Artemia

Adult zebrafish (AB line, ZIRC, Eugene, OR, USA) were kept at 28°C under a 14-h light:10-h dark cycle, and water conditions of environmental quality were maintained according to an established protocol [68 ]. Zebrafish at 3.5 mpf were assigned into two dietary groups (overfeeding and maintenance groups) with approximately five fish per 2-L tank. Zebrafish in the overfeeding group were fed three times per day with freshly hatched Artemia (corresponding to 60 mg cysts/fish/day). Zebrafish in the maintenance group were fed freshly hatched Artemia (corresponding to 5 mg cysts/fish/day) once per day. For calorie restriction, the zebrafish were fed freshly hatched Artemia (corresponding to 2.5 mg cysts/fish/day) for 2 weeks after being overfed for 8 weeks. Commercial flake food (Hikari Tropical Fancy Guppy, Kyorin, Hyogo, Japan) consists of 9% fat, 17% carbohydrate and 59% protein, whereas Artemia nauplii consist of 22% fat, 16% carbohydrate and 44% protein (dry weight basis).
The percentage of consumed Artemia was estimated by counting Artemia before and after feeding. Briefly, 1 mL of water was collected from 1700 mL of water containing freshly hatched Artemia corresponding to 100 or 8.5 mg cysts. The numbers of hatched Artemia were counted three times to determine a mean count. After counting, the samples were returned to the 2-L tank. Then, five fish were transferred to the tank for feeding. After 2 hours, the number of Artemia was counted as before feeding. Zebrafish fed 5 or 60 mg of Artemia per day consumed about 80 or 50% of the provided Artemia, respectively. Zebrafish fed 2.5 mg of Artemia per day consumed almost all of the provided Artemia.

The ambon damselfish Pomacentrus amboinensis and lemon damsel P. moluccensis are common site attached species of damselfish (family Pomacentridae) found throughout the Indo-Pacific on shallow reef habitats at the interface between the live coral and rubble reef edge (Fig. S1). Both species have a similar larval duration after a demersal egg phase and settle at similar sizes (P. amboinensis 17.8 d, 11.2 mm SL; P. moluccensis 19.4 d, 10.7 mm SL; [17] ). Metamorphosis is concomitant with settlement and in these species involves a major change in pigmentation (transparent to coloured) that occurs within hours, but involves little obvious change in shape [36] . However, settlement does involve major changes in physiology [37] and it is likely that marked changes also occur in the sensory systems [38] . A laboratory-based habitat selection experiment has previously shown that both species preferentially settle to healthy live coral [39] . Both species settle naturally to patches of mixed live and dead coral. Both are also planktivores as juveniles and eat a similar array of prey items (Text S1). A tagging study of 295 newly settled P. amboinensis on the continuous reef edge found that fish moved little over the first 3 months after settlement (mean = 0.63 m [40] ). It is likely that P. moluccensis has a similar degree of site attachment (pers. obs.).
Research on newly settled P. amboinensis has shown that fish enter the reef with high variability in their behavioural traits (e.g. boldness, aggression) and these traits are displayed in a manner that is consistent on small time scales of hours to days ([41] (link), [42] (link), Mero, Meekan and McCormick unpublished data]. Establishment of dominance hierarchies occurs within minutes of settlement within the species, which can rapidly lead to the eviction of subordinates from small habitat patches [31] (link). Because of the rapid establishment of territories and the high juvenile mortality, it was decided that 60 min was an ecologically relevant time to use for the establishment of residents in the priority experiments for the present study.
The present datasets were collected at Lizard Island (14° 40′S 145° 28′ E) on the northern Great Barrier Reef, Australia, between October 2007 and March 2010. Both newly metamorphosed juveniles and recently settled juveniles from the reef were used for field experiments. Light traps (see [43] for design; small trap) were used to collect both fish species at the end of their larval phase prior to their settlement to the reef. These newly metamorphosed fish were separated by species and placed into 60 L aquaria with aerated flowing seawater. Fish were kept for 24 h and fed newly hatched Artemia sp. twice per day ad libitum to allow recovery from (or acclimation to) the stress of capture, prior to use in experiments. Juveniles were collected from a shallow fringing reef at the back of Lizard Island using a solution of dilute clove oil and hand nets. All fishes used in the experiments were placed into a small clip-seal bag with a small amount of aerated seawater and measured with calipers (±0.1 mm) and then transferred into individually labeled 1 L clip-seal bags for transport. To reduce transport and handling stress, fish in bags were transported to the field site in a 30 L bin of seawater (to reduce temperature fluctuations) under subdued light conditions.

Embryos were obtained from natural crosses and staged according to Kimmel et al^{74 (link)}.
All fish were housed according to FELASA recommendations^{75 (link)}, in tanks filled with circulating system water at 28 ± 0.2 °C. System water was made up from Reverse Osmosis water, with synthetic sea salt added at an amount such that the conductivity of the water was kept at approximately 800 uS/cm. Average water quality data were as follows: pH: 7.30, general Hardness: 160 mg/l CaCO3, Ammonia: 0 mg/l NH3, Nitrite: 0 mg/l NO2, Nitrate: <10 mg/l NO3- and Conductivity: around 800 µS/cm. Light cycle was 14 h light/10 h dark (Lights on at 08:00, off at 22:00 daily). All fish were in general good health and no specific diseases were observed throughout the colony. The fish were fed Paramecium from 5–15 dpf, followed by Zeigler Larval AP100 powder (MBK Installations, Cat. No. LD150) from day 16–22 dpf. From 23 dpf onwards they were fed Sparos Zebrafeed 200–400 (Sparos) and ZM Brine Shrimp Artemia Cysts Premium 260 grade (ZM Systems). Fish euthanized by schedule 1 killing were given an overdose of an anaesthetic (5 drops of 0.48% MS-222 added to a petridish of water).

Adult double transgenic (mpeg1:mCherry/mpx:eGFPi¹¹⁴) expressing mCherry under the macrophage-specific mpeg1 promotor and GFP under the neutrophil-specific mpx promotor fish were housed and fed as previously described [49 (link)]. Embryos were obtained by natural spawning. Fish were fed as follows: weeks 1 and 2 with rotifers (× 4/day from 5 days post fertilization -dpf-), week 3 with rotifers and Artemia Nauplii 230.000 npg (Nauplii per gram) (Ocean Nutrition Europe, Essen, Belgium) (× 2/day), week 4 with Artemia (× 2/day) and until 40 dpf Artemia and Tetramin Flakes (Tetra, Melle, Germany) (× 2/day). When fish reached the juvenile stage, at 40 dpf [76 (link)], fish were randomly distributed into 6 tanks, 2 per each diet: one was sampled after 1 week and the other tank after 3 weeks of the feeding experiment per each diet. The feeding experiment was performed blind and fish were fed until slightly before satiation twice a day. Each tank received one of the following: a control diet, a saponin-supplemented diet or a butyrate-supplemented diet. Full diet composition is listed in Table 1.Table 1

Formulation of experimental diets analysed once the feed intervention was performed to check which compositions corresponded to the blinded diets

	A: Control diet (%)	B: Butyrate diet (%)	C: Saponin diet (%)
Wheat	7.00	6.99	6.67
Wheat gluten	16.00	16.00	16.00
Sunflower meal	1.68	1.68	1.68
Soy protein concentrate	15.16	15.16	15.16
Fish meal	52.00	52.00	52.00
Fish oil	4.40	4.40	4.40
Rapeseed oil	2.00	2.00	2.00
Vitamin mix	0.35	0.35	0.35
Mineral mix	1.92	1.92	1.92
Butyrate	0.00	0.01	0.00
Saponin	0.00	0.00	0.33
[VOLUME]	100.0	100.0	100.0
Dry matter	92.2	92.0	92.0
Crude protein	56.0	57.4	57.4
Crude fat	13.5	13.8	13.8
Ash	8.9	8.9	8.9

The three diets are similar in composition (dry-matter, protein, fat and ash). 95% ultrapure soy saponin was kindly provided by Trond Kortner NMBU Oslo Norway, origin: Organic Technologies, Coshocton, OH, [40 (link)]