extfieldsofstudy: Medicine Biology

text:
MKRN3 Interacts With Several Proteins Implicated in Puberty Timing but Does Not Influence GNRH1 Expression

Paternally-inherited loss-of-function mutations in makorin ring finger protein 3 gene (MKRN3) underlie central precocious puberty. To investigate the puberty-related mechanism(s) of MKRN3 in humans, we generated two distinct bi-allelic MKRN3 knock-out human pluripotent stem cell lines, Del 1 and Del 2, and differentiated them into GNRH1-expressing neurons. Both Del 1 and Del 2 clones could be differentiated into neuronal progenitors and GNRH1-expressing neurons, however, the relative expression of GNRH1 did not differ from wild type cells (P = NS). Subsequently, we investigated stable and dynamic protein-protein interaction (PPI) partners of MKRN3 by stably expressing it in HEK cells followed by mass spectrometry analyses. We found 81 high-confidence novel protein interaction partners, which are implicated in cellular processes such as insulin signaling, RNA metabolism and cell-cell adhesion. Of the identified interactors, 20 have been previously implicated in puberty timing. In conclusion, our stem cell model for generation of GNRH1-expressing neurons did not offer mechanistic insight for the role of MKRN3 in puberty initiation. The PPI data, however, indicate that MKRN3 may regulate puberty by interacting with other puberty-related proteins. Further studies are required to elucidate the possible mechanisms and outcomes of these interactions.

INTRODUCTION
The onset of puberty is dependent on the reactivation of hypothalamic GnRH secretion, which leads to increased gonadotropin and sex steroid secretion, secondary sex characteristics and the attainment of adult height and fertility (1,2). In patients with central precocious puberty (CPP), puberty starts before the age of 8 years in girls (Tanner breast stage 2) and 9 years in boys (Tanner genital stage G2) (1,3,4). Premature central activation of GnRH secretion may be pathological due to intracranial lesions, or idiopathic when no underlying cause can be identified (4). In girls, the annual incidence of CPP varied from 15 to 29 per 1,00,000 (5), and the overall risk and incidence is higher in girls than in boys (2,3,6).
The genetic etiology of idiopathic CPP is not completely understood, but the clinically most relevant gene underlying CPP is makorin ring finger protein 3 gene (MKRN3) (7)(8)(9)(10)(11). MKRN3 is a single-exon, maternally imprinted gene, which is expressed only from the paternal allele (12). Consequently, only paternally-inherited loss-of-function mutations in MKRN3 cause CPP (7,11). MKRN3 belongs to the makorin family of ubiquitin ligases together with MKRN1 and MKRN2. MKRN3 is a close relative of MKRN1, being an intronless retrocopy of the latter (12,13). Similar to other makorin family members, MKRN3 too consists of three zinc finger domains (C3H), one zinc RING finger domain (C3HC4) and one MKRN3-specific Cys-His domain (CH) and is expressed ubiquitously in human fetal and adult tissues (13,14). Based on its structure, MKRN3 is predicted to function as a putative E3-ubiquitin ligase and it potentially affects gene expression, targeted protein degradation and protein function modulation via its E3 ligase activity (12)(13)(14)(15). Interestingly, MKRN3 is expressed in the mouse and human hypothalamus (7,8), but the mechanistic processes by which a paternally inherited loss-of-function MKRN3 mutation causes CPP are currently unclear.
Human pluripotent stem cells (hPSCs) have the indefinite capability of self-renewal and they can be differentiated into specialized cell types (16). To this end, we have recently described a protocol for the differentiation of GNRH1-expressing neurons from hPSCs (17), which offers a possibility to investigate the role of genetic factors in the regulation of GNRH1 in humans. Clustered Regularly Inter Spaced Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) has become a prevailing technology in the area of gene editing (18). In brief, the CRISPR/Cas9 approach comprises of a short guide RNA (crRNA) fused to bacterial-specific trans-activating crRNA called tracrRNA which processes the crRNA, the fusion of crRNA and tracrRNA forms tracr:cr RNA complex which directs the Cas9 enzyme to a specific locus on DNA, generating double strand breaks (18). These double strand breaks are naturally repaired by an error-prone non-homologous end joining in the absence of a donor template (19). We utilized this technique to generate bi-allelic MKRN3 deletions in hPSCs and differentiated the knock-out (KO) cell lines into GNRH1-expressing neurons by employing our recently described protocol comprising dual SMAD inhibition, FGF8 treatment and Notch inhibition (17).
Most cellular proteins do not function in isolation, but perform their biological functions by interacting with each other (20). Protein-protein interactions (PPIs) form a network that operates in a coordinative manner to bring up physiological Abbreviations: CPP, central precocious puberty; MKRN3, makorin ring finger protein 3; hPSCs, human pluripotent stem cells; WT, wild type; KO, knockout; CRISPR, clustered regularly interspaced palindromic repeats; Cas9, CRISPR associated protein 9; bps, base pairs; gRNA, guide RNA DNA template; GFP, green fluorescent protein; PPI, protein-protein interaction; HCI, high-confident interactions; CHH, congenital hypogonadotropic hypogonadism. activities within a living cell (21). Mass spectrometry (MS) has evolved into an efficient technique in analyzing and characterizing PPIs (20,22). To further investigate the putative mechanisms by which MKRN3 may affect the timing of puberty, we investigated MKRN3 PPI partners in HEK cells by employing our recently described MS-based approach, which allows the detection of stable and transient interaction partners for the protein of interest (20). This cell line was selected as a model, since HEK cells share certain characteristics of neurons (23), are widely available to researchers, and are frequently employed in biomedical research.

Human Pluripotent Stem Cells
Human pluripotent stem cell line H9 [46,XX,(Wicell)] was used in this study (24). hPSCs were maintained on Matrigel R (BD Biosciences) coated dishes with StemPro (Thermo Fisher Scientific) or Essential 8 medium (Thermo Fisher Scientific). During maintenance, the cells were split with 0.5 mM EDTA (Thermo Fisher Scientific, MA) and plated in 1:3-1:8 ratios. Medium was changed daily and before the differentiation, cells were plated on Geltrex-coated dishes (Thermo Fisher Scientific) and grown until >90% confluency.

Guide RNA Design and Production
CRISPR guides were designed using https://benchling.com/ to target the only exon of MKRN3. Based on their off-target and specificity scores, two pairs of the best possible guides were selected. Guide RNA DNA templates (gRNAs) were prepared by PCR (gRNA-PCR) amplification based on a method previously published (25). In brief, gRNA templates having 19 bp overhangs on 5 ′ and 3 ′ ends were fused to U6 promoter and terminator sequences (tracr) using PCR mentioned above with Phusion polymerase (Thermo Fisher Scientific). Further information on the gRNA-PCR is given in the Supplementary Material. All the CRISPR related oligos and guide sequences have been listed in Supplementary Table 1.

Generating CRISPR/Cas9 Based MKRN3 KO Cell Lines
For generating MKRN3 KO in hPSCs, two gRNAs targeting different locations of MKRN3 and a plasmid encoding wild type (WT) Streptococcus pyogenes Cas9 (SpCas9, referred as Cas9 hereafter), Green fluorescent protein (GFP) and puromycin resistance gene were electroporated to two million H9 cells with the Neon transfection system according to the manufacturer's instructions (Thermo Fisher Scientific). A total of 4 µg plasmid DNA and 250 ng of each gRNA was used per electroporation, and the electroporated cells were plated on Matrigel R -coated dishes and supplemented with 10 µM ROCK inhibitor (Y-27632 2HCl, Selleckchem) to enhance survival of hPSCs by inhibiting dissociation-induced apoptosis (26). Culture medium was changed every 24 h, with transient selection of surviving clones using 0.12 µg/ml puromycin (Sigma-Aldrich) starting after 48 or 72 h.

Colony-Picking or Fluorescence Activated Cell Sorting (FACS)
After 48 h of puromycin selection, emerging colonies were either manually picked or single cell sorted using a flow sorter (Sony Biotechnology Inc.). Manual picking of the colonies was carried out using a 10 µl pipette and Stereozoom R S4E light microscope (Leica microsystems). The individual colonies were identified under microscope and manually detached. Colonies were plated in a single well of a Matrigel R coated 96-well tissue culture plate (Sarstedt) containing E8 cell culture medium, supplemented with 10 µM ROCK inhibitor.
For cell sorting, Sony SH800 flow sorter was used to sort GFP positive (indicating successful entry of Cas9 plasmid) single cells, which were then plated on each well of the 96 well-plates containing E8 cell culture medium supplemented with 10 µM ROCK inhibitor. During both manual picking and cell sorting, medium was refreshed every 48 h (without ROCK inhibitor and puromycin) and the colonies were grown until they reached 70-80% confluency.

PCR-Based Screening of All the Surviving Clones
Genomic DNA (gDNA) from all the surviving colonies originating from single colonies or cells, were isolated using Direct cell PCR lysis buffer (Viagen Biotech) supplemented with 20 µg/ml of Proteinase K (Thermo Fisher Technologies). The gDNA served as a template to identify cell lines with biallelic or mono-allelic deletion using a specific primer pairbased PCR screening with AmpliTaq gold DNA polymerase (Thermo Fisher Scientific). Conditions for screening PCR's are provided in Supplementary Material, primers used are listed in Supplementary Table 2.
Targeted Sequencing of MKRN3 KO Cell Lines gDNA from WT and the MKRN3 KO cell lines were PCR amplified with primers 200 bps upstream and downstream of MKRN3 and the product was purified using A'SAP PCR clean up kit (Arcticzymes) according to the manufacturer's instructions. The purified PCR products were subjected to Next generation sequencing using Nextera DNA library preparation kit (Illumina Technologies) performed at the Institute for Molecular Medicine Finland (FIMM, Helsinki). The targeted sequencing data was analyzed with the Integrative Genomics Viewer (IGV) tool from Broad institute (27). The primers used for targeted sequencing of MKRN3 are listed in Supplementary Table 2.

Differentiation of hPSCs to GNRH1-Expressing Neurons
At >90% confluency, both WT and MKRN3 KO hPSCs were differentiated into GNRH1-expressing neurons as described in Lund et al. (17). Briefly, the cells were treated with 2 µM Dorsomorphin (Selleckchem) and 10 µM SB431542 (Sigma-Aldrich) for 10 days to produce neuro-ectoderm, a further 10 days with 100 ng/ml FGF8 (Peprotech), and differentiation into post-mitotic neurons was stimulated by Notch inhibition using 20 µM DAPT (Sigma-Aldrich). The successful differentiation into GNRH1-expressing neurons was confirmed by morphological analysis, antibody staining to identify GnRH expressing cells and qPCR.

Analysis of GNRH1 Expression
The expression levels of GNRH1 gene were measured by qPCR, normalized to Cyclophilin G (PPIG) and compared to expression in undifferentiated PSCs, as previously described (17). The normal range for GNRH1 mRNA expression in WT cells was determined based on nine differentiation experiments (n = 9). Day 25, was used to characterize GNRH1 expression in our protocol (17), and the same day was selected here for comparison of WT (n = 9) and MKRN3 KO cell lines (n = 6) in all the experiments. Conditions for qPCR are provided in the Supplementary Material, the primers used in qPCR are listed in Supplementary Table 2.

Immunocytochemistry
The cells were fixed using 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized for 7 min in 0.5% Triton X-100 (Sigma-Aldrich) diluted in PBS. Unspecific binding was blocked with UltraVision Protein Block (Thermo Fisher Scientific) for 10 min. Primary antibody incubation was performed overnight at 4 • C and secondary antibody for 1 h at RT. Antibodies were diluted in PBS containing 0.1% Tween 20 (Sigma-Aldrich). Slides were mounted using VECTASHIELD R Mounting Medium with DAPI (Vector laboratories) for counterstaining the nuclei and microscopic images were obtained using ZEISS Axio Imager.Z2 upright epifluorescent microscope (Zeiss) using 10x/NA 0.3 and 20x/NA 0.8 EC PL APO CS2 objectives (Biomedicum Imaging Unit) and analyzed with ImageJ (NIH). All the antibodies and dilutions are listed in Supplementary Table 3.

Generation of Stable Expression Cell Line and Cell Culture for Protein-Protein Interaction Studies in HEKs
For the generation of stable and inducible cell lines, gateway cloning (28) was employed to create MKRN3 expression vector.
MKRN3 PCR-product with flanking site specific attachment sites (attB) and donor vector with site specific attachment sites (attP) were recombined by BP reaction to get the gateway compatible entry clone. Recombination between attB and attP sites results in attL and attR sites which are recombined by LR reaction between the entry clones and the MAC-C destination vector (20)  For biotinylation labeling (BioID), 50 µM additional biotin (Thermo Fisher Scientific) was added at the same time with tetracycline. Cells deriving from 5 × 150 mm fully confluent dishes (approximately 5 × 10 7 cells) were pelleted as one biological replicate. Three biological replicates for each condition and in total six biological replicates for two different conditions (with and without biotin) were collected. Samples were snap frozen at −80 • C until further use.

Affinity Purification of Protein Complexes and Mass Spectrometry (MS)
The cells with only tetracycline induction were used for affinity purification (29). The cells treated with tetracycline and biotin were applied for BioID purification as previously described (20). Purified protein complexes (AP-MS)/proteins (BioID) were processed and digested to peptides for MS analysis. The analysis was performed on Qrbitrap Elite hybrid mass spectrometer using Xcalibur version 2.0.7 SP1 (Thermo Scientific) coupled with an EASY nLC 1000-reverse phase HPLC system via an electrospray ionization sprayer (Thermo Fisher Scientific). MS was performed in data-dependent acquisition mode using Fourier transform mass spectrometry full scan (300-1,700 m/z) resolution of 60,000 and collision-induced dissociation scan of top 20 most abundant ions.

Data Processing
For protein identification, Thermo.RAW files were searched against reviewed selected human UniProtKB/SwissProt database (http://www.uniprot.org/, version 2017-02) with SEQUEST search engine. The decoy database was the reverse of the target database. All data were reported based on 95% confidence for protein identification, as determined by the false discovery rate (FDR) ≤5%. Contaminant Repository for Affinity Purification (CRAPome, http://www.crapome.org/) (30) database and inhouse GFP samples database were used as controls with a cutoff frequency of 20% (411 runs from CRAPome database and 100 runs of in house GFP control samples) for identification of high-confidence interactions (HCIs). HCIs data were imported into Cytoscape 3.4.0 to construct protein interaction network. The known prey-prey interaction data were obtained from IMEx database (http://www.imexconsortium.org/) (31). Gene ontology classification analysis was based on DAVID bioinformatics resource (https://david.ncifcrf.gov/) (32). We also compared the HCI partners of MKRN3 with proteins encoded by 371 genes, that are associated with puberty timing according to large population based studies (33,34). In addition, we investigated the HCIs to proteins encoded by 37 genes implicated in congenital hypogonadotropic hypogonadism (CHH) (35)(36)(37)(38)(39)(40).

Statistics
The relative GNRH1 expression in WT H9 and MKRN3 KO cell lines Del 1 and Del 2 was tested with unpaired t-test after logarithmic transformation to normalize the distributions. Pvalue < 0.05 was accepted to indicate statistical significance.

CRISPR/Cas9 Based Generation of MKRN3 KO Cell Lines in hPSCs
To investigate the impact of MKRN3 deficiency in the differentiation of GNRH1-expressing neurons, we generated two hPSC lines with bi-allelic deletions of MKRN3 using CRISPR/Cas9. Bi-allelic KO cell lines were generated, as heterozygous MKRN3 mutations cause CPP only when inherited from the father, and we could not selectively target the desired deletion to the paternal allele. The schematic indicates the guide targeting areas and deletions in these cell lines ( Figure 1A). In brief, two pairs of RNA guides spanning different lengths on the only exon of MKRN3 together with Cas9 resulted in deletions of either −174 or −901 bps. For further studies, we chose two apparently homozygous KO lines Del 1 (small deletion, −174 bps), and Del 2 (large deletion, −901 bp), which were validated to carry desired deletions by PCR ( Figure 1B) and targeted sequencing Supplementary Figures 1, 2). The absence of functional MKRN3 expression was further validated by performing PCR with cDNAs from the WT and MKRN3 KO cell lines as template and with primers flanking the deletion regions (Figure 2A). The length of the transcripts produced from the KO clones corresponded to the size of deletions seen at DNA level ( Figure 2B), consistent with the bi-allelic deletion in the coding region of MKRN3.

Differentiation of MKRN3 KO and WT hPSCs to GNRH1-Expressing Neurons
We next differentiated MKRN3 KO cell lines Del 1 and Del 2 into GNRH1-expressing neurons, according to our recently described protocol (Figure 3A) (17). Both cell lines could be differentiated into neuronal progenitors and further into GNRH1-expressing neurons (Figure 3B). At day 25 of differentiation the relative GNRH1 mRNA expression between the MKRN3 KO cell lines and WT H9 cells (Figure 3C), did not differ (P = NS). The mRNA expression levels of OTX2, a transcriptional regulator of GNRH1 (41), together with OTX1, both of which are enriched in GnRHneurons of mice (42) were not significantly different between WT and Del 2 (n =3) cells at day 25 of the differentiation protocol (Supplementary Figure 3).  1-Del 4). The same pair of guide RNAs, designed to delete ∼900 bps, was used for editing Del 2 and Del 3 cell lines, whereas two different gRNA pairs were used to edit Del 1 and Del 4 cell lines. The PCR products were visualized on a 1.5% agarose gel.   (17). In brief, dual SMAD inhibition by Dorsomorphin and SB431542 was applied for the first 10 days, followed by 10 days of FGF8 treatment and final stage of neuronal maturation for 5-7 days by combining FGF8 with Notch inhibition by DAPT. This differentiation methodology has been adapted from a previously published protocol and the figure has been modified from the original publication by Lund et al. (17). (B) Immunocytochemistry in WT and Del 2 derived day 25 GnRH-positive neurons (green) nuclear stained with DAPI (blue), scale bars represent 100 µM. (C) The relative expression of GNRH1 in WT H9 cells (n = 9) and in two bi-allelic MKRN3 KO cell lines (Del 1 and Del 2) following three independent repeats (n = 6) of differentiation into GNRH1-expressing neurons (17). Expression levels are relative to day 0 hPSCs.

MKRN3 and Its Protein Interaction Partners
Using MS analysis, we identified 104 HCIs including both transient and stable interactions for MKRN3 (Supplementary Table 4). These 104 HCIs total for 81 unique HCIs (Figure 4), were classified into functional groups according to their annotation categories involved in various pathways regulating cellular processes, mainly the regulation of insulin secretion, cell-cell adhesion, TP53-regulated cell metabolism, transcriptional repression and RNA activity related interactors (32). Importantly, our MS data revealed that MKRN3 physically interacts with as many as 19 proteins (including LIN28B) encoded by genes, which have been previously associated with the age at menarche (33,34). In addition, MS data identified OTUD4, a protein implicated in CHH (43), as HCI partner for MKRN3.

DISCUSSION
In mice, expression of Mkrn3 decreases during postnatal development, and in humans, circulating MKRN3 levels decrease toward the onset of puberty in both sexes (44,45). However, investigation of peripheral MKRN3 levels does not allow for mechanistic explanation of the role of MKRN3 in regulation of puberty in humans. Therefore, we employed our recently described differentiation protocol generating GNRH1-expressing neurons (17) and genetically engineered hPSCs to investigate if MKRN3 is required for GnRH neuron differentiation in this model. In addition, we investigated PPI partners of MKRN3 in HEK cells and paid special attention to possible interactors encoded by puberty-related genes.
Although several studies have reported that paternally inherited mutations in MKRN3 cause CPP (46), the underlying mechanisms still remain to be explained (1,2). In our study, we first examined the role of MKRN3 on GNRH1 expression by using two bi-allelic MKRN3 KO clonal cell lines (Del 1 and Del 2), generated with CRISPR/Cas9 technology. These cell lines were subsequently differentiated into GNRH1-expressing neurons through our recently described differentiation protocol, which is based on dual SMAD inhibition, FGF8 treatment and Notch inhibition (17). Our results show that both the Del 1 and Del 2 cell lines could be successfully differentiated into GNRH1expressing neurons, suggesting that MKRN3 is dispensable for the differentiation. Intriguingly, GNRH1 expression of Del 1 and Del 2 cell lines did not differ from the controls, which suggests that MKRN3 does not directly alter GNRH1 expression. However, there are many environmental and metabolic signals postnatally, that may regulate further maturation and function of GnRH neurons (47,48). While MKRN3 does not affect the GnRH neuron differentiation or their ability to express GNRH1 in our model, there are other potential mechanisms by which MKRN3 may regulate GnRH neuron function. For example, major regulation of GnRH neuron activity is carried out by Kisspeptin and KNDy neurons in the hypothalamus, and putative role of MKRN3 on these neurons is not known. The possibility of other MKRNs compensating for the loss of MKRN3 in Del 1 and Del 2 cell lines cannot be ruled out. On the other hand, hPSCbased disease modeling is the closest available approach to model human neuronal disorders (49), and with regard to MKRN3, murine models have not yet been reported. What is known so far, however, is that MKRN3 is not significantly enriched or depleted in mouse GnRH neurons (42).
Given that we could not find a mechanistic cue for the role of MKRN3 in GNRH1 expression in our stem cell model, we considered alternative approaches to gain insight on how this putative E3-ligase might affect puberty timing. In brief the method investigating PPIs employs a MAC-tag system that contains affinity tags for affinity purification-mass spectrometry (AP-MS) as well as the biotin ligase BirA * for BioID. Therefore, a single construct is sufficient to identify both stable and transient interactions (20). We did not carry out PPI studies in our stem cell model, since the efficient cloning of all different tagged MKRN3 constructs to hPSCs was beyond the scope of this work. While this is a potential caveat, it is important to note that HEK cells, one of the most widely employed human cell lines in biomedical research (50), are not unsuitable for our purpose since they exhibit some neuronal properties (23). In addition, our laboratory is extremely experienced in using HEK cell linebased platform, which allows rapid and efficient generation of expression constructs and subsequent data analysis. Based on the list of HCI proteins for MKRN3, we found that MKRN3 might be involved in cell-cell adhesion, RNA metabolism and insulin secretion. Insulin has been suggested to modify GnRH neuron function in humans, and insulin sensitivity is thought to be an important factor in the initiation of puberty (1,51,52). MKRN3 interacts with a group of proteins encoded by metabolic genes, which in turn, are regulated by TP53, a tumor suppressor gene involved in several cancers and implicated in puberty (53). Other PPI partners of MKRN3 are involved in RNA transport and metabolism, which remains less explored areas in terms of puberty.
In addition, we found that MKRN3 interacts with 20 proteins encoded by puberty timing-associated genes of which LIN28B is one of the most established puberty-related genes in genetic studies (54,55) and OTUD4 represents the only puberty-related disease gene (43). Although LIN28B has been associated with the age at menarche, adult height and childhood growth (56)(57)(58)(59)(60), and is known to be a negative regulator of let-7 class of microRNAs (61), the mechanism by which it regulates puberty timing is unknown (1,62,63). In rats and non-human primates Lin28b expression declines in the hypothalamus at puberty (64), and in mice, expression of both Lin28b and Mkrn3 is reduced prior to puberty (7,65). Our findings provide evidence of physical interaction between human LIN28B and MKRN3 and it is tempting to speculate that they may act in concert to regulate the timing of puberty. We also examined MKRN3 interactions among 37 genes implicated in CHH (35)(36)(37)(38)(39)(40), and found that MKRN3 interacts with OTUD4. OTUD4 is a deubiquitinase coding gene (43), and it is intriguing to hypothesize that it may regulate the timing of puberty by counteracting the effects of MKRN3. Further studies are required to confirm this hypothesis. Finally, MKRN3 interacts with other zinc finger genes (ZNFs) which are transcriptional repressors and have an important role during puberty (66), however, we could not observe MKRN3's interaction with ZNFs critical for puberty onset, such as ZNF573 and GATAD1 (66). Even so, the interactions observed invite the possibility of MKRN3 being an epigenetic transcriptional repressor involved in puberty timing.
In conclusion, our results suggest that MKRN3 is dispensable for the GnRH neuron differentiation process and GNRH1 expression during differentiation from human pluripotent stem cells. Although the mechanism by which MKRN3 regulates humans sexual maturation remains unclear, our results suggest that MKRN3 may act in concert with previously identified puberty-related proteins, and it is enticing to hypothesize that this interaction is translated to functional cooperation in terms of puberty timing. Further studies are required before we understand how and why the HPG axis activates prematurely in children with a paternally inherited loss-of-function mutations in MKRN3.

AUTHOR CONTRIBUTIONS
VY, SV, TT, and TR planned the project. TT, MV, and TR supervised the project. VY, CL, KP, and KL carried out the stem cell work. XL and MV designed the protein interaction study. XL and JK carried out the protein interaction work. VY wrote the manuscript and TR edited the manuscript.

FUNDING
This work was supported by the Academy of Finland, Foundation for Pediatric Research, Sigrid-Juselius Foundation, Novo Nordisk Foundation, Jalmari Ja Rauha Ahokkaan Säätiö, University of Helsinki, and Helsinki University Central Hospital.


HO-1 protects the nerves of rats with cerebral hemorrhage by regulating the PI3K/AKT signaling pathway

Objective: This study aimed to investigate the neuroprotective effect of heme oxygenase-1 (HO-1) on the PI3K/AKT signaling pathway in rats with cerebral hemorrhage. Materials and methods: Adult male Sprague-Dawley rats were randomly divided into: a sham group, a model group and an HO-1 inhibitor group (ZnPP group). Functional defects after surgery were scored according to the Longa5 standard. Hemotoxylin and eosin staining was used to detect whether the model was constructed successfully. Superoxide dismutase (SOD) vitality and malondialdehyde (MDA) content were calculated by the xanthine oxidase method and thiobarbituric acid method, respectively. Blood-brain barrier permeability was measured by Evans Blue. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. The expression of Bcl-2 and BAX was evaluated by immunohistochemistry and the expression of PI3K, p-PI3K, AKT and p-AKT was tested by Western blotting. Results: The rat intracerebral hemorrhage model was successfully constructed. Compared with the model group, the bleeding in the ZnPP group was more serious, the cell edema and deformation were aggravated, and the neurological deficit score in the rat was significantly increased. In addition, the content of Evans blue, MDA, the number of apoptotic cells, the water content of brain tissue and the expression of BAX were significantly increased, while the SOD activity and the expressions of Bcl-2, p-PI3K and p-AKT protein were decreased. Conclusion: HO-1 could protect the nerves of rats with cerebral hemorrhage by regulating the PI3K/AKT signaling pathway.

Introduction
Spontaneous intracerebral hemorrhage (ICH) refers to primary non-traumatic intracerebral hemorrhage, which accounts for approximately 15% of stroke. 1 It is a common clinical neurological disease with high incidence, high disability rate and high mortality, and seriously threatens human health. 2,3 Cerebral hemorrhageinduced neuronal apoptosis and secondary cerebral edema play an important role in neurological impairment. 4 However, the pathophysiological mechanism of ICH has not been fully elucidated.
Previously, many researches have studied the neuroprotective effects and made major breakthroughs. For instance, Laabich et al have found that AIP acts as a neuroprotective agent against N-methyl-D-aspartate-induced retinal neuronal cell death. 5 Also, the estrogen receptor selective modulator tamoxifen citrate has been confirmed to have neuroprotective effects in an acute cerebral ischemia model. 6 Moreover, erythropoietin has neuroprotective effects on immature cerebral oxygen-induced cell death. 7 Multiple pathways have also been found to play an important role in neuroprotection. Estradiol preconditioning could promote dopamine neuron survival by activating ERα and increasing Akt and GSK3-β phosphorylation. 8 Besides, naringenin could play a neuroprotective role in stroke rats by suppressing the NF-κB signaling pathway. 9 Recently, many scholars have found that heme oxygenase-1 (HO-1) has neuroprotective effects. [10][11][12] However, the mechanism of HO-1 and related pathways in cerebral hemorrhage have not been thoroughly studied. Therefore, we constructed the rat model ICH. The expression of HO-1 in the cerebral hemorrhage model was studied, and the related mechanisms of protein, pathway and neurological function were studied by inhibiting HO-1 expression.

Materials and methods Regents
DMSO and ZnPP regents were purchased from Sigma Aldrich (St. Louis, MO, USA). Trizol reagent was purchased from Invitrogen (Thermo Fisher ScientificWaltham, MA, USA) and PCR kit was also purchased from Thermo Fisher Scientific. Hematoxylin and eosin (HE) staining kit, EB kit, DAB kit and BCA Protein Assay Kit were purchased from Beyotime Biotechnology (Shanghai, China).

Experimental design and groups
A total of 45 adult male Sprague Dawley rats (about 260 g) were purchased from Beijing Weitong Lihua Experimental Animal Technology Co, Ltd. Rats were placed at 23±1°C with lights on from 6:00 to 18:00. 13 The rats were separated into a sham group, a model group (DMSO group) and an HO-1 inhibitor group (ZnPP group) with 15 rats in each group. All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals, and all experiments were approved and performed according to the guidelines of the Ethics Committee of the Affiliated Yantai Yuhuangding Hospital of Qingdao University.

ICH models
Rats were anesthetized with pentobarbital (40 mg/kg). All operations were carried out under aseptic conditions. Rats were placed in a stereotactic frame (Kopf Instruments, Tujunga, CA, USA). A stereotactically guided needle was placed into the right basal ganglia (3.5 mm beside to bregma, depth 5.5 mm below the surface to midline). Then 100 μL of fresh autologous non-heparinized arterial blood was infused (10 μL/min) using a microsyringe pump. After completing the injection, the needle was removed. The burr hole was sealed with bone wax and the scalp wound was sutured. 14 A sustained release pump was placed in the abdominal cavity of each rat after successful ICH modeling. A slowrelease pump with 2 ml DMSO solution containing ZnPP (0.01 mg/kg) was used in the HO-1 inhibitor group. A slow-release pump with 2 ml of DMSO solution was used in the model group. Similar surgical procedures were processed for sham-operated animals (sham group) except that no blood was injected.
qRT-PCR assay for the expression of HO-1 The expression level of HO-1 was examined by qRT-PCR. The brief process was introduced as follows. The brain tissue was rapidly ground in liquid nitrogen, then trizol reagent was added. Then, the sample was transferred to the EP tube, and total RNA was extracted. The RNA concentration was measured and adjusted. The cDNA was obtained by reverse transcription. The quantitative PCR program was started at 95°C for 5 mins, and the amplification procedure was 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 50 seconds for a total of 40 cycles. Glycerol phosphate dehydrogenase (GAPDH) was used as an internal reference. The primers were as follows: HO-1, (upstream) 5ʹ-CACGGCCATGAC-CACTTTC-3ʹ, (downstream) 5ʹ-GGAACAGAGTGGCTCC-AACAA-3ʹ; GAPDH, (upstream) 5ʹ-CCTGCAAATGAGC-CCCAGCCTTC-3ʹ, (downstream) 5ʹ-TGCTGGCGCTGAT-ACGTCGT-3ʹ. 15 Neurological score After awakening, the symptoms of neurological deficits, including emotion, cognition and movement were observed in the ischemic animal models. Postoperative functional deficits were scored according to the Longa 5-point scale ( Table 1). The model with more than 1 point was regarded to be successfully built. 16 Pathological observation of brain tissue The rats were anesthetized and underwent intracardiac perfusion with 4% paraformaldehyde in physiological saline. The brain was taken out and fixed in 4% paraformaldehyde, dehydrated, permeabilized, and encased in paraffin. The brains were then placed in OCT embedding compound and 2-4 mm thick sections were obtained on a cryostat. Then the sections were stained with hematoxylin and eosin (HE) and observed under the microscope. 17 Determination of Evans blue (EB) content in brain tissue One hour before sacrifice, 2% EB (4 mL/kg) was injected into the femoral vein. Then normal saline was rapidly perfused from the left ventricle to the right atrium until the effluent clarifies. Finally, right nucleus accumbens and the caudate nucleus brain tissue after decapitation were collected.
After weighing, brain tissue was placed in 50% trichloroacetic acid solution for homogenization. The solution was centrifuged, and then 1 mL supernatant was taken and mixed with 3 mL absolute ethanol. Fluorescence values were measured by a fluorescence spectrophotometer with an excitation wavelength of 620 nm and emission wavelength of 680 nm. The content of EB in brain tissue was measured by EB Standard to detect the permeability of the blood-brain barrier at 72 hours after surgery. 18 Evaluation of brain edema Brain water content was evaluated at 72 hours after ICH. After weighing the wet weight, the tissue was dried for 24 hours at 90°C. The weight of brain tissue was immediately reweighed by analytical balance to yield the dry weight. The percentage of brain water content was calculated using the following formula: Determination of superoxide dismutase (SOD) and MDA content in brain tissue All rats were killed by decapitation and the whole brain was removed quickly. The cerebral cortex was taken, and 10% cerebral cortex homogenate was prepared with physiological saline, and the supernatant was taken after centrifugation. Absorbance values of cerebral cortex supernatants were measured using UV-1700 UV-Vis spectrophotometer at 550 nm. SOD vitality was detected by the xanthine oxidase method. Then SOD activity in the tissue was calculated. The absorbance values of the cerebral cortex supernatant were measured at 532 nm. Malondialdehyde (MDA) content was determined by thiobarbituric acid method. 20 Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay Rats were anesthetized by intraperitoneal injection with 10% chloral hydrate (300 mg/kg). They were washed twice with saline and fixed with cardiac perfusion with 4% paraformaldehyde. 21 Paraffin sections of brain tissue were made and washed twice with saline. The labeling solution were added to the sections and incubated at 37°C for 2 hours. Slices were washed twice with saline. Then biotin was added and incubated for 30 minutes. Slices were colored in DAB solution and observed under a microscope. Six high-power fields were selected for each section (400×).

The expression of Bcl-2 and BAX was detected by immunohistochemistry
After successful modeling, the rats were deeply anesthetized with pentobarbital (40 mg/kg, ip) and rapidly perfused with stroke-physiological saline solution and 4% paraformaldehyde. After being cut, the brain tissue was immersed in a neutral formaldehyde solution. Then, they were blocked in a saline solution containing 5% normal goat serum. The membranes were incubated with Bax antibody, Bcl-2 antibody (purchased in Fuzhou Maixin Biotech Co., Ltd, all diluted 1:100) at 4°C overnight. After that, the membranes were incubated with ready-touse rapid immunohistochemistry MaxVisionTM secondary antibody (Fuzhou Maixin Biotech Co., Ltd). Positive cells were observed and counted using grid counting. 22

Western blot
Brain tissue was lysed in ice-cold cell lysis solution and the homogenate was centrifuged at 4°C for 15 mins. After quantifying by BCA protein assay kit (Shanghai Biyuntian Bioengineering Co., Ltd.), the concentration of total protein was adjusted to 30 μg/μL. The protein was separated by SDS-PAGE, and then transferred onto PVDF membranes. The membranes were incubated with p-PI3K (Santa Cruz Biotechnology Co., Ltd, Dallas, TX, USA), p-AKT (Santa Cruz Biotechnology Co., Ltd.), PI3K (Bioworld) and AKT (Bioworld), respectively, at 4°C overnight. After that, the membranes were incubated with alkaline phosphataselabeled goat anti-rabbit IgG secondary antibody (Beijing Zhongshang Jinqiao Biotechnology Co., Ltd) at room temperature for 1 hour. β-actin was used as an internal reference. The protein was visualized with an imager. 23

Statistical analysis
All experiments were repeated three times. All experimental data were processed with SPSS 21.0 (IBM Corporation, Armonk, NY, USA). Data representation of mean values ± standard deviation was used in this study. The comparison of data between the two groups was carried out by Kruskal-Wallis test, while one-way ANOVA was used to analyze the difference among multiple groups. When the P-value was less than 0.05, the result was considered as statistically different.

Successful establishment of the ICH mode and expression of HO-1
The pathological morphology results showed that the nerve cells were in a regular pattern with the same size, the nucleus of nerve cells was observed clearly, and cell swelling was not observed in the sham group. In the model group, the hemorrhagic area and peripheral nerve cells were obviously swollen and deformed, cells were autolysis and necrosis with vacuole, the nucleus was pyknotic and the nucleolus disappeared.
There was a lot of red blood cell exudation in the interstitial, and the tissue structure was loosening. Compared with the model group, the bleeding in the ZnPP group was more serious, the edema and deformation of the cells were aggravated. The membrane and nucleus had become more blurred. The number of red blood cells bleeding was increased and the organizational structure was more uneven ( Figure 1A). As shown in Figure 1B, the expression of HO-1 was significantly lower after surgery. In addition, compared with the model group, the level of HO-1 was decreased in the Znpp group. We inferred that there were inhibitory effects of ZnPP against HO-1 in the injured brain tissues.

Inhibition of HO-1 aggravated neuronal damage, the content of EB and brain water
After surgery, the neural function was judged according to the Longa5 criteria. Rats in sham group had no symptoms of neurological deficit, while in model group and ZnPP group had different degrees of neurological defects. Compared with the model group, the neurological deficit scores of the ZnPP group were significantly increased (P<0.05, Figure 2A). The results indicated that neuronal damage was aggravated after   Figure 2B). In addition, the content of EB in the ZnPP group was higher than that in the model group (P<0.05). Compared with the sham operation group (67.49±2.59%), the brain water content in the model group (72.42±3.73%)and ZnPP group (77.63±2.65%) was significantly higher (P<0.05). The brain water content of the ZnPP group was significantly higher than that of the model group (P<0.05, Figure 2C). This result indicated that the inhibition of HO-1 affected the blood-brain barrier permeability and brain tissue water content of rat brain tissue.
Inhibition of HO-1 increased SOD activity and decreased MDA content As shown in Figure 3, the SOD activity in the brain tissue of the model group was significantly decreased, while MDA content was significantly increased (P<0.05). Compared with the model group, the SOD activity was significantly decreased, while MDA content was significantly increased in ZnPP group (P<0.05).

Inhibition of HO-1 increased neuronal apoptosis after cerebral hemorrhage
At 48 hours after operation, the apoptosis of neuronal cells was detected by TUNEL. Sporadic, sporadic and few TUNEL positive cells were found in the brain tissue of the sham group. The number of apoptotic cells in the model group and ZnPP group were significantly increased. Compared with the model group, the number of apoptotic cells in the ZnPP group was significantly increased (Figure 4).
Bcl-2 and BAX were slightly expressed in the sham group, but the bcl-2/BAX ratio was always close to 1. Compared with this group, the expression levels of Bcl-2 and BAX in the model group were significantly increased (P<0.05). Compared with the model group, the positive expression of Bcl-2 in the ZnPP group was significantly decreased (P<0.05), while the expression of BAX was significantly increased (P<0.05), and the Bcl-2/BAX ratio was much less than 1 ( Figure 5). The results further indicated that inhibition of HO-1 increased apoptosis in brain tissue of rats with cerebral hemorrhage.

Inhibition of HO-1 decreased the expression of PI3K, p-PI3k
The protein expression level of PI3K, AKT, p-PI3K and p-AKT were detected at 72 hours after surgery. Compared with the sham group, there was no significant change in the protein expression level of PI3K and AKT in the model and ZnPP groups, while the protein expression level of p-PI3K and p-AKT decreased significantly (P<0.05). Compared with the model group, there was no significant change in the protein content of PI3K and AKT in the ZnPP group, while the protein expression level of p-PI3K and p-AKT decreased significantly (P<0.05, Figure 6). All of these suggested that HO-1 could alleviate neuronal damage after cerebral hemorrhage by regulating the PI3K/AKT signaling pathway.

Discussion
HO-1 is an inducible low molecular weight stress protein that provides cellular and tissue protection in a variety of injury and disease models. 19 In this study, the rat ICH model was constructed successfully. After inhibiting HO-1, the bleeding was more serious, the cell edema and deformation were aggravated, and the neurological deficit score in the rat was significantly increased. In addition, the expression of p-PI3K and p-AKT was decreased. Therefore, we speculated that HO-1 might have neuroprotective effects on rats with cerebral hemorrhage by regulating the PI3K/AKT signaling pathway.
As shown in a previous study, heme oxygenase (HO) has neuroprotective effects on oxidative stress in HT22 cells transfected with HO-1 siRNA. 11 Nrf2/HO-1 could mediate neuroprotective effects of mangiferin on early brain injury after subarachnoid hemorrhage by attenuating mitochondria-associated apoptosis and neuroinflammation. 10 Some scholars also have found that D163/HO-1 positive microglia  Figure 6 The expression of PI3K, AKT, p-PI3K and p-AKT protein in rat brain tissue. *p<0.05, compared with the sham group. # p<0.05, compared with the model group. and invasive macrophages show anti-inflammatory and neuroprotective phenotype after traumatic brain injury. 24 Moreover, it has been confirmed that the neuroprotective effect of melatonin on ischemia is partly caused by α-7 nicotinic receptor regulation and HO-1 overexpression. 25 Similarly, the neuroprotective effect of Acanthopanax senticosus is caused by HO-1 signaling in hippocampus and microglia. 26 Interestingly, ischemia-induced upregulation of heme oxygenase-1 protects against apoptotic cell death and tissue necrosis. 27 Moreover, ischemia-induced upregulation of heme oxygenase (HO)-1 is also critical for protecting critical perfusion flaps from apoptotic cell death and tissue necrosis. 28 In addition, the cytoprotective effect of HO-1 is found after silencing HO-1 by siRNA. 29 After inhibiting HO-1, the expression of BAX was significantly increased, while the expression of Bcl-2 was significantly decreased. The results further indicated that inhibition of HO-1 increased apoptosis in brain tissue of rats with cerebral hemorrhage.
Our study also found that HO-1 was involved in PI3K/AKT signaling pathway. Several articles have reported the relationship between HO-1 and PI3K/AKT signaling pathways. For instance, Feng et al 30 have demonstrated that the PI3K/Akt and ERK pathways are involved in OA-induced HO-1 expression by activating Nrf2 in VSMC. 30 Besides, the brain-protected part of sevoflurane post-treatment is confirmed to up-regulate the expression of HIF-1α and HO-1 via the PI3K/Akt pathway. 31 Moreover, baicalein protects 6-OHDAinduced neurotoxicity by activating Keap1/Nrf2/HO-1 and participating in PKCα and PI3K/AKT signaling pathways. 32 It is well known, the PI3K/Akt pathway has been shown to be involved in the process of degradation of extracellular matrices and chondrocyte death. 33 Moreover, hypoxia confers protection against apoptosis through the PI3K/Akt pathway in endothelial progenitor cells. 34 Also, Tibes et al 35 have confirmed that the PI3K/ Akt pathway plays an important role in acute myeloid leukemia. 35 As one of the signal pathways for cell survival, PI3K/Akt signal transduction pathway plays an important role in cell proliferation, differentiation and inhibition of neuronal apoptosis. 36 Thereby, HO-1 may protect the nerves of rats with cerebral hemorrhage by regulating PI3K/AKT signaling pathway.
By inhibiting the expression of HO-1, brain water content in cerebral ischemia rats was increased, SOD activity was down-regulated and MAD content was upregulated in this study. The structure of the blood-brain barrier can make brain tissue less susceptible to or even harmful to harmful substances in circulating blood. 37 Therefore, it could maintain the basic stability of the brain tissue environment, which has important biological significance for maintaining the normal physiological state of the central nervous system. 38 SOD and MDA are important indicators for measuring oxidative damage in brain tissue. 39 The main function of SOD is disproportionation of oxygen free radicals. 40 The activity could reflect the body's ability to scavenge oxygen free radicals. 41 The free radicals attack the unsaturated lipids in the membrane to produce lipid peroxidation products MDA, which could reflect the degree of damage to the body caused by free radicals in the body. 42 However, there were some limitations in this study. No special region of brain was researched. In the future, this will be our main research direction.

Conclusion
HO-1 can play a neuroprotective role by regulating PI3K/ AKT signaling pathway and reducing apoptosis in rats with cerebral hemorrhage. It might provide a new target for protecting the nerves of patients with cerebral hemorrhage.

Ethics approval and consent to participate
This study was conducted after obtaining local ethical committee approval of The Affiliated Yantai Yuhuangding Hospital of Qingdao University and we confirm that all experiments were performed following relevant named institutional and national guidelines and regulations.


extfieldsofstudy: Geography Medicine

text:
Effects of Regulating Visitor Viewing Proximity and the Intensity of Visitor Behaviour on Little Penguin (Eudyptula minor) Behaviour and Welfare

Simple Summary Some studies on zoo penguins have shown that the presence of visitors may be fear-provoking. To manage zoo visitor–animal interactions, it is important to determine what it is about visitors that affects zoo penguins. Consequently, we investigated the effects of regulating both zoo visitor viewing proximity to the penguin enclosure and the intensity of visitor behaviour on the behaviour and stress physiology of little penguins. A physical barrier was used to increase visitor viewing distance, by 2 m away from the enclosure, and signage and uniformed personnel was used to attempt to regulate the intensity of visitor behaviour. Signage and uniformed personnel had no effect on visitor number, noise level or visitor behaviour. However, the physical barrier reduced the frequency of potentially threatening visitor behaviours such as banging on enclosure features, looming over the pool, physical contact with the pool’s water and sudden movement. When visitors were further away from the enclosure, fewer penguins were huddling, vigilant, and retreating, and more penguins were close to the visitor viewing area, surface swimming and preening in the water. There were no effects of visitor viewing proximity or signage and uniformed personnel on the stress physiology of the little penguins. While there was no effect of signage and uniformed personnel in moderating visitor behaviour, the viewing proximity of visitors to the enclosure affected both visitor and penguin behaviour, where potentially threatening visitor behaviours were decreased and little penguins showed less avoidance behaviour when visitors were further away. Abstract We examined the effects of regulating the viewing proximity of visitors and the intensity of visitor behaviour on behaviours indicative of fear and stress physiology of 15 zoo-housed little penguins (Eudyptula minor). A 2 × 2 factorial fully randomised design was used to examine the effects of regulating: (1) the viewing proximity of visitors to enclosure, ‘normal viewing distance’ and ’increased viewing distance’ (using a physical barrier set up 2 m away from the enclosure), and (2) the intensity of visitor behaviour, ‘unregulated visitor behaviour’ and ‘regulated visitor behaviour’ (using signage and uniformed personnel). In addition, a treatment consisting of closing the enclosure to visitors was included. Penguin behaviour, visitor numbers and visitor behaviour were recorded by CCTV video recordings and direct observations, respectively. Penguin faecal glucocorticoid metabolites were also analysed as a measure of stress physiology. We found that increased viewing distance reduced (p < 0.05) all visitor behaviours except for loud vocalisations and tactile contact with penguins. However, there were no direct effects of signage and uniformed personnel on visitor behaviour (p > 0.05). As the regulation of viewing proximity increased from a closed exhibit to an open exhibit with increased viewing distance, and then to an open exhibit with normal viewing distance, this increased the proportion of penguins huddling (p = 0.0011), vigilant (p = 0.0060) and retreating (p = 0.00013), and decreased the proportion of penguins within 1 m of the visitor viewing area (p = 0.00066), surface swimming (p = 0.00091) and preening in the water (p = 0.042). There were also limited effects of regulating visitor behaviour on penguin behaviour. No treatment effects were found on faecal glucocorticoid metabolites (p > 0.05). These results indicate that regulating visitor viewing proximity affects penguin behaviours indicative of fear and visitor behaviour. This suggests that close visitor contact can be fear-provoking for little penguins but increasing the distance between visitors and penguins can reduce fear responses of penguins by regulating both viewing proximity and visitor behaviour. However, it is unclear whether these changes in penguin behaviour are due to the increased separation between visitors and penguins and/or specific visitor behaviours associated with close viewing proximity to the enclosure, such as leaning over the enclosure or tactile contact with the pool, which are impeded when visitors are further away.

Introduction
Understanding human-animal relationships in zoos has been an area of growing importance because of the effects that humans, especially visitors, can have on different zoo species such as non-human primates, big cats and ungulates, varying from negative, positive and neutral effects [1]. However, the manner in which visitors affect zoo animals remains unclear, especially for a rarely studied taxa such as penguins [1,2]. Research on the human-penguin relationship has primarily been conducted on wild penguins, with growing evidence that human contact can negatively affect various wild penguin populations. For example, Adelie penguins (Pygoscelis adeliae) have been found to have reduced breeding success as a result of repeated human approaches and nest checks [3]. Yellow-eyed penguins (Megadyptes antipodes) at sites exposed to unregulated tourism have been found to have lower breeding success and fledging weights [4,5]. Also, sites frequently visited by tourists dramatically increased heart rate and reduced reproductive success of Humboldt penguins (Spheniscus humboldti) [6], and increased vigilance and agonistic behaviours in King (Aptenodytes patagonicus), Gentoo (Pygoscelis papua) and Royal penguins (Eudyptes schlegeli) in response to human approach [7,8]. Furthermore, when exposed to high levels of human visitation, wild little penguin (Eudyptula minor) populations in Australia increasingly avoid nesting areas [9]. In contrast, some penguin species in the wild have been found to habituate to human contact, depending on previous experience and the amount of exposure to humans [10][11][12]. Despite the range of research on wild penguins, there is limited research investigating the effects of visitors on zoo-housed penguins.
To our knowledge, there have been only four studies to date that have investigated the effects of visitor presence on zoo-housed penguins. Two of these studies found negative effects of visitors, where one study showed that African penguins (Spheniscus demersus) reduced their pond use [13], and another study found that little penguins displayed increased aggression, huddling and avoidance behaviours [14] in response to visitor presence. In contrast, as visitor numbers increased, Humboldt penguins were found to spend more time swimming underwater at the underwater viewing window [15], and aquarium-housed Gentoo penguins increased their behavioural diversity and pool use [16]. These latter two studies suggest a potential positive effect of visitors. However, results from these four studies need to be interpreted with caution as three of the above-mentioned studies examined correlations between visitor numbers and penguin behaviour [13,15,16], and only one study was under experimental conditions [14]. Also, this variation in penguin behaviour related to visitor contact may be due to species or individual animal differences, such as past experiences and coping style, enclosure design or the nature of the visitor interactions [17]. Consequently, it remains unclear what aspect of visitor contact affects penguin behavioural responses.
In zoos, animals may be exposed to a range of stimuli from visitors including auditory, visual, tactile, olfactory and vibratory stimuli that may affect an animal's perception of visitors, resulting in different behavioural responses [2,18]. If visitors are perceived as fear-provoking, knowledge about the effects of regulating visitor contact, such as visitor behaviour management to reduce stimuli that animals may perceive as threatening, could be useful in reducing visitor effects on animal fear. Behavioural responses to threats, which may be indicative of fear, in penguins commonly include immobility, retreat or avoidance, vigilance, huddling, reduced preening and aggression [7,8,[19][20][21][22][23]. Meanwhile, the main physiological stress responses to threatening stimuli are via the sympatho-adrenal medullary system and the hypothalamo-pituitary-adrenal (HPA) axis, resulting in the secretion of catecholamines and glucocorticoids, respectively, when activated [24,25]. Physiological stress in zoo animals is commonly measured through faecal glucocorticoid metabolite (FGM) concentrations because of its non-invasive sampling technique [26]. Consequently, by utilising both animal behavioural and physiological measures, and experimentally regulating visitor contact, we can identify approaches to manage visitors to reduce their behaviours that may be perceived as fear-provoking for penguins, and consequently improve penguin welfare.
In this study, as a follow-up to Sherwen, Magrath, Butler and Hemsworth [14], we jointly examined the regulation of two aspects of visitor contact on little penguin behaviours indicative of fear and stress physiology: (1) visitor viewing proximity, by regulating how close visitors could approach the enclosure, using physical barriers, and (2) intensity of visitor behaviour, by including visual reminders to visitors for them to reduce noise, startling and interactive behaviours. We also added a treatment where the exhibit was closed to visitors.

Materials and Methods
This study received ethical approval from the Zoos Victoria Animal Ethics Committee (approval number ZV15022) and was conducted at the Melbourne Zoo penguin exhibit. It housed a breeding group of 15 little penguins (ten females, five males; aged between 5 months to 13 years) in an outdoor, naturalistic 330 m 2 enclosure consisting of sand and vegetation areas, and a large swimming pool (from shallow waters near ramps, to amaximum depth of 3 m) with current flow throughout the water ( Figure 1). The exhibit walls were 1.2 m in height and the visitor pathway ran along three sides of the exhibit, with the main penguin viewing positions being along the length of the pool, side A, but opportunities to view penguins also occurred on the short ledge of the pool, side B ( Figure 1). The penguins were fed twice a day (09:00 and 15:30). Husbandry including the monitoring of animals, cleaning and feeding of penguins, followed normal routines and remained consistent throughout the course of the study.

Design & Treatments
A 2 × 2 factorial treatment arrangement was used to examine the combined effects of regulating both viewing proximity and the intensity of visitor behaviour on penguins: 1.
Viewing proximity of visitors to enclosure at two levels: a.
'Increased viewing distance'-a barrier was set up 2 m from the enclosure to increase the distance between visitors and the enclosure ( Figure 2). This allowed unrestricted viewing of the enclosure but was a strong impediment to visitors physically interacting with the glass windows of the pool, pool water and other enclosure features. b.
'Normal viewing distance'-no barrier was in place and visitors could approach to the edge of the pool (i.e., visitors could approach within 2 m of the enclosure).

2.
Intensity of visitor behaviour at two levels: a.
'Regulated visitor behaviour'-the objective of this treatment was to attempt to reduce the intensity of visitor behaviour using signs requesting visitors to be quiet, move slowly in the exhibit area and avoid physically interacting with the penguins (Figure 3). Also, for this treatment, the researcher was dressed in zoo uniform. This procedure has been successfully used in research on meerkats to reduce the intensity of visitor behaviours [27].

Design & Treatments
A 2 × 2 factorial treatment arrangement was used to examine the combined effects of regulating both viewing proximity and the intensity of visitor behaviour on penguins: 1. Viewing proximity of visitors to enclosure at two levels: a. 'Increased viewing distance'-a barrier was set up 2 m from the enclosure to increase the distance between visitors and the enclosure ( Figure 2). This allowed unrestricted viewing of the enclosure but was a strong impediment to visitors physically interacting with the glass windows of the pool, pool water and other enclosure features. b. 'Normal viewing distance'-no barrier was in place and visitors could approach to the edge of the pool (i.e., visitors could approach within 2 m of the enclosure).
2. Intensity of visitor behaviour at two levels: a. 'Unregulated visitor behaviour'-visitor behaviours were uncontrolled. b. 'Regulated visitor behaviour'-the objective of this treatment was to attempt to reduce the intensity of visitor behaviour using signs requesting visitors to be quiet, move slowly in the exhibit area and avoid physically interacting with the penguins (Figure 3). Also, for this treatment, the researcher was dressed in zoo uniform. This procedure has been successfully used in research on meerkats to reduce the intensity of visitor behaviours [27].   In addition to this factorial arrangement, a treatment consisting of closing the enclosure to visitors ('closed exhibit') was included. This fifth treatment can be used to confirm that the presence of visitors increased behaviours indicative of fear in penguins. Alternatively, it can be considered as an extreme level of regulating visitor proximity.
Thus, there were five treatments studied: 1. Normal viewing distance and unregulated visitor behaviour-no barrier in place and visitor behaviour was uncontrolled. 2. Increased viewing distance and unregulated visitor behaviour-barrier was in place and visitor behaviour was uncontrolled. 3. Normal viewing distance and regulated visitor behaviour-no barrier in place and visitor behaviour was attempted to be controlled. 4. Increased viewing distance and regulated visitor behaviour-barrier was in place and visitor behaviour was attempted to be controlled. 5. Closed exhibit-penguin exhibit closed to the public.
Each treatment was randomly imposed for 2-day periods, two treatments per week with one day break in between (Mon-Tues and Thurs-Fri), with three replicates of each treatment (total of 30 study days) using a fully randomised factorial design. The study was conducted from the end of February to May 2016 (Summer/Autumn) over 9 weeks, and was only conducted on school working days, to avoid the normal systematic variation in visitor numbers that occurs on weekends and during school holiday periods. Two out of the 9 weeks had treatments with no day break in between. This was due to public holidays occurring on the Monday one week and Friday the other week.

Animal Behavioural Observations
Four CCTV cameras placed around the enclosure, covering the main areas utilised by the penguins and recording continuously each day, were used to transcribe penguin behaviour at the group level ( Figure 4). Observations were only conducted on the birds visible on camera.
Penguin behavioural data were transcribed during the playback of the video footage and were overlayed to watch all four cameras on one screen using the VLC Media Player 2.2.1 ( Figure 4). Observations were conducted on all study days in 3 × 1 h observation blocks (10:00-11:00, 11:30-12:30, 14:00-15:00). A combination of instantaneous point sampling and one-zero sampling was used for behaviour patterns that were considered states (of an appreciable duration) and events (relatively short durations), respectively [28] (see Table 1 for a description of these two types of behaviours). Instantaneous point sampling at 3 min intervals was used to record the number of visible penguins in each behavioural state. Furthermore, one-zero sampling every 30 s, within each 3 min interval, was used to record the number of visible penguins performing each of the behavioural events. In addition to this factorial arrangement, a treatment consisting of closing the enclosure to visitors ('closed exhibit') was included. This fifth treatment can be used to confirm that the presence of visitors increased behaviours indicative of fear in penguins. Alternatively, it can be considered as an extreme level of regulating visitor proximity.
Thus, there were five treatments studied:

1.
Normal viewing distance and unregulated visitor behaviour-no barrier in place and visitor behaviour was uncontrolled.

2.
Increased viewing distance and unregulated visitor behaviour-barrier was in place and visitor behaviour was uncontrolled.

3.
Normal viewing distance and regulated visitor behaviour-no barrier in place and visitor behaviour was attempted to be controlled.

4.
Increased viewing distance and regulated visitor behaviour-barrier was in place and visitor behaviour was attempted to be controlled.

5.
Closed exhibit-penguin exhibit closed to the public.
Each treatment was randomly imposed for 2-day periods, two treatments per week with one day break in between (Mon-Tues and Thurs-Fri), with three replicates of each treatment (total of 30 study days) using a fully randomised factorial design. The study was conducted from the end of February to May 2016 (Summer/Autumn) over 9 weeks, and was only conducted on school working days, to avoid the normal systematic variation in visitor numbers that occurs on weekends and during school holiday periods. Two out of the 9 weeks had treatments with no day break in between. This was due to public holidays occurring on the Monday one week and Friday the other week.

Animal Behavioural Observations
Four CCTV cameras placed around the enclosure, covering the main areas utilised by the penguins and recording continuously each day, were used to transcribe penguin behaviour at the group level ( Figure 4). Observations were only conducted on the birds visible on camera.
Penguin behavioural data were transcribed during the playback of the video footage and were overlayed to watch all four cameras on one screen using the VLC Media Player 2.2.1 ( Figure 4). Observations were conducted on all study days in 3 × 1 h observation blocks (10:00-11:00, 11:30-12:30, 14:00-15:00). A combination of instantaneous point sampling and one-zero sampling was used for behaviour patterns that were considered states (of an appreciable duration) and events (relatively short durations), respectively [28] (see Table 1 for a description of these two types of behaviours). Instantaneous point sampling at 3 min intervals was used to record the number of visible penguins in each behavioural state. Furthermore, one-zero sampling every 30 s, within each 3 min interval, was used to record the number of visible penguins performing each of the behavioural events.     Locomotion (land) Upright position, moving from one location to another either by walking or running.

States
Surface swimming Moving or floating on the surface of the water, with head erect or in the water.

Diving
Swimming under the water surface.

Events
Preening (on land) Running bill through plumage on land.
Preening (in water) On the surface of the water and running bill through plumage.

Allopreening
Running bill through the plumage of another bird(s).

Flee
Diving or moving rapidly away from current position in response to a direct approach or interaction by human.

Retreat
Swimming or locomoting (on land) slowly away from human approach or presence.

Social interaction
On land: Approaching another individual with flippers back, pushing individual in a circular motion, nibbling and/or preening head or neck of the individual.
In water: approaching another individual in the water, in a circular motion, nibbling and/or preening head or neck of the individual.

Manipulating inanimate object
Using bill to peck or nibble at an inanimate object such as a plant, stick, rock, grass, etc.

Chasing insect
Following a flying insect.

Interaction with keeper/staff
Approaching keepers and engaging in play, feeding and/or agonistic behaviours toward keepers.

Penguin Faecal Sampling and Analysis: Faecal Glucocorticoid Metabolites (FGM)
Faecal samples were collected by the principal investigator (SJC) at the end of the first and second day of each replicate of each treatment between 15:30 and 16:30, and samples were immediately stored in a freezer at −20 • C. Samples were collected on both days because previous research on this population has found that the median excretion lag time is 7 h and peaked on average at 10.8 h after a health check [29]. However, Sherwen and Fanson [29] found that three of the four penguins studied showed a peak response 5-7 h after the stressor. Consequently, we would expect that if some individuals on the first day of each replicate of a treatment in our experiment were exposed in the morning to fear-provoking bouts of visitor contact, these stress responses would be reflected in FGM concentrations in samples late in the afternoon. Due to limitations of collection, it was not always possible to identify which faecal samples came from which bird. Therefore, we collected all samples deposited over an hour after their feed with the aim of obtaining a good representation of the whole population. An average of 13 samples (range 7-20) were collected per day for the group of 10-15 birds, for an average of 91% of the penguins sampled each day. When more than one sample was knowingly collected from the same individual on the same day (n = 17 samples), the average FGM value for that individual was used. A total of 397 faecal samples were collected during the study. From those samples, 372 samples (96%) were then used to calculate final concentrations for analysis (after samples with a sample coefficient of variation (CV) greater than 20% and sample weights less than 0.01 g were removed); 83, 82, 74, 73 and 60 samples were collected in treatments 1, 2, 3, 4 and 5, respectively.
Faecal samples were dried in a lyophilizer (TFD series, ilShin Biobase, Amsterdam, Netherlands) at −80 • C for 24 h. They were then pounded and sifted to remove debris. One mL of 60% methanol was added to 0.10 g of dried faecal powder in polypropylene tubes. Samples were mixed on a vortex until samples were homogenised (~10 s) and then shaken on an orbital shaker for 1 h then centrifuged for 10 min at 2000 rpm (Hettich Universal 320 R, Tuttlingen, Germany; RCF = 2500). Supernatant was poured into 1.5 mL microcentrifuge tubes and stored in a freezer at −20 • C. FGM was measured and analysed using a validated double-antibody cortisone enzyme-immunoassay for little penguins [29]. Furthermore, this assay was similar to an assay made by the same manufacturer that has been validated for Adelie penguins [30,31]. Assay procedures have been previously described [29]. All samples were assayed in duplicates and data are expressed as ng/g dried faeces for final concentrations. The intra-assay CV was less than 15%. Inter-assay CVs were 4.2% and 9.1% for low and high controls, respectively (n = 11 plates).

Visitor Behavioural Observations
Visitor observations were directly conducted by the principal investigator (SJC) using instantaneous point sampling at 3 min intervals in seven 30 min blocks between 09:30 and 15:15 to record visitor numbers (number of adults and children within 1 m of enclosure). Within each 3 min interval, the frequency of visitor behavioural events (described in Table 2) were recorded continuously in each observation block. These observations were made from the visitor viewing area in the furthest corner of the exhibit area to reduce the effect of researcher presence on the penguins and visitors. Ambient noise level was recorded every 3 min using a sound level data logger (Standard ST-173) which was placed inside the penguin exhibit ( Figure 1).

Behaviour Description
Tactile contact with enclosure features Tapping or banging on the glass windows, barriers, or enclosure features.
Loud vocalisations Shouts, screams, loud whistles to attract the animals' attention.

Looming
Leaning on or over the exhibit barriers to view animals in the water or on land. Penguins > 0.5 m-penguins greater than 0.5 m away from looming position. Penguins < 0.5 m-penguins less than 0.5 m away from looming position.
Tactile contact with water Touching/flicking/slapping the water with the hand(s), which creates ripples in the water.
Tactile contact with penguin Touching penguin with the hand(s).
Sudden movement Running, waving or jumping towards or at the penguin(s) and/or exhibit.

Data Analysis
Measurements were based on the proportion of penguins visible on the video footage and were averaged over the 2-day period treatment to obtain a single summary value for each 2-day period. For animal behavioural states, the proportion of penguins visible per sample point per day was calculated and averaged in order to calculate the proportion of visible penguins observed displaying each behaviour for each day. Animal behavioural events were handled in a similar manner whereby the average proportion of visible birds within each 30 s observation interval per day, observed displaying each behavioural event was calculated. In relation to visitor variables, the numbers of adults and kids were summed to give the visitor number, noise level (dB) was averaged and visitor behaviours were totaled for each day (the average number of times each behaviour was observed in each of the seven 30 min observation blocks) and subsequently averaged over the 2-day period treatment replicates to obtain a single summary value. FGM was also averaged over samples obtained in the 2-day period treatment replicates to obtain a single summary value. The 2-day summary values were used as the unit of analysis in all statistical analysis.
Preliminary analysis indicated that the strongest observed effects could be considered as an ordered function of the allowed viewing proximity of visitors (normal viewing distance vs. increased viewing distance vs. no visitors (exhibit closed)). Thus, measurements were analysed using a single error analysis of variance (ANOVA) with terms for (i) visitor viewing proximity regulation (normal viewing distance; increased viewing distance; exhibit closed), (ii) visitor behaviour regulation (regulated; unregulated) when exhibit was open and (iii) interaction between visitor viewing proximity and visitor behaviour regulation when exhibit was open. These terms had 2, 1 and 1 degrees of freedom, respectively. No visitor measurements were recorded when the exhibit was closed, since most visitor measurements are, by definition, zero (e.g., when the exhibit is closed there are no visitors, thus the frequency of each visitor behaviour is always zero). Thus, the analysis of visitor measurements was modified to have only 2 levels for the visitor proximity term (normal viewing distance; increased viewing distance). In these cases, the analysis simplified to a 2 by 2 (visitor viewing proximity by visitor behaviour regulation) factorial ANOVA. Prior to statistical analysis, the values for animal behaviour (state and events) were angularly transformed and the values for visitor behavioural events were square root transformed so that the residual variation was similar in all treatments. No transformation was required for FGM.
Meteorological data (maximum and minimum daily temperatures), zoo visitor gate numbers and the proportion of male penguins, all measurements that are unlikely to be affected by the treatment applied, were used as covariates if they were significant (p < 0.05). This improved, sometimes substantially, the precision of some of the statistical analyses. The proportion of males was considered as a covariate since by the end of the study only females were exhibited after two males were removed for medical reasons and three males were removed to prevent early breeding.
For visitor data, the p values, of each term, were calculated using the F distribution with 1 or 8 degrees of freedom, except for analyses that included covariates, where an F distribution with 1 or 7 degrees of freedom was used. Some visitor behaviours only occurred with the normal viewing distance, and in these cases p values were calculated using non-parametric permutation tests since the large number of zero values can cause over-sensitivity in the analyses. Also, in these cases, standard errors of difference were calculated from analyses only including normal viewing distances, so as to avoid severe underestimation of these standard errors.
For animal behaviour data and FGM, p values for proximity effects were calculated using the F distribution with 2 or 10 degrees of freedom, except for analyses that included covariates, where an F distribution with 1 or 9 degrees of freedom was used. For regulation effects and interactions, p values were calculated using the F distribution with 1 or 10 degrees of freedom, except for analyses that included covariates, where an F distribution with 1 or 9 degrees of freedom was used. Analyses were carried out using the ANOVA directive and APERMTEST procedure of GenStat 16th Edition statistical program (software for biosciences).  Table 3). However, there was no corresponding effect (p > 0.05) on ambient noise, loud vocalisations, looming behaviour with penguins less than 0.5 m from looming position or tactile contact with penguins. Increased viewing distance was also associated with a 30% reduction in the number of visitors to the exhibit (F 1,7 = 0.51, p = 0.0047; Table 3). There was no evidence at the 5% level that the attempted regulation of visitor behavior affected actual visitor behavior, ambient noise or the number of visitors to the exhibit (Table 4). Nevertheless, the best estimate for the effect of the attempted regulation of visitor behaviour is the frequency of tactile contact with enclosure features (the third highest frequency of the recorded behaviours) which was reduced by about 40%, although the p value was 0.30 (Table 3). Table 3. Effect of treatment on visitor variables measured. The means shown are the mean frequency of visitor behaviours (per seven 30 min observations blocks per 2-day period) considered as events and ambient noise level per 2-day period. All measurements were square root transformed, except for ambient noise level and the number of visitors (*). Back-transformed means are presented in parentheses.

Behavioural States
As the viewing proximity of visitors (with unregulated visitor behaviour) increased from a closed exhibit to an open exhibit with increased viewing distance, and then to an open exhibit with normal viewing distance, the proportion of penguins huddling increased from 25% to 69% (F 2,10 = 14.56, p = 0.0011), the proportion vigilant increased from 3% to 22% (F 2,10 = 8.89, p = 0.0060), the proportion of penguins within 1 m of side A (F 2,10 = 16.63, p = 0.00066) and B (F 2,10 = 11.66, p = 0.0024) decreased from 28% to 1% and 3%, respectively, and the proportion surface swimming decreased from 39% to 9% (F 2,10 = 15.27, p = 0.00091; Table 4). When the exhibit was open, regulating the intensity of visitor behaviour (using signage and uniformed personnel) reduced huddling by about 20% (F 1,10 = 15.95, p = 0.0025) but had no effect on vigilance, proximity to side A or B of the visitor viewing area or swimming ( Table 4). The proportion of penguins huddling in an open exhibit with regulated behaviour and increased viewing distance (25%) was similar to the proportion huddling when the exhibit was closed to visitors ( Table 4).
The effect on locomotion appears more problematic; especially considering the s.e.d.s were considerably lower than those of other behavioural states (Table 4), the significant (p < 0.05) effects would disappear if there were similar s.e.d.s to other behavioural states, and the fact that it was the only behaviour in which a significant interaction (p < 0.1) between visitor viewing distance and regulated visitor behaviour was found. No other effects of regulating visitor behaviour or interactions were found on penguin behaviour (p > 0.05; Table 4). There were no treatment effects (p > 0.05) on the proportion of penguins visible, resting, idle and diving (Table 4).

Behavioural Events
Evidence (p < 0.05) of visitor viewing proximity on penguin behavioural events only occurred for preening in the water (F 2,10 = 4.44, p = 0.042) and retreating ((F 2,9 = 28.56, p = 0.00013; Table 4). The occurrence of retreating was very low when viewing distance was increased (with unregulated visitor behavior) or when the exhibit was closed (Table 4). On the other hand, preening in the water mostly occurred with visitors at increased viewing distance (with unregulated visitor behaviour; Table 4). Also, there was some evidence (p < 0.05) that regulating visitor behaviour decreased the occurrence of allopreening and pecking regardless of viewing distance (Table 4). However, it should be noted that the proportion of penguins engaging in the behavioural events in any 30 s period was, in general, very small.

Little Penguin FGM Concentrations
There was no evidence (p > 0.05) of any treatment effect on FGM concentrations (Table 4).

Discussion
The viewing proximity of visitors was the only factor that had a statistically significant effect on visitor behaviour, whereby the 2 m separation (using a physical barrier) between the visitor viewing area and the penguin enclosure decreased the frequency of tactile contact with enclosure features, looming over the enclosure ledge, tactile contact with the pool's water and sudden movement. This indicates that the physical barrier not only regulated visitor viewing proximity but also visitor behaviour. Park et al. [32] supports our results, since they found that a physical fence was a more effective strategy to keep visitors on maintained trails at Acadia National Park, USA, compared to educational signage. Similarly, we found no direct evidence that regulating the intensity of visitor behaviour using signage and uniformed personnel was effective, which was aimed at regulating noise, sudden movement and interaction with the penguins. These results suggest that signage and uniformed personnel were much less effective in regulating visitor behaviour than regulating viewing proximity. This is consistent with Acevedo-Gutiérrez et al. [33], who found that posted signs were unsuccessful in managing interactions between tourists and seals. In comparison, other studies have found that signs can effectively regulate visitor behaviour [27,34]. Nevertheless, our findings highlight that physical barriers may be an effective management strategy to manage visitor-penguin interactions in zoos.
The primary objective of our experiment was to identify aspects of visitor contact involved in eliciting behaviours indicative of fear, such as increased avoidance behaviour, huddling and vigilance, in little penguins. The study by Sherwen, Magrath, Butler and Hemsworth [14] found that the presence of visitors was fear-provoking but not specifically what aspect of visitors was responsible for this effect. We found that as the regulation of visitor viewing proximity increased from a closed exhibit to an open exhibit with increased viewing distance, and then to an open exhibit with normal viewing distance, more little penguins were observed huddling and vigilant, and less penguins were observed close to the visitor viewing area and surface swimming. This indicates that the study penguins may have perceived visitors as threatening when in close proximity, with the increased vigilance and huddling behaviour reflecting increased alertness, and the increased distance from the visitor viewing area reflecting avoidance. These results are consistent with Sherwen, Magrath, Butler and Hemsworth [14], who studied the same enclosure but with a different mix of individual little penguins, and studies on wild penguins that have found close human proximity affects their avoidance behaviour. For example, the close proximity of humans to penguin landing sites has been shown to delay the landing times on beaches of Yellow-eyed penguins returning from their daily forage at sea [35], elicit avoidance responses in Adelie [36] and Humboldt penguins [6] and increase heart rates in nesting Gentoo [37] and Adelie penguins [38]. Wild little penguins also prefer to nest further away from visitor paths or areas of high human disturbance [39,40]. Furthermore, in our experiment, the similarity between the effects of increased viewing distance and the exclusion of visitors from the exhibit suggests that a physical barrier to push visitors 2 m away from the enclosure can closely parallel visitor absence for penguins. Consequently, this provides evidence that a physical barrier may be an effective management strategy that results in reduced fear responses in little penguins.
It should be recognised that the increase in behaviours indicative of fear in little penguins, which was elicited by the close proximity of visitors, may have also been influenced by the opportunity for visitors to perform behaviours such as looming, whereby visitors could lean on or over the enclosure ledge, that may have been fear-provoking. This behaviour was the most frequently recorded visitor behaviour and was eliminated by restricting visitors from closely approaching the enclosure by erecting the barrier 2 m from the enclosure. Looming by visitors was often accompanied by tactile contact with the water and sudden movements towards the enclosure. Thus, looming and these accompanying behaviours by visitors when in close proximity are likely to be perceived by penguins as a predatory threat, similar to how an aerial predator would prey upon little penguins. This is supported by Giese [38] that found a human standing lower to nesting Adelie penguins' horizon represented less of a threat on the basis of reduced heart rate, compared to a human looming over nesting penguins. Furthermore, little penguins are the smallest species of penguins, that grow to an average height of 30 cm and weigh about 1 kg [41][42][43]. They are commonly predated upon by terrestrial predators such as red foxes (Vulpes vulpes), cats (Felis catus) [44,45] and water rats (Hydromys chrysogaster) [46], aerial predators such as sea eagles (Haliaeetus spp.) and large gulls [43], and aquatic predators such as long-nosed fur seals (Arctocephalus forsteri) [47]. It is therefore not surprising that little penguins may perceive visitors as threatening, particularly when close visitor contact may be intense, unpredictable and possibly unrewarding. In addition to the effects on avoidance behaviour and alertness in little penguins, an interesting finding with the effects of regulating visitor viewing proximity was an increase in the number of birds observed preening in the water when visitors were further away.  anecdotally reported that penguins perform increased preening while swimming. Presumably, comfort behaviours such as preening are generally more likely to occur when the animal is not threatened [19,[48][49][50]. Furthermore, the deprivation of a presumably highly motivated behaviour such as swimming is a welfare concern [51][52][53]. Wild little penguins spend most of their day(s) foraging out at sea [43,54]. However in the zoo, penguins do not need to forage as their food is provided to them [14]. Yet not surprisingly, we found the penguins still spent most of their time swimming when visitors were not present or when visitors were further away, suggesting it is a highly motivated behaviour and possibly an indicator of a positive welfare state.
While the present experiment and that of Sherwen, Magrath, Butler and Hemsworth [14] show evidence that the penguins perceived visitors as fear-provoking based on behavioural changes, there are several possible explanations for a lack of treatment effects on FGM in our study. There is an extensive body of evidence demonstrating the importance of both predictability and control in determining the behavioural and physiological effects of aversive stimuli [55][56][57]. A degree of both predictability of visitor contact and control over visitor contact, by hiding from and avoiding fear-provoking visitor interactions, may have been an effective adaptive response in ameliorating the activation of the HPA axis in the penguins when visitor viewing proximity and behaviour were uncontrolled (i.e., standard zoo conditions) [58,59]. Another possibility may be that the study period was insufficient to elicit a measurable change in FGM. Our study exposed penguins to some treatments that should have been less stressful than their normal exposure to zoo visitors, and it may take longer for the HPA axis to lower glucocorticoid production than it does to mount a stress response. Nakagawa, Möstl and Waas [30] found that FGM concentrations for Adelie penguins after an adrenocorticotropic hormone (ACTH) challenge can take up to 48 h to return to baseline levels. Consequently, a more sustained period of treatment, that is greater than a 2-day period, may be required to demonstrate increased stress arising from uncontrolled visitor contact.
There were limited effects of attempting to regulate the intensity of visitor behaviour, through use of signage and uniformed personnel, on penguin behaviours indicative of fear. While fewer penguins were observed huddling, allopreening and pecking, there were no effects of attempting to regulate visitor behaviour on the number of penguins observed close to the visitor viewing area, surface swimming and vigilant. These limited effects on penguin behaviours are not surprising, since this treatment had no observed effect on reducing visitor behaviours that intuitively may be fear-provoking such as tactile contact with enclosure features, loud vocalisations, looming over the enclosure ledge, physically touching the pool's water and suddenly moving towards the enclosure. However, there may be a precision issue with our results regarding the regulation of visitor behaviour, as the frequency of tactile contact with enclosure features, loud vocalisations and physical contact with the pool were about halved when signage and uniformed personnel was used. Despite not being statistically significant, it could explain why there were still some effects of attempting to regulate visitor behaviour on some penguin behaviours which provides evidence that signage and uniformed personnel must have affected some relevant aspect of visitor behaviour. On the other hand, a possible reason that the signage had limited effectiveness is that some of the potentially fear-provoking visitor behaviours were not specifically targeted in the signage. Several studies have shown signs that have clear instructions that target a specific behaviour [60], justification [61] and appeal to visitors' emotions [34], are more effective in moderating visitor behaviour. The signs, in our study, requested visitors to be quiet, move slowly and not interact with the penguins in the exhibit area, but there was no mention of avoiding specific behaviours such as looming and tactile contact with the water, which were effectively reduced or eliminated by regulating viewing proximity. Thus, in addition to close proximity per se, these visitor behaviours that are possible when close to the enclosure such as leaning on or over the enclosure ledge and physical contact with the water, may be fear-provoking and may be more effectively managed by using a physical barrier rather than signage. Clearly, further experimentation is required to fully appreciate visitor behaviours that are fear-provoking for penguins.
The decreased number of visitors found when visitor viewing distance was increased suggests a potential reduction in visitor dwell times and possibly reduced visitor viewing experience at the penguin enclosure. This is consistent with Sherwen, Magrath, Butler, Fanson and Hemsworth [18] who found that reducing the visual contact of capuchins with visitors, by using a one-way visual barrier, reduced visitor numbers. In contrast, Sherwen, Magrath, Butler, Phillips and Hemsworth [27] found no effect of signs on visitor numbers. Further investigation on the effects of manipulating visitor behaviour on visitor experience would be valuable for future focus.
Interestingly, no treatment effects were found on the proportion of penguins visible despite the evidence that visitors may be perceived as threatening for penguins with close visitor contact. Visitor gate numbers was a statistically significant covariate for this behaviour. The more visitors that came through the zoo gates, the less penguins were visible. This suggests that penguins may utilise different behavioural strategies depending on the type of visitor contact. For example, high numbers of visitors in the zoo would increase noise levels, but not direct interactive behaviours that would occur at the exhibit. It would be valuable to further investigate this relationship.

Conclusions
This study provides evidence that regulating the viewing proximity of visitors, either by the installation of a physical barrier or by closing the exhibit to visitors, strongly reduces little penguin behaviours indicative of fear and strongly increases swimming, which is likely impeded by fear. Regulating visitor behaviour by signage was much less effective in changing penguin behaviour, but it is unknown to what extent this was caused by having signage that insufficiently targeted specific visitor behaviours. Regulating visitor viewing proximity or visitor behaviour did not affect little penguin FGM, which may suggest that penguin behavioural responses such as avoidance may have been an effective adaptive response in ameliorating stress arising from close visitor contact, or that the treatment period was insufficient to elicit a measurable change in FGM. However, whether it is visitors being further away per se and/or the reduction of some visitor behaviours such as leaning over the enclosure or tactile contact with the pool that are impeded when visitors are further away, remains unclear.