and its association with aggressive behaviors in a Death Valley pupfish. Hormones and Behavior. 50, 183-193.
Ludwig, 1998. Dendritic release of vasopressin and oxytocin. Journal of Neuroendocrinology. 10, 881-895.
Maruska, K.P., Mizobe, M.H., Tricas, T.C., 2007. Sex and seasonal co-variation of arginine vasotocin (AVT) and gonadotropin-releasing hormone (GnRH) neurons in the brain of the halfspotted goby. Comparative Biochemistry and Physiology a-Molecular & Integrative Physiology. 147, 129-144.
Maruska, K.P., 2009. Sex and temporal variations of the vasotocin neuronal system in the damselfish brain. General and Comparative Endocrinology. 160, 194-204.
McIlwain, J.L., Jones, G.P., 1997. Prey selection by an obligate coral-feeding wrasse and its response to small-scale disturbance. Marine Ecology Progress Series. 155, 189-198.
Meek, J., Nieuwenhuys, R., 1998. Holosteans and Teleosts. In: The central nervous system of vertebrates. Vol. 1, R. Nieuwenhuys, H.J. Donkelaar, C. Nicholson, ed.^eds. Springer Verlag.
Miranda, J.A., Oliveira, R.F., Carneiro, L.A., Santos, R.S., Grober, M.S., 2003. Neurochemical correlates of male polymorphism and alternative reproductive tactics in the Azorean rock-pool blenny, Parablennius parvicornis. General and Comparative Endocrinology. 132, 183-189.
53
Moore, F.L., Lowry, C.A., 1998. Comparative neuroanatomy of vasotocin and vasopressin in amphibians and other vertebrates. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology. 119, 251-260.
Nakashima, Y., Sakai, Y., Karino, K., Kuwamura, T., 2000. Female-female spawning and sex change in a haremic coral-reef fish, Labroides dimidiatus. Zoological Science. 17, 967-970.
Nelson, R.J., Trainor, B.C., 2007. Neural mechanisms of aggression. Nature reviews. Neuroscience. 8, 536-46.
Newman, S.W., 1999. The medial extended amygdala in male reproductive behavior. A node in the mammalian social behavior network. Annals of the New York Academy of Sciences. 877, 242-257.
Noë, R., 2006. Cooperation experiments: coordination through communication versus acting apart together. Animal Behaviour. 71, 1-18.
Nowak, M.A., 2006. Five Rules for the Evolution of Cooperation. Science. 314, 1560-1563.
Parhar, I.S., Tosaki, H., Sakuma, Y., Kobayashi, M., 2001. Sex differences in the brain of goldfish: gonadotropin-releasing hormone and vasotocinergic neurons. Neuroscience. 104, 1099-1110.
Pavlidis, M., Sundvik, M., Chen, Y.-C., Panula, P., 2011. Adaptive changes in zebrafish brain in dominant-subordinate behavioral context. Behavioural Brain Research. 225, 529-537.
Perreault, H.A.N., Semsar, K., Godwin, J., 2003. Fluoxetine treatment decreases territorial aggression in a coral reef fish. Physiology & Behavior. 79, 719-724.
Perrone, R., Batista, G., Lorenzo, D., Macadar, O., Silva, A., 2010. Vasotocin actions on electric behavior: interspecific, seasonal, and social context-dependent differences. Frontiers in Behavioral Neuroscience. 4.
Perrott, M.N., Carrick, S., Balment, R.J., 1991. Pituitary and plasma arginine vasotocin levels in teleost fish. General and Comparative Endocrinology. 83, 68-74.
Raihani, N.J., Grutter, A.S., Bshary, R., 2010. Punishers benefit from third-party punishment in fish. Science. 327, 171.
Raihani, N.J., Pinto, A.I., Grutter, A.S., Wismer, S., Bshary, R., 2012. Male cleaner wrasses adjust punishment of female partners according to the stakes. Proceedings of the Royal Society B: Biological Sciences. 279, 365-370.
Ros, A.F.H., Vullioud, P., Bshary, R., 2012. Treatment with the glucocorticoid antagonist RU486 reduces cooperative cleaning visits of a common reef fish, the lined bristletooth. Hormones and Behavior. 61, 37-43.
Rose, J.D., Kinnaird, J.R., Moore, F.L., 1995. Neurophysiological effects of vasotocin and corticosterone on medullary neurons - implications for hormonal control of amphibian courtship behavior. Neuroendocrinology. 62, 406-417.
Sachs, J.L., Mueller, U.G., Wilcox, T.P., Bull, J.J., 2004. The evolution of cooperation. Quarterly Review of Biology. 79, 135-160.
Saito, D., Komatsuda, M., Urano, A., 2004. Functional organization of preoptic vasotocin and isotocin neurons in the brain of rainbow trout: Central and neurohypophysial projections of single neurons. Neuroscience. 124, 973-984.
Santangelo, N., Bass, A.H., 2006. New insights into neuropeptide modulation of aggression: field studies of arginine vasotocin in a territorial tropical damselfish. Proceedings of the Royal Society B-Biological Sciences. 273, 3085-3092.
Santangelo, N., Bass, A.H., 2010. Individual behavioral and neuronal phenotypes for arginine vasotocin mediated courtship and aggression in a territorial teleost. Brain Behavior and Evolution. 75, 282-291.
Semsar, K., Kandel, F.L.M., Godwin, J., 2001. Manipulations of the AVT system shift social status and related courtship and aggressive behavior in the bluehead wrasse. Hormones and Behavior. 40, 21-31.
54
Semsar, K., Godwin, J., 2003. Social influences on the arginine vasotocin system are independent of gonads in a sex-changing fish. Journal of Neuroscience. 23, 4386-4393.
Semsar, K., Godwin, J., 2004. Multiple mechanisms of phenotype development in the bluehead wrasse. Hormones and Behavior. 45, 345-353.
Semsar, K., Perreault, H.A., Godwin, J., 2004. Fluoxetine-treated male wrasses exhibit low AVT expression. Brain Research. 1029, 141-147.
Soares, M.C., Bshary, R., Cardoso, S.C., Cote, I.M., 2008. The meaning of jolts by fish clients of cleaning gobies. Ethology. 114, 209-214.
Soares, M.C., Bshary, R., Fusani, L., Goymann, W., Hau, M., Hirschenhauser, K., Oliveira, R.F., 2010. Hormonal mechanisms of cooperative behaviour. Philosophical Transactions of the Royal Society B-Biological Sciences. 365, 2737-2750.
Soares, M.C., Bshary, R., Mendonça, R., Grutter, A.S., Oliveira, R.F., in review. Arginine vasotocin regulation of interspecific cooperative behavior in a cleaner fish. PLoS ONE.
Srivastava, R., Cornett, L.E., Chaturvedi, C.M., 2010. Age-dependent expression of AVT and its oxytocic-like receptor VT3 in the shell gland of Japanese quail, Coturnix coturnix japonica. General and Comparative Endocrinology. 165, 47-52.
Strand, F.L., 1999. Neuropeptides: regulators of physiological processes, Vol., MIT Press, Cambridge, Mass.
Tebbich, S., Bshary, R., Grutter, A.S., 2002. Cleaner fish Labroides dimidiatus recognise familiar clients. Animal Cognition. 5, 139-145.
Thompson, R.R., Walton, J.C., 2004. Peptide effects on social behavior: Effects of vasotocin and isotocin on social approach behavior in male goldfish (Carassius auratus). Behavioral Neuroscience. 118, 620-626.
Trivers, R.L., 1971. The evolution of reciprocal altruism. Quarterly Review of Biology. 46, 35-57.
Waldie, P.A., Blomberg, S.P., Cheney, K.L., Goldizen, A.W., Grutter, A.S., 2011. Long-Term Effects of the Cleaner Fish Labroides dimidiatus on Coral Reef Fish Communities. PLoS ONE. 6, e21201.
Warne, J.M., Balment, R.J., 1997. Changes in plasma arginine vasotocin (AVT) concentration and dorsal aortic blood pressure following AVT injection in the teleost Platichthys flesus. General and Comparative Endocrinology. 105, 358-364.
Wassermann, G.J., Afonso, L.O.B., 2002. Validation of the aceto-carmine technique for evaluating phenotipic sex in nile tilapia (Oreochromis niloticus) fry. Ciência Rural, Santa Maria. 32, 133-139.
West, S.A., Griffin, A.S., Gardner, A., 2007a. Evolutionary Explanations for Cooperation. Current Biology. 17, R661-R672.
West, S.A., Griffin, A.S., Gardner, A., 2007b. Social semantics: altruism, cooperation, mutualism, strong reciprocity and group selection. Journal of Evolutionary Biology. 20, 415-432.