Amphiprion ocellaris (Fautin & Allen, 1992)

Nguyen, Hai-Thanh T., Zhao, Min, Wang, Tianfang, Dang, Binh T., Geffen, Audrey J. & Cummins, Scott F., 2024, Sea anemone-anemonefish symbiosis: Behavior and mucous protein profiling, Journal of Fish Biology 105 (2), pp. 603-618 : 609-613

3.2 | Analysis of juvenile A. ocellaris skin mucous proteins

To determine whether the profile of anemonefish mucous proteins was impacted by the observed symbiosis, a proteomics analysis was performed across symbiosis intervals (I – III), as well as compared to the NC and PC groups. PCA scatter plots of skin mucous proteins from pooled samples (N = 8) of the experimental treatments revealed three main clusters: the PC group, the NC group, and the E (experimental) group (Figure 4). At the first interval (I), when fish lived with a natural host in both EXP and PC group, the protein profiles between these groups seemed tightly clustered. The protein composition of fish living with rubber anemones (NC) did not change significantly between the first (I) and the second (II) intervals. The mucous proteins from fish in E did change over time as the unnatural host anemone were removed from the tank. At the last interval (III), there was variation in the protein composition of the NC fish, whereas PC and E fish were separated in two clusters. The two-way ANOSIM indicated significant changes in protein profiles between intervals (File S2a, two-way ANOSIM, factor time) and between treatments (File S2a, two-way ANOSIM, factor treatment, p = 0.0054). The hierarchical clustering analyses performed on the PCA loadings for protein profiles over treatments versus intervals corroborated these clusters (File S2b; Figure 4).

A total of 348 nonredundant proteins were identified from all skin mucous samples combined, of which the PC group contained the highest proportion of proteins (Figure 5a; File S3). The

Anemone Experiment hosts Host characteristics interval

Stichdactyla Natural host, short tentacles Interval I gigantea (60 – 69 DPH)

Acclimation Fish that contacted Survival (%) mean ± SD behavior duration anemone (%) (of two experimental runs)

<10 min 100 (24/24) 100 (42/42) Stichodactyla Cogeneric with natural host, Interval II haddoni short tentacles (70 – 78 DPH)

Heteractis Unnatural host, long tentacles Interval III crispa (79 – 92 DPH) 1 h

3 h

100 (24/24) 94 ± 6.0 (40 ± 1/42)

20.8 (5/24) 80.1 ± 11.5 (32 ± 1/39) Heteractis Unnatural host, long tentacles malu

Macrodactyla Unnatural host, long tentacles doreensis

Entacmaea Unnatural host, long tentacles quadricolor

No symbiosis 0

No symbiosis

No symbiosis

Note: S. gigantea was removed after interval I, and S. haddoni was removed after interval II, leaving only H. crispa , H. malu , M. doreensis , and E. quadricolor in the experimental tanks during interval III. Survival of fish (%) was measured in two separate runs of the experiment. DPH, days post-hatch.

total number of proteins identified increased progressively within the EXPs, from intervals I to III. The PC group samples contained the most unique proteins (117) when compared to all other samples, whereas 63 proteins were common in all experimental and control samples (Figure 5a); this provided a reference list of proteins that are present in A. ocellaris skin mucus, with or without symbiosis. The ANOSIM analysis also indicated that the mucous proteins in the EXP were significantly different between intervals (File S2c; ANOSIM, 1E-3E p = 0.0439 <0.05), whereas those differences were not significant in the NC and PC groups (File S2c; ANOSIM, 1NC – 3NC p = 0.2031 and 1PC – 3PC p = 0.1001, respectively). Gene ontology analysis of these common proteins at the biological process level found that most proteins were associated with cytoskeleton organization, protein-containing complex assembly, signal transduction, translation, and cellular protein modification (Figure 5b). At the molecular function level,

Best

Description Number e -value

14-kDa apolipoprotein 1 3.23E- 48

14-3-3 Protein beta/alpha-B-like 1 9.21E- 175

14-3-3 Protein zeta-like 2 1.08E- 165

Actin, alpha skeletal muscle 1 6.60E- 74

Actin, cytoplasmic 1 3 0

Acyl-CoA-binding domain-containing 1 2.31E- 55 protein 7

ADP-ribosylation factor GTPase-activating 1 8.28E- 45 protein 1-like

Apolipoprotein A-I-like 1 3.80E- 167

Calpain-2 catalytic subunit-like 1 0 Calpastatin isoform X17 1 0

Cystatin-B-like 1 4.26E- 64

Dentin matrix acidic phosphoprotein 1-like 1 8.80E- 11

DNA polymerase alpha subunit B 1 0 Elongation factor 1-alpha 2 0

Eukaryotic translation initiation factor 5A- 1 4.44E- 131 1-like

GATOR complex protein NPRL3 isoform 1 0

X1

Glutathione S -transferase A-like 5 4.50E- 167

Glyceraldehyde-3-phosphate 1 0 dehydrogenase 2

Haemoglobin subunit beta-like 1 2.22E- 106

Histidine triad nucleotide-binding protein 1 1 5.71E- 88

Histone H1-like 1 1.97E- 62

Description Number Best e -value

Histone H2A-like 4 4.25E- 118

Histone H2B 1/2-like 1 2.03E- 59

Histone H 3 1 5.32E- 152

Ictacalcin-like isoform X 2 1 9.46E- 20

Induced stolen tip protein TUB8-like 1 1.28E- 67

Keratin, type I cytoskeletal 13-like 3 0

Keratin, type II cytoskeletal cochleal-like 1 0

Leukocyte elastase inhibitor-like 1 0

Myosin light polypeptide 6 2 5.15E- 127

Neuroblast differentiation-associated protein 1 0

AHNAK-like isoform X1

Non-histone chromosomal protein HMG-14-like 2 2.00E- 38

Peptidyl-prolyl cis–trans isomerase-like 1 5.16E- 118

Polyubiquitin-B 3 0 Profilin-1-like 1 4.16E- 100

Protein S100-A1-like 4 1.35E- 74

Protein STPG 4 1 9.06E- 144

Prothymosin alpha-B-like 1 3.70E- 05

Thioredoxin-like 1 6.58E- 74

Thymosin beta- 12 1 2.84E- 33

TRAF2 and NCK-interacting protein kinase-like 1 0

isoform X2

Ubiquitin-40S ribosomal protein S27a 1 4.78E- 83 Ubiquitin-60S ribosomal protein L 40 1 6.48E- 89 the majority of these proteins were involved in ion binding and DNA binding. Examples of proteins that match with other proteins with a confident annotation (e -value cutoff:10-3) are summarized in Table 4, including proteins associated with defense (e.g., apolipoprotein A-I, 14-3-3 protein, peptidylprolyl cis–trans isomerase [PPI], thymosin beta-12) and cell structure (e.g., keratin, myosin light polypeptide 6, actin). To our knowledge, this is the first report on protein S100-A1 identified in the fish skin mucus. Utilizing a S. haddoni transcriptome-derived protein database, we found that 39 proteins could be confidently (e - value cutoff: 10-3) designated as nonfish, of which 7 were anemone-like (with highest similarity to Exaiptasia pallida ) and absent from NC group samples (Table 5; File S4).

Further comparative analysis of skin mucus in EXPs (interval stages I – III), using only proteins present in all biological replicates, showed the presence of unique proteins in interval I (sciellin protein), whereas interval III samples contained an abundance of histone and ribosomal proteins that were absent from other interval samples (Figure 6a; Table 6; File S3). No unique proteins were identified at interval II. Additionally, proteins related to cellular response to stress, cell death, and response to abiotic stimulus were found. Comparative analysis of skin mucous proteins between the NC and PC groups found that most unique proteins were present within the PC group at the final stage (3PC) (Figure 6b). Gene ontology analysis of those proteins for biological processes showed a prominence of cellular and metabolic processes and, with the involvement in translation, small-molecule metabolic process and response to stress. The high proportion of proteins involved in translation is primarily derived from the presence of numerous 40S and 60S ribosomal proteins. Similarly, histones and actins were prevalent in PC group samples.

Protein match and species e -Value

Interval I Interval II Interval III PC NC

Note: Gray shading represents intervals where proteins were present in mucous samples. See Supplementary File S4 for details of protein sequences. Abbreviations: NC, negative control; PC, positive control.