Nonapeptide levels in male cleaner fish brains during interactions with unfamiliar intra and interspecific partners

  • Murilo S. Abreu
  • Ewa Kulczykowska
  • Sónia C. Cardoso
  • Gonçalo I. André
  • Marta Morais
  • Magdalena Gozdowska
  • Marta C. Soares
Original Article


In nature, cleaner fish face complex situations arising from a highly demanding socio-environment that includes conspecific and mutualistic relationships. The nonapeptide arginine vasotocin (AVT) is known to play a relevant role in the expression of mutualistic behaviour in the Indo-Pacific bluestreak cleaner wrasse (Labroides dimidiatus). However, the role of the second nonapeptide isotocin (IT) in mediating social behaviour has yet to be established. Here we investigated whether there is a link between the distribution of active nonapeptides across brain regions and cleaners’ social behaviour. Male cleaner fish were exposed to different social contexts: (i) an unfamiliar male conspecific (conspecific context), (ii) an unfamiliar interspecific partner (blond naso tang, Naso elegans; mutualistic context) and (iii) a ball (non-social context). Furthermore, to distinguish between physicochemical and visual stimulations, cleaners were also exposed to an unfamiliar conspecific or an unfamiliar interspecific partner placed in a separate, smaller aquaria while inside the experimental tank. We then measured biologically available AVT and IT levels in distinct brain macro-areas, forebrain, optic tectum, cerebellum and brain stem, using high-performance liquid chromatography with fluorescence detection. We did not find any association between the levels of AVT and social contexts. On the other hand, we found lower levels of IT in the forebrain of cleaners in contact with a conspecific compared to those introduced to a client and a client inside another aquarium. Additionally, cleaners displayed aggression but only towards other conspecifics. These findings indicate that (i) IT in cleaners’ forebrain is linked with mutualistic engagement between cleaners and clients, (ii) stimulation of IT pathways in the forebrain is probably linked with the visual recognition of potential clients and (iii) physicochemical signals are essential to trigger aggressive displays in male conspecifics.

Significance statement

The visual stimulation of IT pathways in the forebrain of male cleaner wrasses while introduced to sympatric clients is associated with the establishment of cleaner-client mutualistic relationship; physicochemical signals are important to trigger aggressive displays in male cleaners introduced to conspecifics.


Mutualisms Labroides dimidiatus Communication Visual contact Physicochemical interactions Nonapeptides 



We thank the principal curator of Oceanário de Lisboa (Núria Baylina) and staff for logistical support. Thanks are also due to José R. Paitio for his help in the maintenance of the fish facilities during experimental procedures. We would like to thank the reviewers for their valuable comments.

Authors’ contribution

MCS designed the study. SCC and GIA ran experimental procedures. GIA prepared the samples for HPLC. MG ran HPLC analysis. MM analysed behavioural videos. MSA analysed the data. MSA, EK and MCS wrote the paper. All authors discussed results and commented on the manuscript.


This study was supported by the Portuguese Foundation for Science and Technology-FCT (grant PTDC/MAR/105276/2008 given to MCS). MSC is currently supported by SFRH/BPD/109433/2015. HPLC analyses of AVT and IT analyses were partially financed by the National Science Center grant 2012/05/B/NZ4/02410 and by Institute of Oceanology of the Polish Academy of Sciences statutory research task IV.2.2 (given to EK).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

Animal procedures used in this study were approved by the Portuguese Veterinary Office (Direcção Geral de Veterinária, license # 0420/000/000/2009) and were carried out in accordance with the approved guidelines.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Graduation Program in PharmacologyFederal University of Santa Maria (UFSM)Santa MariaBrazil
  2. 2.Bioscience InstituteUniversity of Passo Fundo (UPF)Passo FundoBrazil
  3. 3.Genetics and Marine BiotechnologyInstitute of Oceanology of the Polish Academy of SciencesSopotPoland
  4. 4.CIBIO, Centro de Investigação em Biodiversidade e Recursos GenéticosUniversidade do PortoVairaoPortugal

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