Arginine Vasotocin, Isotocin and Nonapeptide Receptor Gene Expression Link to Social Status and Aggression in Sex-Dependent Patterns S

Arginine Vasotocin, Isotocin and Nonapeptide Receptor Gene Expression Link to Social Status and Aggression in Sex-Dependent Patterns S. C. Lema, K. E. Sanders and K. A. Walti Biological Sciences Department, Center for Coastal Marine Sciences, California Polytechnic State University, San Luis Obispo, CA, USA..

Journal of Nonapeptide hormones of the vasopressin/oxytocin family regulate social behaviours. In mam- Neuroendocrinology mals and birds, variation in behaviour also is linked to expression patterns of the V1a-type receptor and the oxytocin/mesotocin receptor in the brain. Genome duplications, however, expand the diversity of nonapeptide receptors in actinopterygian fishes, and two distinct V1a- type receptors (v1a1 and v1a2) for vasotocin, as well as at least two V2-type receptors (v2a and v2b), have been identified in these taxa. The present study investigates how aggression con- nected to social status relates to the abundance patterns of gene transcripts encoding four vasotocin receptors, an isotocin receptor (itr), pro-vasotocin (proVT) and pro-isotocin (proIT)in the brain of the pupfish Cyprinodon nevadensis amargosae. Sexually-mature pupfish were maintained in mixed-sex social groups and assessed for individual variation in aggressive behaviours. Males in these groups behaved more aggressively than females, and larger fish exhibited higher aggression relative to smaller fish of the same sex. Hypothalamic proVT transcript abundance was elevated in dominant males compared to subordinate males, and correlated positively with individual variation in aggression in both social classes. Transcripts encoding vasotocin receptor v1a1 were at higher levels in the telencephalon and hypothalamus of socially subordinate males than dominant males. Dominant males exhibited elevated hypothalamic v1a2 receptor transcript abundance relative to subordinate males and females, and telencephalic v1a2 mRNA abundance in dominant males was also associated positively with individual aggressiveness. Transcripts in the telencephalon encoding itr were elevated in females relative to males, and both telencephalic proIT and hypothalamic itr transcript abundance varied with female social status. Taken together, these data link hypothalamic proVT expression to aggression and implicate forebrain expression of the V1a-type receptor v1a2 as potentially mediating the effects of vasotocin on behaviour in male fish. These findings also illustrate how associations between social status, Correspondence to: aggression and gene expression within the VT and IT nonapeptide systems can be contingent on S. C. Lema, Biological Sciences behavioural context. Department, California Polytechnic State University, San Luis Obispo, CA Key words: AVT, vasopressin, receptor, social behaviour, network, dominance, dominant, 93407, USA (e-mail: slema@calpoly. subordinate, mRNA, hypothalamus, fish edu). doi: 10.1111/jne.12239

The social lives of animals commonly require complex, and some- arginine vasotocin (VT) and the oxytocin (OT)/mesotocin (MT)/isoto- times rapid, shifts in agonistic behaviours (e.g. aggressive displays, cin (IT) families have emerged as key regulators of social behaviour retreats) and affiliative behaviours (e.g. pair-bonding, parental care) in vertebrates (2–6). Across vertebrate taxa, the production of VP/VT as organisms cope with changing social conditions. Given the mul- and OT/MT/IT nonapeptides occurs primarily within the hypothalamus, tifarious and context-dependent nature of sociality, social behav- from which the nonapeptides are transported to other regions within iours have evolved to be regulated by a variety of peptide and the brain or to the neurohypophysis for release into circulation. There steroid hormones that act on the central nervous system to alter is now extensive evidence that changes in forebrain nonapeptide sensory perception, social recognition and cognition (1). concentrations can alter behaviour (7–12). In addition, endogenous Neurohypophysial nonapeptides of the arginine vasopressin (VP)/ variation in the hypothalamic expression of nonapeptides has been Nonapeptide system gene expression links to aggression 143 linked to individual, sex and species variation in social behaviours receptor can vary with individual social status (44) or between (6,13–17). For example, in mammals, VP has been demonstrated to species exhibiting different mating systems (46), although no study mediate social recognition, pair-bonding behaviour and aggressive has yet explored whether brain expression of the other teleost VT behaviours associated with social status (18,19), whereas the mam- receptors might also link to behavioural variation. In the present malian counterpart of VP, namely OT, regulates filial bonding, study, we begin to investigate that question by examining whether maternal behaviours and sexual behaviours (20,21). VT or IT receptor gene expression in the brain relates to individual Previous studies of nonapeptide regulation of social behaviour variation in aggression in the pupfish C. nevadensis. In brief, we have revealed that variation in behaviour does not consistently maintained adult pupfish in the laboratory in mixed-sex social associate with nonapeptide production but, instead, often relates to groups comprising male and female pupfish of defined body size variation in the distribution and abundance of nonapeptide recep- categories. Pupfish exhibit high rates of aggression both in natural tors in the brain (15,22,23). The model that has emerged from stud- habitats and in the laboratory, and body size is a strong determina- ies of mammals is that social behaviours are influenced by patterns tion of social rank in pupfish of both sexes (47). Variation in body of expression for both the V1a-type receptor for VP and the oxyto- size within each sex therefore fostered individual variation in social cin receptor in the brain (21,23). For example, the administration of rank-associated aggression among same sex pupfish in an experi- pharmacological antagonists for the V1a-type receptor alters mental context that represents the mixed sex conditions of a natu- aggression in mammals (24,25) and the brain distribution of the ral environment. The behavioural phenotypes of individual fish were V1a-type receptor has been linked to species differences in social then documented, and relative levels of gene expression for pro- behaviours in voles, mice and other mammalian taxa (22,26,27). In vasotocin (proVT), pro-isotocin (proIT), four VT receptor subtypes addition, several studies using transgenic technologies to alter V1a- (v1a1, v1a2, v2a and v2b) and a single IT receptor (itr) in the telen- type receptor expression patterns in mice have established experi- cephalon, hypothalamus and hindbrain of socially dominant and mentally that the brain distribution of the V1a-type receptor relates subordinate pupfish of both sexes were quantified using a quantita- directly to aggressive phenotype (28,29,30,31). tive real-time reverse-transcriptase polymerase chain reaction (qRT- The expression of nonapeptide receptors in the brain also PCR). This experimental design thereby allowed for statistical isola- appears to play an important role in regulating social behaviours in tion of the effects of sex and social rank (as represented by body non-mammalian vertebrates, such as birds and fish (32). For exam- size differences) on gene transcript abundance, as well as the test- ple, several studies have now administered V1a-type receptor ing of relationships between individual variation in aggression and antagonists to actinopterygian fishes and observed changes in gene expression by examining associations between these variables aggression and courtship behaviours (12,16,33,34). Although such among individuals within each sex-size category. VT had previously evidence points to the behavioural effects of VT in fish as being been demonstrated to regulate aggression in males of this pupfish mediated by a V1a-type receptor, recent investigations in actinop- species (43) and variation in hypothalamic VT expression has been terygian fishes have revealed that the diversity of nonapeptide linked to population variation in aggression in allopatric populations receptors in these taxa is broader than that of mammals. Konno of C. nevadensis (48,49). However, because only three VT receptors et al. (35) reported the identification of the first fish V2-type recep- (v1a1, v1a2 and v2a) had been identified previously from C. nevad- tor in the sarcopterygian African lungfish Protopterus annecens in ensis pupfish (36,50), we also amplified and sequenced a partial 2009. Shortly after that discovery, V2-type receptors were identified cDNA encoding the v2b receptor from this species before evaluating in several actinopterygian fishes (36,37). Lema (36) also identified how VT or IT receptor gene expression in the brain links to aggres- two distinct V1a-type receptor genes (v1a1 and v1a2) in the pup- sion. fish Cyprinodon nevadensis, providing evidence that V1a-type receptor diversity in Actinopterygii could be greater than in tetrapod vertebrates. Subsequent studies of other taxa have now con- Materials and methods firmed the presence of two V1a-type receptors in several actinopterygian fishes (38–41), indicating that at least some actin- Identification of a partial cDNA encoding a V2b-type opterygian fishes have also evolved two or more V2-type receptors receptor for VT (40,41). This greater diversity of nonapeptide receptors in actinopterygian fishes appears to be a result of genome duplications dur- Total RNA isolation and reverse transcription ing the evolutionary history of these taxa (42). Although the Whole brain tissue was dissected from an adult male Amargosa pupfish Actinopterygii still appear to lack a V1b-type receptor, genes encod- (Cyprinodon nevadensis amargosae) (body mass: 3.99 g; standard length: ing multiple IT receptors have also now been identified in some 46.90 mm), flash frozen in liquid nitrogen and stored at 80 °C. Total RNA actinopterygian species (40,41). was extracted from the tissue with TriReagent (Molecular Research Center, In light of the newly-identified diversity of nonapeptide receptors Inc., Cincinnati, OH, USA) using bromochloropropane as the phase separation in actinopterygian fishes, it remains unclear which of these V1a- reagent. Total RNA was DNase I treated (TURBO DNAfree Kit; Life Technolo- gies, Grand Island, NY, USA) and quantified using a P300 NanoPhotometer type and V2-type receptors mediates the effects of VT on social (Implen, Inc., Westlake Village, CA, USA). Total RNA was then reverse tran- behaviour that have been demonstrated experimentally in a variety scribed in 20-ll reactions by incubating 5 lg of RNA template (4.0 ll) with of fishes (33,43–45). Recent evidence from experiments with cichlid 1.0 ll of random primers (random hexadeoxynucleotides; Promega Corp., fishes indicates that brain gene expression for the V1a-type v1a2 Madison, WI, USA) at 70 °C for 10 min. The above mixture was then

combined with 4.0 ll59 buffer, 3.0 ll of MgCl2 (25 mM), 1.0 ll of deoxy- were fed ad lib. a diet of 1 : 1 mixed spirulina (Pentair Aquatic Ecosystems, nucleotide triphosphates (dNTPs, 100 mM; Promega Corp.), 0.25 of ll recom- Sanford, NC, USA) and brine shrimp (San Francisco Bay Brand, Newark, CA, binant RNasin ribonuclease inhibitor (20 U/ll; Promega Corp.), 0.5 llof USA) ﬂake feeds. Each tank contained rocks of similar size and dimensions

GoScript reverse transcriptase and 5.5 ll of nuclease-free H2O, and then placed in approximately the same locations to provide structural complexity. reverse transcribed under a thermal profile of 25 °C for 5 min and 42 °C Three male and three female pupfish were then placed in each 114-litre for 1 h, followed by 70 °C for 15 min, in accordance with the protocol of tank (n = 14 replicate tanks); these fish were selected for variation in body the GoScript Reverse Transcription System (Promega Corp.). size, so that the social group in each tank contained one fish in each of the following sex-size categories: large male: 40–44 mm; 2.00–3.40 g body mass; medium male: 35–41 mm, 2.00–3.10 g; small male: 31–40 mm, 0.90– Amplification and sequencing of partial cDNA encoding v2b 3.40 g; large female: 35–44 mm, 1.00–2.70 g; medium female: 33–41 mm, PCR was performed using nested sets of degenerate primers designed to 1.00–2.00 g; and small female: 31–39 mm, 0.80–1.90 g. This variation in consensus regions of the open reading frame region of genes encoding a body size was selected to increase the probability of fish of both sexes V2b-type receptor from threespined stickleback (Gasterosteus aculeatus; exhibiting variation in aggression within each tank. Prior to moving fish to ENSGACG00000000754), medaka (Oryzias latipes; ENSORLG00000001494), these experimental tanks, each fish was anaesthetised in tricaine methane- pufferfish (Takifugu rubripes; ENSTRUG00000001010), and zebrafish (Danio sulfonate (MS222; Argent Chemical, Redmond, WA, USA), weighed rerio; ENSDARG00000076797). Nucleotide sequences for these degenerate ( 0.01 g), measured (standard length; 0.05 mm), and marked s.c. with a primers were: (forward1) 50-TTYTTCCARGTKTGCCCKCAGCT-30, (forward2) visible implant elastomer tag (Northwest Marine Technologies, Seattle, WA, 50-ATDTGGGACRTMACYGACMGMTT-30, (forward3) 50-CARGTBSTCGGSATGTTTK USA). Fish in each tank were fed to satiation twice daily. CRTC-30, (reverse3) 50-TGAACAGMAGGTARATGCAKGGRTT-30, (reverse2) 50-AG After transfer of fish to the experimental tanks, fish were given 5 days to CARCATSARGAYGGTGAARGT-30, and (reverse1) 50-CTGSACRGTRAAGAANGG acclimate before commencing behavioural observations. Focal behavioural MGC-30. First-strand cDNA was amplified in 50-ll reactions containing observations were recorded over 10-min intervals on days 5, 10, 18 and 30

25 ll of GoTaq Master Mix (Promega Corp.), 21 ll of RNase-free H2O, 2 ll after the introduction of fish. For each tank, observations were made on of cDNA template and 1 ll each of forward and reverse primers (50 mM). four of the six fish: the largest male and female and the smallest male and Each PCR was run on a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) female. Quantified aggressive behaviours included aggressive ‘charges’, ‘nips’, using a thermal profile of 95 °C for 2 min followed 34 cycles of 95 °C for and ‘displays’ as described previously (43,50) and the individual that initiated 30 s, 50 °C for 30 s and 72 °C for 1 min, and then ended with 72 °C for and received the aggression was recorded for each agonistic interaction for 5 min. Resulting PCR products were examined on 1.2% ethidium bromide a total of nine behaviour variables (see listed in Table 1). Feeding behaviour agarose gels, and cDNA from any bands of predicted product size was puri- was also recorded for all fish observed. fied (QIAquick PCR Purification Kit; Qiagen, Valancia, CA, USA) and then Sanger sequenced (MacrogenUSA, Rockville, MD, USA). The resulting Tissue collection sequences were aligned using SEQUENCHER, version 4 (GeneCodes Corp., Ann Arbor, MI, USA) to generate a cDNA sequence, which was then used in On day 32, 2 days after the last behavioural observation, four fish (the larg- BLAST searches of the NCBI database (http://www.ncbi.nlm.nih.gov) to con- est and smallest males and the largest and smallest females) were collected firm the identity of this cDNA sequence. from each tank. All fish were collected between the hours 13.00 h and 16.00 h to eliminate any effects of photoperiod on gene expression patterns in the VT and IT systems (50). Fish were euthanised using MS222 immedi- Phylogenetic analysis ately after capture, weighed and measured [large males: 41.29 0.68 mm, The deduced amino acid sequence of the partial cDNA from C. n. amargosae 2.54 0.17 g (mean SEM); small males: 35.56 1.04 mm, was aligned to select sequences for VT/VP receptors and IT/MT/OT receptors 1.55 0.67 g; large females, 39.06 0.81 mm, 1.75 0.10 g; and small females: 34.21 0.64 mm, 1.26 0.08 g]. The brain tissues of each fish from other vertebrates using CLUSTALX (51). This alignment was used to gener- were dissected, flash frozen with liquid nitrogen and stored at 80 °C. ate a phylogenetic tree in MEGA, version 5 (52) via the Neighbor-joining method (53) with a p-distance model and pairwise deletion of sequence Brain tissue was subdivided macroscopically for the analysis of relative gaps. Confidence values for each node were determined using bootstrap gene transcript abundance in three regions: telencephalon, hypothalamus, tests (1000 replicates). and hindbrain/brainstem. The telencephalon was dissected first by severing the tissue with a scalpel along the natural commissures that distinguish the olfactory bulbs from the telencephalon, and the telencephalon from the Associations between aggression and gene expression in diencephalon. The telencephalon region therefore contained the pars parvo- the VT and IT systems cellularis cells of the nucleus preopticus (i.e. parvocellular neurones of the proptic area; POA) (54), which has been shown to exhibit positive immunoreactivity for VT in C. nevadensis pupfish (48). The hypothalamus was then Individual behavioural variation within mixed-sex social dissected by inverting the brain and removing the ventrolaterally extending groups hypothalamic lobes and diencephalon tissue below the optic tectum (55,56). The hypothalamic subdivision therefore contained the pars magnocellularis Adult male and female pupfish (C. n. amargosae) were collected by minnow (magnocellular and gigantocellular POA neurones) that occurs posterodorsal- trap on 9 September 2012 from the Amargosa River, San Bernardino County, ly within the nucleus preopticus (54). Lastly, the hindbrain was removed by California, USA (35°50.9760N, 116°13.8500W). Fish were transported to Califor- cutting the brain ventrally below the boundary between the optic tectum nia Polytechnic State University, San Luis Obispo, CA, USA, where they were and cerebellum. maintained in 208-litre closed system tanks at 24-26 °C and 0.4 p.p.t. salinity. All experimental procedures were approved by the Animal Care and Use Com- mittee of California Polytechnic State University, San Luis Obispo. Quantitative real-time RT-PCR assays Social groups of sexually-mature pupfish were established in fourteen replicated 114-litre tanks (length 90 cm, width 45 cm, height 30 cm) under Total RNA was extracted from the subdivided brain tissues using TriReagent conditions of 25.64 0.34 °C (mean SD) and 0.4 p.p.t. salinity. All fish as described above. The resulting RNA was DNase I treated (TURBO DNA-

free Kit; Ambion, Austin, TX, USA) and quantified using a P300 NanoPho- two-factor ANOVA models. ANOVA models initially included tank as a factor, tometer (Implen, Inc., Westlake Village, CA, USA) (260 : 280 ratios > 1.98). although this factor was found to be nonsignificant in all statistical compar- Total RNA was then reverse transcribed in 16-ll reaction volumes contain- isons and so was removed from the ANOVA models. Post-hoc pairwise com- ing 8 ll of RNA (15 ng/ll), 0.8 ll of dNTPs (Promega), 0.8 ll of random parisons between groups were subsequently calculated using Tukey’s primers (500 lg/ml; Promega), 0.0625 ll of recombinant RNasin ribonucle- honestly significant difference (HSD) tests when a significant effect of factor ase inhibitor (40 u/ll; Promega), 0.1375 ll of nuclease-free H2O, and 3.2 ll or interaction was identified. of 5 9 buffer, 2.4 ll of MgCl2 and 0.6 ll of GoScript reverse transcriptase Because many of the behaviours measured might correlate with one (Promega). Reverse transcription reactions were run under a thermal profile another (e.g. charges by focal fish, nips by focal fish), inter-relationships of 25 °C for 5 min and 42 °C for 1 h, followed by 70 °C for 15 min to between behavioural variables were exampled using principal components inactivate the reverse transcriptase. analysis (PCA) to identify underlying multivariate structure among the mea- qRT-PCR was conducted according to the guidelines outlined by Bustin sured behaviours. The number of principal components (PCs) retained was et al. (57). Primers for Taqman probe qRT-PCR assays were designed to pro- determined using two criteria: (i) via Kaiser’s criterion where only PCs with ei- tein coding regions of the partial cDNAs for each gene (see Supporting genvalues > 1.0 were retained for subsequent analyses and (ii) by examination information, Table S1) (36,50). Primers and probes designed to partial cDNAs of slope changes in the resulting Scree plot. Both criteria led to identical con- encoding elongation factor-1a (ef-1a) (GenBank accession number clusions on how many PCs to retain. The resulting PCs were then used as inde- EU906930) and 60S ribosomal protein L8 (rpl8) (KJ719257) from Amargosa pendent variables to examine the relationships between behaviour and relative pupfish were used as endogenous control genes. Primers and probes were mRNA abundance in specific brain regions, as described below. synthesised by Integrated DNA Technologies (Coralville, IA, USA) and, when Gene transcript abundance data that failed to conform to the assump- possible, primers were designed to span an intron boundary. All qRT-PCR tions of normality were ln transformed prior to analysis. Effects of Sex and reactions were conducted as 16-ll reaction volumes containing 4.9 llof Body Size Category on the relative abundance of each transcript were exam- nuclease-free water (Sigma, St Louis, MO, USA), 8 ll of iQ Universal Probes ined using two-factor ANOVA models for each brain subdivision separately. If Supermix (Bio-Rad, Hercules, CA, USA), 0.8 ll each of forward and reverse a significant main factor or interaction was found, a Tukey’s HSD test was primers (10 lM), 0.8 ll of probe (45 lM) and 1.5 ll of reverse-transcribed then performed to assess pairwise differences among Sex–Body Size catego- cDNA template. All assays were run on a 7300 Real-Time PCR System ries. The relative mRNA value for v1a1 in the telencephalon of one large (Applied Biosystems, Foster City, CA, USA) using a thermal profile of 50 °C female was identified as an outlier (60) and was removed from the analyses. for 2 min, 95 °C for 10 min, 42 cycles of 95 °C for 15 s and 59 °C for Lastly, Pearson’s correlations were used to examine whether individual 1 min. For each gene, a standard curve was made from a pool of RNA from behaviour PC values obtained from the PCA showed any relationship with samples representing all body size categories and sexes. Each standard was individual variation in relative gene transcript abundance in specific brain serially diluted and assayed in triplicate. DNA contamination was assessed subdivisions. To reduce the probability of Type I statistical errors, correla- for each gene by analysing RNA samples that were not reverse-transcribed, tional associations were only examined for gene transcripts that showed a and each qPCR run included two samples without cDNA template to further significant difference in relative expression levels among fish ‘Sex–Body Size’ control for contamination. The specificity of each primer set was confirmed categories using the two-factor ANOVA model analyses described previously. by cloning and Sanger sequencing select PCR products. For each gene, cor- To further correct for multiple correlational testing, we controlled the false- relation coefficients (r2) for the standard curve were > 0.97. PCR efficiencies discovery rate (FDR) at 0.05 using the sequential P value threshold method for each gene were calculated using the equation: % efficiency = described by Benjamini and Hochberg (61) and Benjamini et al. (62). Because [10(1/slope)–1]9 100 and are provided in the Supporting information the original joint data set of behaviours was normally distributed, behaviour (Table S1). Relative levels of ef-1a (one factor ANOVA models, P = 0.602– PCs were considered independent when calculating modified P value thresh- 0.912 depending on brain subdivision) and rpl8 (P = 0.743–0.920) did not olds. Separate brain regions were also treated as independent when calcu- vary among the four sex and body size categories. For each gene, relative lating FDR corrections. Because this statistical approach is conservative and mRNA levels were subsequently calculated using the standard curve and may increase the likelihood of making Type II errors, we report the exact P normalised to the geometric mean of transcript abundance values for ef-1a values for each correlation at the same time as stating explicitly which and rpl8; the use of these transcripts for normalisation was confirmed by relationships were regarded as statistically significant after correction to their stabilisation values (M) as calculated with geNorm software (Q+BASE, maintain the family-wise FDR at 0.05. version 2.6.1; BioGazelle NV, Gent, Belgium) (58,59). Finally, expression of each gene of interest was expressed as a relative level by dividing all resulting values for a given tissue by the lowest mean value calculated Results for any treatment group in that tissue. The mean intra-assay percentage coefficient of variation (% CV) for the qPCR reactions was 0.49%, and the Identification of a partial cDNA encoding v2b from pupfish interassay % CV between plates was 2.91%. Degenerate primer RT-PCR amplified a 912-bp partial cDNA sequence encoding a putative v2b mRNA from C. n. amargosae Statistical analysis (GenBank accession number KJ668598). BLAST analysis of this cDNA

All statistical analyses were conducted using JMP PRO, version 11 software fragment indicated that the deduced polypeptide from this cDNA (SAS Institute, Cary, NC, USA). Behavioural data that failed to conform to shared high nucleotide sequence identities (81–89%) with V2b-type the equal variances assumption of parametric statistics were transformed as VT receptors from other teleost fishes. Alignment of the deduced ln(behaviour + 1) to homogenise variances prior to the statistical analyses. amino acid sequence of this partial cDNA confirmed closest struc- Preliminary analyses using repeated measures analyses on the four behavio- tural similarity to V2b-type receptors from teleost fishes and V2- ural sampling times (i.e. days 5, 10, 18 and 30) found no significant effects type receptors from birds. Figure 1 shows phylogenetic relationships of time on any behaviour measured, and so the analyses were performed on mean values of each behaviour calculated across the four sampling periods based on amino acid sequences for this new partial cDNA, select for each individual fish. Effects of Body Size Category, Sex and Body Size VT/VP and IT/MT/OT receptors from other vertebrates, and full- Category 9 Sex interaction on measured behaviours were determined using length cDNAs encoding v1a1 (GenBank accession number

IT/MT/OT-type teleost ITR V1b-type

firebelly newt V1b V1b human c mouse V1b hicken V1b

human V1a prairie vo ITR sucker white pupfish ITR (GQ981415) zebrafish ITR

bullfrog MTR hicken OTR c le V1a cow V1a cow OTR mouse OTR human OTR chicken V1a 100

bluehead wrasse V1a2 99 pupfish V2b (KJ668598) teleost 100 100 stickleback V2b V2b-type medaka V1a2 100 96 100 zebrafish V2b 100 100 stickleback V1a2 90 100 100 77 d V1a2 100 chicken V2 rockhin 100 teleost zebra finch V2 V1a2-type 98

tilapia V1a2 98 firebelly newt V2 lungfis pupfish V1a2 (GQ981413)rockhind V1a1 h V2

mous e V2 h uman V2 stickleb cow V2 pufferfish V1a1 bluehead wrasse V1a1 pufferfish V2a stickleback V2a2 stickleback p

stickleback V1a1 upfish V2a (GQ981414) tilapia V1a1 tilapia V1a-type medaka V1a1 ack V2a1

teleost V1a1-type (GQ981412) V1a1 pupfish V2-type teleost

octopus OPR V2a-type

0.05

Fig. 1. Phylogenetic tree based on the alignment of deduced amino acid sequences for nonapeptide receptors from select teleost fishes and other vertebrates. The tree was assembled using a Neighbor-joining method with pairwise deletion of gaps. Bootstrap values (1000 replicates) are displayed only at nodes for major clades of receptors within the tree. Coloured clades denote groupings of the V1a1, V1a2, V2a, V2b receptors for vasotocin (VT) and the teleost receptor for isotocin (IT) within the tree. Boxes indicate the deduced polypeptide encoded by the newly-identified v2b cDNA from the pupfish Cyprinodon nevadensis amargosae, as well as previously identified v1a1, v1a2, v2a and itr cDNAs from this same species. The octopressin receptor (OPR) from octopus was used to root the tree. GenBank accession numbers for each pupfish cDNA are provided in parentheses within the boxed sequences. GenBank accession nos. for all other sequences within the tree are provided in the Supporting information (Table S2).

GQ981412), v1a2 (GQ981413), v2a (GQ981414) and itr (GQ981415) Pupfish aggression varies with sex and body size from C. n. amargosae pupfish (36). This phylogeny illustrates the presence of multiple V1a-type and V2-type nonapeptide receptors The frequency of aggression within the mixed-sex social groups varied in actinopterygian fishes and confirms that the newly-identified with both body size category and sex of the focal fish. Large focal partial cDNA is part of the clade of V2b-type receptors of fishes. males were highly aggressive, and many of the aggressive charges

(Body Size Category 9 Sex interaction: F1,54 = 8.430, P = 0.005) and Body Size: F1,54 = 16.440, P = 0.0002) and nips (Body Size: nips (Body Size Category 9 Sex interaction: F1,54 = 4.082, P = 0.048) F1,54 = 5.241, P = 0.026) was also observed. Small focal females that were observed came from large focal males charging and nipping received both more aggressive charges from females and more nips other males of smaller body size (Fig. 2A). Large and small focal males from females (Fig. 3B). Taken as a whole, these observations indi- both showed high frequencies of aggressive charges (Sex: cate that males initiated more aggression and received signiﬁcantly

F1,54 = 76.658, P < 0.0001) and nips (Sex: F1,54 = 25.138, P < 0.0001) less aggression than females, and that larger fish were more toward both large and small females (Fig. 2B). Focal females of large aggressive than smaller fish of the same sex. Feeding behaviour did and small body sizes exhibited very little aggression directed toward not vary with sex (F1,54 = 0.135; P = 0.715) or body size males (Fig. 2A) and directed aggression at other females at rates that (F1,54 = 0.192, P = 0.663) category. were approximately 25% lower than rates by focal males (Fig. 2B). This elevated aggression by males was also apparent in the num- Relationships among measured behaviours reveal composite ber of times focal fish were recipients of aggression. Small focal behavioural phenotypes females, large focal females and small focal males all received charges (Fig. 3A) (Body Size Category 9 Sex interaction: PCA reduced the nine measured categories of agonistic behavioural

F1,54 = 7.856, P = 0.007) and nips (Sex: F1,54 = 10.113, P = 0.0024; interactions down to two PC axes (Table 1), which together

Body Size: F1,54 = 6.998, P = 0.011) from other males more fre- accounted for 61.11% of the variation observed in agonistic behav- quently than did large focal males. Signiﬁcant variation in the fre- iour (Fig. 4). Behaviour PC1 (eigenvalue = 4.2438) explained 47.15% quency of aggressive charges (Sex: F1,54 = 19.707, P < 0.0001; of the behavioural variation observed. All quantiﬁed offensive

(A) Focal aggression toward males 25 (A) Male aggression toward focal Female - small 25 Female - large Female - small 20 Male - small a Female - large 20 b Male - large a Male - small 15 a Male - large 15 10 10 5 a a' Behaviours per 10 min b' a 5 b a' a'

a' Behaviours per 10 min a a' a' b' 0 0 Charges Nips Charges Nips Aggressive behaviour Aggressive behaviour (B) Focal aggression toward females 25 b (B) Female aggression toward focal 25 20 b 20 15 15 10 a b' 10 b' a 5 a Behaviours per 10 min b a' a' 5 b Behaviours per 10 min a' b' a',b' a',b' 0 b Charges Nips 0 Aggressive behaviour Charges Nips Aggressive behaviour Fig. 2. Aggression toward male (A) and female (B) pupfish varied with sex and body size of the focal fish. Large focal males performed aggressive Fig. 3. Frequencies of aggression directed toward focal individuals from charges and nips toward other males at a higher frequency than did either nonfocal males (A) and females (B). Focal male pupfish of large body size small males or females of large or small body size (A). Males of both large received less aggression from nonfocal males than did small focal males or and small body sizes were both observed to initiate aggressive charges and focal females of either small or large size (A). Aggression from nonfocal nips toward females at rates three- to four-fold greater than that of females was directed more frequently toward other females of small body females (B). All data are shown as the mean SEM. Lowercase letters size (B). Data are shown as the mean SEM. Lowercase letters indicate sig- denote significant pairwise difference (Tukey’s honestly significant difference nificant pairwise difference (Tukey’s honestly significant difference tests) tests) between sexes or size categories within each behaviour category. between sexes or size categories within each behaviour category.

included the frequencies of aggressive charges and nips received from Female - small nonfocal females. The frequency of nips by focal females also showed a Female - large less strong positive loading on PC2. Examination of variation in PC2 val- Male - small 5.0 ues among sex and body size categories revealed that PC2 represented Male - large variation in female behaviour, with small females exhibiting higher PC2 values (mean SEM: 0.401 0.358) and large females having lower 2.5 PC2 values (0.640 0.220); in contrast, PC2 values varied little between small males (0.092 0.315) and large males (0.078 0.231). We therefore interpret behaviour PC2 as representing the strength and 0.0 sign of social subordination to female aggression in the mixed-sex social groups. Focal ﬁsh with higher PC2 values were recipients of

PC2 (13.96%) greater female aggression and were therefore considered socially subordinate to females with lower PC2 values. –2.5

Transcript abundance for proVT and proIT are inﬂuenced by –5.0 social status –5.0 –2.5 0.0 2.5 5.0 The relative abundance of gene transcripts encoding proVT in the telen- PC1 (47.15%) cephalon (Sex: F1,46 = 8.882, P = 0.0046) and hypothalamus

Fig. 4. Principal components analysis (PCA) for the nine aggressive behav- (Sex 9 Body Size Interaction: F1,46 = 19.679, P < 0.0001) varied in iours observed during agonistic interactions of focal pupfish. PCA reduced patterns dependent on sex and social status. Transcripts encoding the nine behaviours into two independent principal component (PC) vari- proVT were more than three-fold more abundant in the hypothalamus ables. PC1 explained 47.15% of the behavioural variation observed, whereas of large males than in small males or in large females (Fig. 5A). In the PC2 explained 19.86% of the behavioural variation. Clustering of individual telencephalon, proVT mRNA levels were higher in small males com- focal fish by sex and body size is apparent along the PC1 axis. pared to small females; although not statistically significant, proVT transcript abundance also trended toward being elevated in small, aggressive behaviours (e.g. charging, nipping) performed toward socially subordinate males compared to dominant, large males (Fig. 5A). males and females by focal fish loaded positively on PC1, whereas Patterns of proIT transcript abundance varied from that of proVT. aggression toward the focal fish (e.g. charges by males at focal, nips Transcripts encoding proIT were only approximately one-third as by males at focal) loaded negatively to PC1 (Table 1). The finding that abundant in the telencephalon of small female pupfish compared PC1 represents a combination of strong positive loadings of aggres- to large females or to males of either small or large body size sion by the focal fish and negative loadings for aggression by nonfo- (Sex 9 Body Size Interaction: F1,46 = 5.338, P = 0.0254) (Fig. 5B). cal individuals points to PC1 as an indicator of overall ‘social No differences in proIT gene transcript abundance were observed in dominance’ in the mixed sexes experimental testing design, with high the hypothalamus. PC1 values denoting fish of higher social dominance and a low PC1 value being indicative of an individual of lesser social status. Variation in VT and IT receptor mRNA levels with social Behaviour PC2 (eigenvalue = 1.2581) explained an additional 13.96% status and sex of the behavioural variation observed. Behaviours that loaded to PC2 In the telencephalon, mRNAs encoding VT receptors v1a1, v1a2 and itr varied with sex and body size (Fig. 6A). Transcripts encoding the Table 1. Factor Loadings for Principal Components (PC) Analysis Performed v1a1 receptor were greater in small females and small males than on Measured Agonistic Behaviours. in large fish of the same sex (F1,46 = 13.093, P = 0.0007). Gene Behaviour PC1 PC2 transcripts encoding v1a2 in the telencephalon, however, were at highest relative abundance in large, socially dominant male pupfish,

Focal charge at male 0.7480 0.1607 and lowest in small, female pupﬁsh (F1,46 = 8.882, P = 0.0046). In Focal nip at male 0.7910 0.1445 addition, females exhibited greater itr mRNA levels in the telen-

Focal charge at female 0.8362 0.2126 cephalon than males (F1,46 = 12.354, P = 0.0010). Focal nip at female 0.6543 0.4434 The abundance of transcripts encoding the v1a1 receptor was Male charge at focal 0.8458 0.0434 greater in the hypothalamus of small males compared to large Male nip at focal 0.5621 0.0917 males (Sex 9 Body Size Interaction: F1,46 = 9.328, P = 0.0038) Female charge at focal 0.6729 0.5127 (Fig. 6B). Transcripts for v1a2 in the hypothalamus also varied Female nip at focal 0.3313 0.8248 among pupﬁsh categories, and were observed to be almost four- Display by focal 0.5809 0.1193 fold greater in the hypothalamus of large, socially dominant male Loadings shown in bold indicate the axis of strongest loading for the pupﬁsh compared to small males or large females (Sex 9 Body Size measured variable. Interaction: F1,46 = 10.554, P = 0.0022). Hypothalamic itr mRNAs

Female - small Female - small Female - large Female-large Male - small Male - small (A) Male - large Male - large proVT A Telencephalon 6 b' ( ) 3.5 a a" 3.0 b a 4 a',b' a",b" 2.5 b'

a,b 2.0 a',b' n.d. a,b a' 2 a,b a',b' n.d. a a' 1.5 b" b b" Relative mRNA level Relative mRNA level a' 1.0 0 Telencephalon Hypothalamus 0.5 v1a1 v1a2 v2a v2b itr B proIT ( ) 6 (B) Hypothalamus 6.0 b' b b 4 a,b 5.0 n.d. 4.0 b a 2 3.0 b" Relative mRNA level a,b a',b' Relative mRNA level 2.0 a,b n.d. a",b" a' n.d. 0 a a' a" a",b" Telencephalon Hypothalamus 1.0

Fig. 5. Differences in relative gene transcript abundance for proVT (A) and v1a1 v1a2 v2a v2b itr proIT (B) in the telencephalon and hypothalamus were observed to be dependent on sex and body size category, which is linked directly to social (C) Hindbrain status. Transcripts encoding proVT mRNAs were observed at levels four times 10 a greater in the hypothalamus of large males than in small males (A). In the telencephalon, however, proVT transcript abundance was greater in small 8 males than in females. Although proIT mRNAs did not exhibit any differ- a' ences in expression in the hypothalamus, proIT transcripts were reduced in the telencephalic tissue of small females relative to large females or males 6 of either small or large size (B). All data are shown as the mean SEM a,b (n = 12–13 fish per sex and body size category). Lowercase letters indicate b 4 n.d. pairwise differences between sex and body size categories (Tukey’s honestly a',b' a',b' difference significant tests). Relative mRNA expression values for each tran- Relative mRNA level n.d. n.d. 2 script are shown normalised against the lowest abundance value of that b b' transcript within the same tissue. n.d., no difference. 0 v1a1 v1a2 v2a v2b itr were at greater relative levels in large females than in small Fig. 6. Variation in relative abundance of gene transcripts encoding the females but at similar levels in both size categories of males vasotocin (VT) receptors v1a1, v1a2, v2a, and v2b, and the isotocin (IT) 9 = = (Sex Body Size Interaction: F1,46 5.811, P 0.0200). receptor itr. Data for the telencephalon (A), hypothalamus (B) and hindbrain In the hindbrain, variation in relative expression levels of nona- (C) are plotted separately. Transcript abundance for v1a1 and v1a2 varied in paptide receptor mRNAs could be attributed to sex differences. both the telencephalon (A) and hypothalamus (B) in patterns linked to body Female pupfish exhibited higher relative levels of transcripts encod- size and/or social status. Transcript abundance for itr in the telencephalon was also observed to be approximately two-fold greater in relative levels in ing receptors v1a1 (F1,46 = 18.561, P = 0.001), v1a2 (F1,46 = females compared to males (A). Relative levels of transcript encoding the VT 6.0246, P = 0.0287), v2a (F = 4.596, P = 0.0374) and itr 1,46 receptors v1a1 and v2a were also found to be elevated in the hindbrain of (F = 4.242, P = 0.0451) (Fig. 6C), although not all of these dif- 1,46 small female pupfish (C). All data values are shown as the mean SEM. ferences were of sufficient effect size to be statistically significant Lowercase letters indicate pairwise differences difference (Tukey’s honestly in Tukey’s post-hoc comparisons. Transcript abundance for v2a also significant difference tests) between sex and body size categories. Relative varied in patterns dependent on body size (Fig. 6C)(F1,46 = 7.044, mRNA expression values are normalised against the lowest mean abundance P = 0.0109) and small, socially-subordinate female pupfish value for that transcript within the same tissue. n.d., no difference.

Journal of Neuroendocrinology, 2015, 27, 142–157 © 2014 British Society for Neuroendocrinology 150 S. C. Lema et al. exhibited relative v2a mRNA levels almost five-fold higher than the (r = 0.613, P = 0.026). In small females, hypothalamic proVT levels observed in large, dominant males. mRNA levels were correlated negatively with the behaviour PC2 value (Fig. 7D)(r= 0.719, P = 0.0056), indicating that hypothalamic proVT gene transcripts were more abundant in the hypo- Correlations between behaviour and nonapeptide system thalamus of small female pupfish receiving less aggression from gene expression other females. Significant correlative relationships were observed between hypo- A correlation between individual nonapeptide receptor gene thalamic proVT mRNA levels and behaviour PC1 values in both expression and aggressive behaviour was also observed for tran- small male (Fig. 7A)(r= 0.841, P = 0.0004) and large male scripts encoding the v1a2 receptor in the telencephalon of large (Fig. 7B)(r= 0.727, P = 0.0074) pupfish. These relationships male pupfish. Large male pupfish with higher telencephalic v1a2 revealed that males that were more aggressive within either body mRNA levels were more aggressive than large males with lower size category also exhibited higher hypothalamic proVT transcript v1a2 levels (Fig. 8A)(r= 0.742, P = 0.0057). Although again not abundance. Although not considered statistically significant follow- statistically significant under the modified P value thresholds ing the stepwise P value threshold procedure used to control the used here, our data did suggest that female pupfish of both family-wise FDR (61,62), our data provide preliminary evidence large (Fig. 8B)(r= 0.653, P = 0.0213) and small body size that relative mRNA levels for proVT in the telencephalon of small, (Fig. 8C)(r= 0.575, P = 0.0398) may exhibit positive relationships subordinate males may relate positively individual aggressiveness, between v1a2 expression in the telencephalon and behaviour PC2 as indicated by an individual’s behaviour PC1 value (Fig. 7C) values indicative of the amount of female aggression received.

(A)(6 Male - small B) 6 Male - large

4 4

2 2 Behaviour PC1 Behaviour PC1 0 0 r = 0.841 r = 0.727 P = 0.0004 P = 0.0074 –2 –2 0246 0 5 10 15 20 25 Hypothalamic Hypothalamic proVT mRNA expression proVT mRNA expression

(C)(D) 6 Male - small 4 Female - small

4 2 r = –0.719 P = 0.0056 2

0 Behaviour PC1 0 Behaviour PC2 r = 0.613 P = 0.026 –2 –2 0204060 0 510152025 Telencephalic Hypothalamic proVT mRNA expression proVT mRNA expression

Fig. 7. Aggressive behaviour principal components (PC1 or PC2) plotted against relative mRNA levels for proVT in the hypothalamus or telencephalon of pupfish. Data indicate statistically significant positive correlations between aggressive behaviour PC1 and hypothalamic proVT gene transcript abundance in both small male (A) and large male (B) pupfish. Nonsignificant relationships were observed between telecephalic proVT mRNA abundance and aggression PC1 in small male pupfish (C), and between aggression PC2 and hypothalamic proVT gene transcript abundance in small female pupfish (D). Correlation statistics are also provided. Statistically significant differences were determined using a sequential P value threshold procedure (61,62) to control the false-discovery rate.

Even though relative gene expression for other nonapeptide (A) 6 Male - large receptors (e.g. v1a1, v2a, itr) varied among sex and body size categories in some brain regions (Fig. 6), no statistically signiﬁ- 4 cant relationships were detected between individual variation in behaviour and variation in mRNA levels for these other receptors. 2

Behaviour PC1 Discussion 0 r = 0.742 Brain VT nonapeptide production has been linked to variation in social behaviours in several fish species (16,49,63–71) and pharma- P = 0.0057 –2 cological blockage of VT signaling in the brain can inhibit 0.0 0.5 1.0 1.5 2.0 2.5 aggression in male fishes (33,72). However, it is not known which VT receptors mediate these behavioural effects. Even less is known Telencephalic v1a2 mRNA expression about the importance of IT receptors for fish behaviour despite recent evidence showing that IT can modulate behaviours associ- (B) 2 Female - large ated with aggression and submission during agonistic exchanges (12,73–75). The discovery that multiple V1a-type, V2-type and IT r = 0.653 receptors arose via genome duplications in the evolutionary history 1 P = 0.0213 of actinopterygian fishes points to the possibility that several nonapeptide receptors could be involved in VT and IT regulation of social behaviours in these taxa. 0 In the present study, we maintained male and female adult C. n. amargosae pupfish in mixed-sex social groups in the laboratory Behaviour PC2 to generate individual variation in aggression that could resolve rela- –1 tionships between aggression and gene expression within the neural VT and IT systems. Our observations of behaviour in these social –2 groups indicated that pupfish males performed aggression at higher 0.0 0.5 1.0 frequencies than females, and larger pupfish of either sex behaved Telencephalic more aggressively (but received less aggression) than smaller individ- v1a2 mRNA expression uals of the same sex. Cyprinodon nevadensis pupfish are highly aggressive both in laboratory settings (43,49) and in their wild habi- (C) 4 Female - small tats (43,76), and variation in aggression associated with sex and body size is typical of C. nevadensis and other pupfishes (43,47,76,77). r = 0.575 P = 0.0398 2 Hypothalamic proVT mRNA abundance varies with social status and individual aggression We found that the abundance of transcripts encoding proVT in the telencephalon and hypothalamus varied in patterns related to both

Behaviour PC2 0 sex and social status. Small, socially subordinate males were observed to have elevated telencephalic proVT mRNA levels, and proVT mRNAs were approximately four-fold more abundant in the –2 hypothalamus of large, socially-dominant male pupfish than in the 0.0 0.2 0.4 0.6 0.8 1.0 hypothalamus of small, socially-subordinate males or of large Telencephalic females, suggesting that aggression by males is linked to higher v1a2 mRNA expression relative levels of hypothalamic proVT gene expression. Supporting Fig. 8. Aggressive behaviour principal components (PC1 or PC2) plotted this idea, we also observed that the relative level of hypothalamic against the relative abundance of transcript encoding vasotocin (VT) receptor proVT mRNAs correlated positively with an individual male’s v1a2 in the telencephalon. Aggression PC1 was correlated positively with rela- behaviour PC1 score, which serves as a composite indicator of tive levels of v1a2 mRNAs in the telencephalon of large male pupfish (A). Addi- aggressiveness. Such positive correlations between hypothalamic tional, nonsignificant correlations were also observed between aggression PC2 proVT transcript abundance and behaviour PC1 were observed for and v1a2 mRNA levels in the telencephalon in both large female (B) and small female (C) pupfish. Correlation statistics are also provided. Statistically signifi- both large, dominant males and small, subordinate males. cant differences were determined using a sequential P value threshold proce- Variation in neural expression of proVT transcripts has previously dure (61,62) to control for false discoveries (Type I errors). been linked to social status in male teleost fishes. Renn et al. (78),

Journal of Neuroendocrinology, 2015, 27, 142–157 © 2014 British Society for Neuroendocrinology 152 S. C. Lema et al. for example, observed that dominant, territorial male Astatotilapia behaviours associated with social dominance, although this failed burtoni cichlids exhibited higher proVT mRNA levels in whole brain to increase in response to experimental elevation of 11-ketotestos- than subordinate, nonterritorial males. Greenwood et al. (79) terone androgen concentrations (70). similarly found that male A. burtoni cichlids varied in preoptic By contrast to evidence for magnocellular and gigantocellular VT proVT mRNA expression in patterns linked to social status and neuronal phenotype being related to reproductive function, VT pro- accompanying aggressive behaviours. In that work, dominant male duction in parvocellular neurones within the POA of teleost fishes cichlids were observed to have higher proVT mRNA abundance has been linked to experience with environmental stressors. VT has in the gigantocellular neurones of the preoptic area as assessed by a synergistic action with corticotrophin-releasing hormone in pro- in situ hybridisation, whereas subordinate males had greater proVT moting the release of adrenocorticotrophic hormone from the pitui- expression in parvocellular neurones. Because the hypothalamic tary (87), and parvocellular proVT gene expression has been shown brain subdivision in the present study conducted in pupfish would to increase in fish after exposure to acute environmental stressors have included both the magnocellular and gigantocellular POA neu- (88). Whole brain proVT mRNAs and VT peptide have also been rones, our results support the hypothesis that aggression by male observed to become elevated after exposure to stressors such as fish is positively associated with magnocellular and/or gigantocellu- pathogen challenge (89) or high social densities (90). lar proVT expression (16,70,79). Our data support a link between parvocellular VT and stress in that There is evidence that these relationships between brain proVT subordinate male pupfish exhibited elevated proVT mRNA levels in the gene transcript abundance and behaviour may be generated in part telencephalon, the brain subdivision containing the parvocellular pre- from differences in behavioural experience associated with belong- optic neurones. Those smaller, subordinate males were recipients of ing to different social classes. In birds, social interactions have been high rates of aggression from the larger, dominant male pupfish in shown to influence the neural VT-immunoreactive phenotype (80), their tanks. The finding by Greenwood et al. (79) that the frequency and recent work in anole lizards demonstrated that the act of of fleeing from agonistic interactions correlated positively with proVT engaging in sexual behaviours itself activates VT-producing neurones mRNA levels in parvocellular neurones among individual, nonterritori- in the paraventricular nucleus (PVN) and preoptic area (81). Huffman al A. burtoni males provides evidence that variation in parvocellular et al. (44) recently found that, although the relative abundance of neurone VT expression may be related to experiences with social proVT mRNAs was similar in the whole brain tissues of subordinate stress. Similarly, prior work with closely-related taxa of C. nevadensis and dominant male A. burtoni fish, male fish transitioning from sub- pupfish linked experience with aggression to the soma size of parvo- ordinate to dominant social status after experimental removal of the cellular VT-immunoreactive (VT-ir) neurones (49). That work found dominant male in a social group exhibited whole brain proVT mRNA that C. n. amargosae and Cyprinodon nevadensis mionectes male levels approximately three-fold greater than the levels observed in pupfish reared as larvae from 15 days post fertilisation until sexual either subordinate or dominant fish of stable social class. Taken maturity (17–23 weeks of age) in environments with higher rates of together, such findings not only suggest that behavioural interactions social aggression (and thus elevated social stresses) had smaller-sized influence neural proVT gene transcription, but also that changes to parvocellular VT-ir neurones (49). When reared from hatching under brain proVT expression may be most apparent when individuals expe- common laboratory conditions, C. n. mionectes males also behaved rience a change in social status. If that is indeed the case, such more aggressively and exhibited larger parvocellular VT-ir neurones behaviourally-induced responses of proVT gene expression may help than C. n. amargosae males (49). Although these subspecies compar- explain why relationships between proVT gene expression and behav- isons were conducted to help explain prior observations of variation iour have been challenging to identify consistently in studies using in neural VT-ir phenotype between wild populations of C. n. mionec- different behavioural testing designs. tes and C. n. amargosae pupfish (48), they also provide evidence It is also possible, however, that relationships between brain supporting a link between parvocellular VT expression and social proVT gene expression and behaviour may be influenced by varia- experience. tion in sex steroid concentrations between the sexes or between Despite the current finding of higher telencephalic proVT gene social classes. Androgens such as testosterone have been found to transcript abundance in small, socially subordinate male pupfish affect VT-immunoreactive phenotype in several vertebrate taxa compared to large, dominant males, individual variation in telence- either directly or via aromatase conversion to oestradiol (82–84). phalic proVT mRNA abundance in these small male pupfish corre- Transcripts encoding the fish v1a1 receptor have recently been lated positively with variation in behaviour PC1 score (Fig. 7C). This found to be present at high levels in the testicular tissues of sev- pattern indicates that small males exhibiting elevated aggression eral fishes (36,37,39,41), and there is evidence to suggest that VT had higher relative proVT transcript abundance in the telencephalon can affect gonadal synthesis of sex steroids (85,86). It is thus possi- compared to other small males that were less aggressive. How can ble that socially mediated changes in hypothalamic VT production small subordinate males generally have higher telencephalic proVT and VT secretion from the neurohypophysis have a regulatory role expression than dominant males, whereas it is also the case that on the gonadal production of sex steroids, which may ultimately individual subordinate males that behaved more aggressively had reinforce male aggression. It is important to note, however, that higher telencephalic proVT mRNA levels than other subordinate studies of the protogynous bluehead wrasse (Thalassoma bifascia- males that were less aggressive? A partial explanation to that tum) found that proVT gene transcript abundance in gigantocellular ostensibly contradictory pattern might be found by looking at VT POA neurones was increased, accompanying an increase in receptor expression.

the process of ascending social status from subordinate to domi- Telencephalic nonapeptide receptor mRNA abundance nant (44). In one study with bluehead wrasse, v1a2 transcripts in relates to social status and aggression both the whole brain and isolated hypothalamus were observed to Current data indicate that Actinopterygii evolved a greater diversity be present at the highest relative levels in terminal phase (TP) of nonapeptide receptors than other vertebrates, and several of males, at intermediate levels in initial phase (IP) males, and at low- these receptors appear to be expressed in the brain. Transcripts est levels in females (39). TP male wrasse perform aggressive encoding V1a-type receptors v1a1 and v1a2 have been found to be behaviours more frequently than IP males or females, again sug- present in the forebrain, midbrain, cerebellum and hindbrain of sev- gesting that brain V1a2 receptor expression in male fish may be eral fishes (36,39,41,50), and v1a2 mRNAs and V1a2 receptor pro- related positively to social dominance. tein appear distributed throughout the brain of fish with Our results also provide limited evidence implicating v1a2 tran- particularly high expression levels in the dorsal telencephalon, ven- script abundance as being associated positively with the behaviour tral telencephalon and POA (38,39,91). Although there has yet to be PC2 score of both small and large females. Although these relation- a detailed examination of V1a1 receptor protein expression in the ships were not statistically significant under the conservative fish brain, preliminary evidence from bluehead wrasse suggests that sequential P value threshold method for controlling the FDR that the distribution of v1a1 mRNAs overlaps with that of v1a2 tran- we used here (61,62), our data suggest that relative v1a2 mRNA scripts in the forebrain and hypothalamus (39). Transcripts encoding levels in the telencephalon of female pupfish, who expressed the V2-type receptors v2a and v2b have also been localised by RT- reduced telencephalic v1a2 mRNA abundance compared to male PCR to the fish brain (36,37,41), although the cellular localisation pupfish, may be elevated in females experiencing higher frequencies patterns for these V2-type receptors in the brain have not been of female-female aggression (Fig. 8B,C). With so much remaining examined. unknown about nonapeptide regulation of behaviour in female fish, In the present study, we looked for associations between behav- future studies should aim to evaluate these suggested relationships iour and the transcriptional abundance of four different teleost VT between v1a2 gene expression and the behaviour observed in receptors and an IT receptor in the brain. Our results point toward female pupfish. the expression of the V1a2 receptor as being related to aggressive Although the findings of the present study only provide an initial phenotype in male fish, although we only provide limited evidence clarification, it appears from these results that several components for associations between aggression and gene expression patterns of the neural VT system, including proVT gene expression within of the other VT receptors or the IT receptor. Dominant male pupfish from specific nuclei of the POA and the relative amount of v1a2 exhibited relative v1a2 mRNA levels that were more than 3-fold receptor gene transcription in other regions of the brain, can vary higher in the hypothalamus than levels in subordinate males or concurrently in patterns that may contribute to behavioural differ- females. Although v1a2 mRNAs in the telencephalon of these same ences among the sexes or among individuals of differing social sta- males were not elevated compared to subordinate males (but were tus. For example, the positive association between telencephalic almost two-fold higher than in females), telencephalic v1a2 tran- proVT mRNA abundance and PC1 observed in small males (Fig. 7C), script abundance in these dominant males correlated positively with but not in large males, may indicate that the higher aggression that aggressiveness as represented by an individual’s behaviour PC1 small males experience acts as a social stress that affects parvocel- value. lular VT neurones at the same time as inhibiting v1a2 transcription Associations between v1a2 gene transcription and social behav- in the telencephalon. The joint effects of such parvocellular VT acti- iour have also been identified recently in other teleost fishes. One vation coupled with diminished forebrain V1a2 receptor density study of two species of North American cichlids (the monogamous might then contribute to the reduced aggression observed in smal- Herichthys cyanoguttatus and polygynous Herichthys minckleyi) ler males. The testing of that hypothesis will require future studies provides evidence that v1a2 receptor expression may be linked to aiming to develop and employ new approaches to co-localise and behaviours associated with territory defence and polygynous repro- quantify protein expression of both hypothalamic VT and VT recep- duction (46). In that study, reproductive active H. minckleyi males tors such as the V1a2 receptor throughout the brain (92). were found to have higher hypothalamic v1a2 gene transcript Even though statistically significant correlative associations with abundance compared to nonreproductive males of this same spe- individual behavioural variation were only observed with the V1a2 cies, whereas reproductively active and inactive males of H. cyano- receptor and not any of the other VT receptors or the IT receptor, guttatus had similar hypothalamic v1a2 mRNA levels (46). our results provide evidence that the abundance of transcripts Reproductively active H. minckleyi males defend large territories of encoding other nonapeptide receptors also varies in the brain with the substrate (typically several meters in diameter) compared to social status or sex. Transcripts encoding v1a1 were at significantly the small territories (approximately 1 m in diameter) of male higher relative levels in the telencephalon of both small female and H. cyanoguttatus, suggesting that the elevated hypothalamic v1a2 small male pupfish (Fig. 6A), as well as in the hypothalamus of mRNAs observed in reproductively active H. minckleyi males may be small males (Fig. 6B). Almost nothing is known about the function associated with behaviours of territory defence and social domi- of this recently discovered actinopterygian V1a1 receptor, although nance related to the polygynous mating system of this species. In a this observed variation in telencephalic v1a1 transcript abundance separate study of the A. burtoni cichlid, v1a2 mRNAs were in both male and female pupfish of differing social status (Fig. 6A) observed to be elevated in the whole brain of males that were in may point to a role for the V1a1 receptor in mediating

Journal of Neuroendocrinology, 2015, 27, 142–157 © 2014 British Society for Neuroendocrinology 154 S. C. Lema et al. physiological responses to stressors including social stress. VT is a In other vertebrates, there is evidence that the behavioural effects multifunctional hormone in fishes and plays important roles in reg- of VT can shift between inhibiting and enhancing aggression ulating hydromineral balance, cardiovascular function, and the depending on social conditions (95,96). If social context influences hypothalamic-pituitary-interrenal axis responses to stressors in not only VT production by the POA, but also the type and relative addition to its role in regulating behaviour (93). Lema (36) previ- abundance of VT receptors expressed in the brain, as appears to be ously found that hypothalamic v1a1 transcript abundance became the case given our finding in the present study that relationships elevated 5 h and 20 h after exposure of C. n. amargosae pupfish between VT receptor gene expression and individual variation in to hyperosmotic challenge, supporting the supposition that hypo- behaviour were dependent on social status and sex, then the thalamic V1a1 expression may be responsive to environmental behavioural effects of VT might be expected to vary depending on stressors. Future studies investigating the functional significance of an individual’s recent social interactions or experience with social this V1a-type receptor both in the regulation of behaviour and in stressors (e.g. losing an agonistic interaction). In addition, in light the physiological responses to stressors are needed. of previous observations suggesting that VT receptor gene expres- Transcript abundance for the IT receptor was also at higher rela- sion in the fish brain can be influenced by other environmental fac- tive levels in the telencephalon of female pupfish compared to tors such as photoperiod and environmental salinity (36,50), such males (Fig. 6A) and at higher abundance in the hypothalamus of complexity might explain why consistent relationships between large, socially dominant females compared to small females neural VT system phenotype and behaviour have been challenging (Fig. 6B). In other fishes, brain levels of IT peptide have been to identify across fish species without a more coordinated examina- reported to change in male fish in patterns related to social status tion of how social interactions, reproductive status and environ- (61) and seasonal variation in reproductive behaviours (65). In addi- mental experiences (e.g. salinity, recent stressor exposure) synergise tion, exogenous treatment with IT has been shown to affect how to jointly influence variation in nonapeptide functioning (97). If the male fish behave during aggressive interactions (12,71). IT receptor behavioural effects of a change in nonapeptide hormone are protein was only recently shown to localise throughout several dependent on the coordinated changes in nonapeptide receptor brain regions, including the forebrain and midbrain, of male fish expression, including the localisation, type and abundance of differ- (91); however, comparable data for female fish are scarce. The find- ent receptors (92,98), future studies will need to integrate assess- ing in the present study of sex variation in telencephalic itr gene ments of the distribution, function and expressional regulation of expression indictates the need for future studies to explore possible nonapeptide receptors concurrently with evaluations of nonapeptide roles for IT in the regulation of female behaviour. hormone production to provide a fuller understanding of how the VT Lastly, our data provide only limited evidence for hindbrain VT system mediates behaviour. receptor v1a1 and v2a gene expression being linked to social status and behaviour. We specifically examined the hindbrain tissue in the Acknowledgements present study because work by Thompson and Walton (12) suggested that the effect of VT on social approach behaviours in male goldfish This research was supported by a New Investigator Award from the California (Carassius auratus) might be mediated via the action of VT on auto- State University Program for Education and Research in Biotechnology nomic regulatory processes in the hindbrain. Walton et al. (94) subse- (CSUPERB) to S.C.L., as well as by College Based Fee awards from CalPoly, San Luis Obispo to K.E.S. and K.A.W. The authors thank Rob Brewster and Doug quently found evidence that gene transcript abundance for the v1a2 Brewster for their assistance with the experimental tank construction, as well receptor might increase in the goldfish hindbrain during that species’ as two anonymous referees whose insightful comments greatly improved the reproductive season. Although our results did not identify any correl- quality of the submitted manuscript. Pupfish were collected with permission ative associations between hindbrain nonapeptide receptor expression of the California Department of Fish and Wildlife (SC-4793) and the US and individual variation in behaviour, we did observe elevated hind- Bureau of Land Management (CA-680.32). brain v1a1 and v2a mRNA levels in small, female pupfish, and future studies should continue to examine how patterns of VT receptor Received 15 June 2014, expression in the hindbrain may relate to social experience. revised 14 November 2014, accepted 20 November 2014

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