1 3 Brain levels of nonapeptides in four labrid fish species with different 4 levels of mutualistic behavior 5 6 7 Ewa Kulczykowska a , Sónia C. Cardoso b,c , Magdalena Gozdowska a , Gonçalo I. André b , José R. Paula b , 8 Marek S ´ lebioda d , Rui F. Oliveira c,e , Marta C. Soares b,⇑ 9 a Genetics and Marine Biotechnology, Institute of Oceanology of the Polish Academy of Sciences, Sopot, Poland 10 b CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Campus Agrário de Vairão, 4485-661 Vairão, Portugal 11 c Unidade de Investigação em Eco-Etologia, ISPA – Instituto Universitário, Lisboa, Portugal 12 d Perlan Technologies Sp. z.o.o., Pulawska 303 St., 02-785 Warszawa, Poland 13 e Champalimaud Neuroscience Programme, Instituto Gulbenkian de Ciência, Oeiras, Portugal 14 15 17 article info 18 Article history: 19 Received 2 April 2015 20 Accepted 9 June 2015 21 Available online xxxx 22 Keywords: 23 Neurohormones 24 Arginine vasotocin 25 Isotocin 26 Liquid chromatography–tandem mass 27 spectrometry 28 Labrid fish 29 Cleaner fish 30 Mutualistic behavior 31 32 abstract 33 There is strong evidence that brain nonapeptides are implicated as modulators of a wide array of social 34 and reproductive behaviors in fishes. However, the question remains, as to whether there is a link 35 between the distribution of active nonapeptides across brain regions and fishes specific behavioral phe- 36 notypes. To explore this link we compared the nonapeptides’ profile across the brains of fishes represent- 37 ing different degrees of mutualistic behavior (here: cleaning behavior). Herein we studied the 38 quantitative distribution of both nonapeptides, arginine vasotocin (AVT) and isotocin (IT), in the brains 39 of four species of fish belonging to the family Labridae: two are obligatory cleaners throughout their 40 entire life (Labroides dimidiatus and Labroides bicolor), one species is a facultative cleaner (Labropsis aus- 41 tralis; juveniles are cleaners and adults are corallivorous), and one is a non-cleaner species, corallivorous 42 throughout its entire life (Labrichthys unilineatus). The biologically available AVT and IT concentrations 43 were measured simultaneously in distinct brain macro-areas: forebrain, optic tectum, cerebellum and 44 brain stem, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). We showed that 45 the levels of both AVT and IT varied significantly across species, as measured in the whole brain or in 46 the specific macro-areas. Significantly higher AVT concentrations in the cerebellum which were found 47 in the obligate cleaners seemed to be related to expression of mutualistic behavior. On the other hand, 48 the higher levels of brain IT in the non-cleaner L. unilineatus suggested that these might be linked to 49 the development of sexual dimorphism, which occurs only in this non-cleaner species. 50 Ó 2015 Published by Elsevier Inc. 51 52 53 54 1. Introduction 55 One of the most important questions to ask when studying 56 social behavior concerns physiological processes that underlie 57 the species-specific behavioral variation. It is not surprising that 58 the integrative approach to behavior and physiology has received 59 considerable attention in fishes (Johnsson et al., 2006). Teleost fish 60 are able to exhibit unparalleled diversity when it comes to social 61 organization and behavioral output thus providing an excellent 62 opportunity to search for patterns of variation in physiological 63 proxies associated with social divergence. For instance, some spe- 64 cies live a solitary life while others tend to aggregate in schools and 65 develop the social skills that serve them well across their lifespan 66 (Bshary, 2001). Additionally, some fishes exhibit cognitive abilities 67 considered to be complex in social settings, which make them rel- 68 evant models to study social behavior and cognition (Bshary et al., 69 2014). There are several categories of physiological factors includ- 70 ing neurotransmitters and neuromodulators in the brain that can 71 be related to the behavioral plasticity of teleost fish (for review: 72 Oliveira, 2006). Among those signaling molecules are the nonapep- 73 tides, arginine vasotocin (AVT) and isotocin (IT), evolutionary pre- 74 decessors of well-known mammalian arginine vasopressin (AVP) 75 and oxytocin (OT). They belong to ancient family of neurohor- 76 mones (nonapeptides) with a well-conserved structure and core 77 functions across vertebrate taxa (Acher and Chauvet, 1995; 78 Goodson and Bass, 2001). There is strong evidence that brain non- 79 apeptides are implicated in a wide array of social and reproductive 80 behaviors in fishes (Godwin and Thompson, 2012), however, the 81 question remains, whether there is a link between the distribution http://dx.doi.org/10.1016/j.ygcen.2015.06.005 0016-6480/Ó 2015 Published by Elsevier Inc. ⇑ Corresponding author. Fax: +351 252 661 780. E-mail address: marta.soares@cibio.up.pt (M.C. Soares). General and Comparative Endocrinology xxx (2015) xxx–xxx Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen YGCEN 12127 No. of Pages 7, Model 5G 18 June 2015 Please cite this article in press as: Kulczykowska, E., et al. Brain levels of nonapeptides in four labrid fish species with different levels of mutualistic behav- ior. Gen. Comp. Endocrinol. (2015), http://dx.doi.org/10.1016/j.ygcen.2015.06.005