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A microalga is better than a commercial lipid emulsion at enhancing live feeds for an ornamental marine fish larva

Basford, Alexander J; Mos, Benjamin; Francis, David S; et al. https://researchportal.scu.edu.au/discovery/delivery/61SCU_INST:ResearchRepository/1268876610002368?l#1368876590002368

Basford, A. J., Mos, B., Francis, D. S., Turchini, G. M., White, C. A., & Dworjanyn, S. (2020). A microalga is better than a commercial lipid emulsion at enhancing live feeds for an ornamental marine fish larva. Aquaculture, 523. https://doi.org/10.1016/j.aquaculture.2020.735203 Document Version: Accepted

Published Version: https://doi.org/10.1016/j.aquaculture.2020.735203

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Please do not remove this page 1 A microalga is better than a commercial lipid emulsion at enhancing live feeds for an

2 ornamental marine fish larva

3

4 Alexander J. Basford ab*, Benjamin Mos a, David S. Francis c, Giovanni M. Turchini c,

5 Camille A. White d, Symon Dworjanyn a

6

7 a National Marine Science Centre, Southern Cross University, Coffs Harbour 2450, Australia

8 b Darwin Aquaculture Centre, Department of Primary Industry and Resources, Northern

9 Territory Government, Channel Island 0822

10 c Deakin University, School of Life and Environmental Sciences, Geelong 3220, Australia

11 d Oceans and Atmosphere, Food Nutrition and Bio-based Products, Commonwealth Scientific

12 and Industrial Research Organization, Hobart 7000, Australia

13 * Corresponding author: [email protected] (A. J. Basford)

14

15 Highlights

16 • Larval Amphiprion latezonatus grew faster and had a higher survival rate when fed

17 live feeds enhanced with the microalga Proteomonas sulcata compared to live feeds

18 enriched using a commercial lipid emulsion.

19 • A. latezonatus fed live feeds enriched with a commercial lipid emulsion had the

20 highest DHA content, but this did not correlate with survival or growth.

21 • DPAn-6 and the ratio of DPAn-6 to DHA in A. latezonatus tissues was positively

22 correlated with larval survival and growth.

23 • High levels of dietary DHA may be less important for some marine fish larvae than

24 previously thought.

25 26 Key words: Amphiprion, anemonefish, cryptomonad, larval rearing, phospholipids, fatty

27 acids, 22:6n-3, 22:5n-6 28 Abstract

29 High mortality during larval rearing is a persistent bottleneck in finfish aquaculture

30 and is often caused by inadequate nutrition. Nutritional deficiencies in live feeds are usually

31 overcome by enrichment with commercial lipid emulsions; however, enriching live feeds

32 using microalgae may be more effective. This study investigated the efficacy of the microalga

33 Proteomonas sulcata as a live feed enhancement for rearing larvae of the wide-band

34 anemonefish, Amphiprion latezonatus. We found A. latezonatus larvae fed live feeds

35 enhanced with P. sulcata had higher survival and better growth at 7 days post hatch (dph)

36 compared to fish fed live feeds enriched with a commercial lipid emulsion or left unenriched.

37 At 14 dph, A. latezonatus initially fed rotifers cultured on P. sulcata for several generations

38 had the highest survival overall. These results may be due to the high phospholipid content in

39 rotifers enhanced on P. sulcata compared to other diets. Survival, length, depth, and eye

40 diameter of 7 dph A. latezonatus was also positively correlated with omega-6

41 docosapentaenoic acid (DPAn-6, 22:5n-6) and high ratios of DPAn-6 to docosahexaenoic

42 acid (DHA, 22:6n-3) in their tissues, highlighting the need to better understand the role of

43 DPAn-6 in larval fish ontogeny. Surprisingly, given their high survival and growth, A.

44 latezonatus larvae fed P. sulcata enhanced live feeds had lower levels of DHA compared to

45 the more poorly performing fish fed the commercial lipid emulsion enriched live feeds; this

46 challenges the paradigm of a positive correlation between DHA and growth and survival that

47 is documented in many other marine fish larvae. This study demonstrates the benefits of

48 using P. sulcata to enhance the nutritional quality of live feeds and highlights the need for a

49 better understanding of the role of n-6 long-chain polyunsaturated fatty acids in the early

50 development of larval fish. 51 1. Introduction

52 The majority of marine fish sold in the ornamental aquarium trade are wild caught

53 from coral reefs (Rhyne et al., 2014). This harvest is an additional stressor for one of the most

54 threatened ecotypes on the planet, but could be alleviated if more marine ornamental fish

55 were produced in closed cycle aquaculture (Ban et al., 2014). The small proportion of

56 cultured finfish in the ornamental trade is largely due to the difficulty in rearing larvae

57 (Calado et al., 2017), similar to the persistent bottleneck caused by high mortality of fish

58 larvae grown for food (Hamre et al., 2013; Rønnestad et al., 2013).

59 An important contributor to mortality during larval rearing is inadequate nutrition in

60 live feeds (Hamre et al., 2013). Rotifers, Brachionus spp., and brine shrimp, Artemia spp., are

61 the most common live feeds used in aquaculture, but contain insufficient amounts of essential

62 nutrients required by fish larvae such as long chain (≥20 chain length) polyunsaturated fatty

63 acids (LC-PUFA) and phospholipids (Rainuzzo et al., 1997; Sorgeloos et al., 2001; Hamre et

64 al., 2008; Hamre et al., 2013). Lipids are the most energy dense nutrients available to marine

65 fish, and larvae require high levels of specific LC-PUFA such as arachidonic acid (ARA,

66 20:4n-6), omega-6 docosapentaenoic acid (DPAn-6, 22:5n-6), eicosapentaenoic acid (EPA,

67 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3), due to their inability to synthesise these

68 fatty acids from more commonly available, shorter-chain and less unsaturated, precursors

69 (Parrish et al., 2007; Glencross, 2009; Tocher 2010; Izquierdo and Koven, 2011). Marine fish

70 larvae also require dietary LC-PUFA in specific ratios that vary among species (Glencross

71 2009). It is generally accepted that more dietary DHA should be provided than EPA and

72 ARA, although there is less clarity about what DHA to DPAn-6 ratios are ideal (Sargent et

73 al., 1997). DHA is preferentially conserved over other LC-PUFA and plays an essential role

74 in larval fish survival and development (Watanabe, 1993; Sargent et al., 1999; Hamre et al.,

75 2013). 76 In aquaculture, DHA and other essential fatty acids are typically delivered to marine

77 fish larvae by enriching live feeds with lipid emulsions (Dhert et al., 2001; Ma and Qin,

78 2014). Commercially available lipid emulsions are typically rich in n-3 LC-PUFA,

79 particularly DHA, and are used to increase the levels of these nutrients in rotifers and

80 Artemia. However, this is primarily in the form of neutral lipids and triacylglycerols

81 (Coutteau and Mourente, 1997; Li et al., 2015). While live feeds with high neutral lipid

82 content can be nutritious (Estévez and Giménez, 2017), larval fish should ideally receive

83 dietary lipids in the form of polar lipids, such as phospholipids, which are more efficiently

84 assimilated (Olsen et al., 2014). In addition, lipid emulsions are expensive, and live feeds

85 egest gut-loaded lipid emulsions, adversely affecting water quality (Høj et al., 2009;

86 Yamamoto et al., 2009).

87 An alternative to lipid emulsions for enhancing the nutritional content of live feeds is

88 microalgae (Ma and Qin, 2014; Rehberg-Haas et al., 2015a; Ferreira et al., 2018). Live feeds

89 raised on microalgae can incorporate desirable nutrients such as LC-PUFA in the form of

90 biologically available phospholipids (Rainuzzo et al., 1994a; Li and Olsen, 2015; Li et al.,

91 2018) and pass these nutrients to their offspring. This can amplify the content of these

92 nutrients in live feeds over generations (Koiso et al., 2009; Li et al., 2015; Sperfeld and

93 Wacker, 2015). Attempts to grow rotifers on microalgae have been hindered by the use of

94 relatively non-motile genera such as Pavlova and Isochrysis, which settle out of culture and

95 degrade water quality (Ferreira et al., 2008; Rehberg-Hass et al., 2015b). Motile microalgae

96 such as cryptomonads remain suspended in the water column and are already used as food for

97 invertebrate larvae (Knuckey et al., 2005; Mos et al., 2011), raising the possibility that these

98 microalgae may be suitable for enhancing the nutritional content of live feeds.

99 This study compared the nutritional quality of live feeds enhanced with the

100 cryptomonad microalga, Proteomonas sulcata, to live feeds enriched with a commercial lipid 101 emulsion. Most studies on improving the nutritional value of live feeds for larval fish have

102 used commercially important food finfish, while similar studies on ornamental species are

103 scarce (Calado et al., 2017). In this study, larvae of the wide-band anemonefish Amphiprion

104 latezonatus were used as a model ornamental fish species. A. latezonatus is a rare and

105 valuable species in the aquarium trade due to restricted wild collection from its small home

106 range (Scott et al., 2011). To gauge the efficacy of mutigenerational and short term

107 enhancement of live feeds with microalgae, we compared the survival and growth of A.

108 latezonatus larvae fed rotifers that were: 1. Cultured on P. sulcata for several generations; 2.

109 Enriched with P. sulcata overnight; 3. Enriched with a commercial lipid emulsion (Selco

110 S.presso); or 4. Left unenriched (Table 1). As larvae grew and became capable of ingesting

111 larger prey, they were switched from rotifers to Artemia that were: 1. Enriched with P.

112 sulcata; 2. Enriched with a commercial lipid emulsion; or 3. Left unenriched (Table 1). The

113 survival, body length, body depth, eye diameter of A. latezonatus larvae, as well as their total

114 lipid, lipid class, and fatty acid composition, were measured at 7 and 14 days post hatch

115 (dph). Relationships between the fatty acid and lipid profiles and the survival and growth of

116 A. latezonatus were tested using distance based linear modelling. The fatty acid and lipid

117 composition of different enrichments, culture media, and live feeds were also examined.

118 2. Materials and methods

119 2.1. Study organism

120 Amphiprion latezonatus larvae originated from brood stock housed in a communal

121 tank at the National Marine Science Centre, Coffs Harbour. Brood stock were fed a mix of

122 fresh fish, squid, and prawn meat. Spawning occurred naturally every 2 – 3 weeks between

123 November and July from a mated pair within the tank. A clutch of A. latezonatus eggs on a

124 terracotta plate was removed from the brood stock tank the night before the eggs were due to

125 hatch. The clutch was placed in a 30-L glass tank with fine aeration over the eggs to mimic 126 water movement by parents and promote high hatching rates. Larvae were used in

127 experiments immediately after hatching.

128 2.2 Experimental design

129 A. latezonatus larvae were fed one of four diets that incorporated differently enhanced

130 rotifers to 7 dph and differently enhanced Artemia from 7 to 14 dph (Table 1). The four diets

131 were: 1. Rotifers cultured on P. sulcata followed by Artemia enriched with P. sulcata (PsC

132 diet), 2. Rotifers and Artemia enriched with P. sulcata (PsE diet), 3. Rotifers and Artemia

133 enriched with the commercial lipid emulsion Selco S.presso (INVE Aquaculture, Salt Lake

134 City, UT, USA) (SsE diet), and 4. Unenriched rotifers and Artemia (Un diet) (Table 1).

135 2.3. Experimental set up

136 Twenty 9-L cylindrical black plastic tanks were kept in heated water baths to maintain

137 a water temperature of ~23 °C (n = 5 tanks per diet). Each tank was fitted with a 50 mm Ø

138 banjo screen with 500 µm mesh that maintained the water volume at 7 L. Newly hatched A.

139 latezonatus larvae were gently scooped from the hatching tank with a small beaker and

140 haphazardly stocked into and were stocked at a density of 5 larvae L-1 (Olivotto et al., 2008).

141 Abnormal larvae that could not maintain their position in the water column or were

142 swimming erratically were not used in experiments. Each tank had an air stone providing fine

143 aeration and gentle upwelling to promote water circulation and distribute prey evenly without

144 causing excessive disturbance to the fish. Nannochloropsis sp. paste (Nanno 3600, Reed

145 Mariculture, Campbell, CA, USA) was added to all tanks daily to a density of 1 × 106 cells

146 mL-1 prior to feeding to prevent phototactic behaviour by A. latezonatus larvae. Tanks were

147 flushed nightly with 1 µm filtered, UV treated seawater (FSW) at a rate of 0.2 L min-1 to

148 remove excess prey and waste. Water quality parameters were always within pH 7.9–8.1, > 7

149 mg L-1 dissolved oxygen, and 35.0–36.0 ppt salinity, measured in all tanks daily using a Hach 150 HQ40d multi-controller fitted with a Hach PHC101 temperature compensated pH probe,

151 Hach LDO101 probe, and Hach CDC101 conductivity probe. Tanks were maintained under a

152 12:12 (light:dark) period, with a light intensity of ~2000 lx at the surface of the water.

153 2.4. Live feed preparation

154 2.4.1. Microalgae

155 Live cultures of Proteomonas sulcata were grown in aerated 20-L carboys filled with

156 FSW at 25–26 °C. Cultures were fertilised with F media (AlgaBoost 2000×, AusAqua Pty

157 Ltd., Wallaroo, SA, Australia) and were placed in front of four 32 W, 4000 K fluorescent

158 lights. P. sulcata cultures were fed to rotifers or Artemia after reaching a cell density of ~4.5

159 × 106 mL-1.

160 To obtain P. sulcata for lipid analysis, 1 L from three separate cultures used during

161 the study were pooled. Microalgae cells were concentrated by repeatedly centrifuging (3000

162 rpm for 5 min) in 50 mL falcon tubes and discarding excess water. Samples were then freeze

163 dried, combined, and stored at -80 ºC prior to lipid analyses.

164 2.4.2. Rotifers

165 Two separate cultures of the rotifer Brachionus sp. (S-strain, acquired from Port

166 Stephens Fisheries Institute, Taylors Beach, NSW, Australia) were established two weeks

167 prior to the start of the experiment. Cultures were maintained at 190–250 ind. mL-1 in 200-L

168 black fibreglass tanks filled with 25–27 ºC FSW. Water was exchanged at a rate of 20 % d-1

169 and oxygen saturation was kept above 7.5 mg L-1. One culture was fed Nannochloropsis paste

170 at a rate of 40 mL d-1. The second culture was fed P. sulcata (cell density ~4.5 × 106 mL-1) at

171 a rate of 20 L d-1.

172 Rotifers were fed to A. latezonatus larvae each day for 7 d, at a density of 10 ind. mL-

173 1. For the P. sulcata cultured treatment (PsC diet, Table 1), rotifers were harvested and rinsed 174 with FSW immediately before feeding to A. latezonatus larvae. For the P. sulcata enriched

175 (PsE diet, Table 1) and Selco S.presso enriched treatments (SsE diet, Table 1), each night 40

176 L of the rotifer culture fed Nannochloropsis paste was harvested, concentrated, rinsed with

177 FSW in a 45µm wet sieve, and divided equally into two 20-L plastic buckets. Rotifers for the

178 PsE diet were enriched with 3 L of P. sulcata for 12 h. Rotifers for the SsE diet were

179 enriched with 5 mL of emulsified Selco S.presso for 12 h. Vigorous aeration was provided to

180 both buckets. Unenriched rotifers (Un diet, Table 1) were harvested directly from the

181 Nannochloropsis paste fed culture and rinsed with FSW immediately before feeding to A.

182 latezonatus larvae. On the first day of the experiment, rotifers from each diet were

183 photographed under a stereo microscope and measured using ImageJ 1.51j8 imaging

184 software. The volume and lorica length of 50 rotifers from each diet were calculated

185 following Hilder et al. (2015).

186 Excess rotifers (~4 × 106 individuals) from each diet were retained for fatty acid

187 analyses each day. Rotifers were centrifuged (3000 rpm for 2 min) to remove excess

188 seawater, freeze dried, and stored at -80 ºC. Samples from each day were pooled for lipid

189 analyses. At the end of the experiment, 2 mL of Nannochloropsis paste and 2 mL of Selco

190 S.presso were freeze dried and stored at -80 ºC prior to lipid analyses.

191 2.4.3. Artemia

192 Each day, 36 h prior to feeding Artemia to A. latezonatus larvae, 5 g of Artemia cysts

193 (EG type, Sep-ArtTM, GSL, INVE, Belgium) were hatched over 24 h in illuminated and

194 aerated 28–30 ºC FSW. Artemia were separated from their cysts magnetically and divided

195 equally into three 20-L buckets. In each respective bucket, Artemia were enriched for 12 h

196 overnight using 4 L of P. sulcata (PsC and PsE diets, Table 1), or 10 mL of emulsified Selco

197 S.presso (SsE diet, Table 1), or held in FSW only (Un diet, Table 1). Vigorous aeration was

198 provided to all buckets. Immediately prior to feeding to the larval fish, Artemia from the three 199 buckets were separately rinsed with FSW in a 70µm wet sieve to remove the enrichment

200 medium, and added to larval rearing tanks each day for 7 d at a density of 0.5 ind. mL-1. On

201 the seventh day of the experiment, the length of 50 Artemia from each diet was assessed

202 using the same method as for the rotifers (section 2.4.2).

203 Excess Artemia (~2 × 105 individuals) from each diet were retained for fatty acid

204 analyses each day. Samples from each diet were condensed into 15 mL falcon tubes, chilled

205 at -20 ºC for 5 min to slow movement, centrifuged (3000 rpm for 2 min) to remove excess

206 seawater, freeze dried, and stored at -80 ºC. Samples from each day were pooled for lipid

207 analyses.

208 2.5. Sampling and analyses

209 2.5.1. Survival

210 Each morning prior to feeding, survival was determined by removing and counting

211 dead A. latezonatus, and calculating the percentage of fish left alive in each replicate. Five

212 fish were subtracted from the initial stocking number in calculations done after 7 dph to

213 account for fish removed for morphometric and biochemical analyses.

214 2.5.2. Morphometrics

215 The growth and morphology of A. latezonatus was measured at 7 and 14 dph,

216 corresponding with the transition from eating rotifers to Artemia and the end of the larval

217 phase respectively (Table 1). At 7 and 14 dph, five living fish were sampled from each tank.

218 Fish were removed in the morning prior to feeding, allowing sufficient time for the previous

219 day’s food to be egested. At 14 dph, all fish were removed from replicates that had five or

220 fewer fish remaining. Fish were anaesthetized (1 ppm AQUI-S®, New Zealand Ltd), placed

221 on a stage micrometre, and photographed using an Olympus DP26 camera mounted on a 222 stereo microscope. A. latezonatus larvae were then washed in Milli-Q water, frozen at -80 ºC,

223 and then freeze dried within 24 h, before being stored at -80 ºC prior to lipid analysis.

224 To gauge differences in the development of A. latezonatus among the four diets, eye

225 diameter, body depth at anus, and standard length were measured from photographs using

226 ImageJ 1.51j8 imaging software following Thépot et al. (2016). Only one fish survived to 14

227 dph in the Un diet, so measurements were not taken for this treatment at this time point.

228 2.5.3 Fatty acid and lipid class analyses

229 Total lipids were extracted by modified Folch et al. (1957) technique using a

230 dichloromethane: methanol extraction (2:1). Extracts were washed with a 0.44% potassium

231 chloride: methanol: water solution (3:1:1). Lipids were recovered and quantified

232 gravimetrically. Following lipid extraction, fatty acids were transesterified into methyl esters

233 using acetyl chloride: methanol (1:10) at 100ºC for 1 h. Briefly, a known aliquot of C23:0

234 was added to each sample as an internal standard (Sigma-Aldrich, Inc., St. Louis, MO, USA)

235 and fatty acid methyl esters were isolated and identified using an Agilent Technologies GC

236 7890A equipped with a DB23 capillary column (30 m, 0.25 mm internal diameter, 0.25 μm

237 film thickness; Agilent Technologies, Santa Clara, California, USA), a flame ionisation

238 detector (FID), an Agilent Technologies 7693 autosampler injector and a split injection

239 system (split ratio 50:1). Fatty acids were identified relative to known external standards

240 (Sigma-Aldrich, Inc., St. Louis, MO, USA, and Nu-Chek Prep, Elysian, MN, USA), and

241 resulting peaks were corrected by the theoretical relative FID response factors for methyl

242 transformation and then quantified relative to the internal standard. Lipid classes were

243 determined by Iatroscan MK 6s thin layer chromatography-flame ionisation detector (TLC)-

244 FID (Mitsubishi Chemical Medience, Tokyo, Japan) according to the method previously

245 described (Conlan et al., 2018). Total lipid was determined gravimetrically and via the sum of

246 peak areas obtained from TLC-FID. 247 2.6. Statistical analyses

248 Data for A. latezonatus survival, eye diameter, body depth, length, fatty acids, and

249 lipid classes at 7 dph and at 14 dph were analysed in Primer 7 with PERMANOVA+

250 extension (v. 7.0.11) with diet as a fixed factor (Anderson et al., 2008). Pairwise comparisons

251 were used as post-hoc tests when the main test indicated a significant difference among diets

252 (p < 0.05). Monte-Carlo values were used when there were fewer than 100 permutations.

253 Multiple linear regressions were used to examine the relationships between survival,

254 eye diameter, body depth at anus, and standard length of A. latezonatus at 7 dph, as well as

255 survival to 14 dph, and individual fatty acids and ratios (Table S4, S5) or lipid classes (Table

256 S6, S7) using mean data for each replicate. Marginal tests and draftsman’s plots were used to

257 check for collinearity among predictors and analyse the relationship between a single fatty

258 acid, ratio, or lipid class variable and survival, eye diameter, body depth at anus, and standard

259 length of A. latezonatus at 7 dph, as well as survival at 14 dph, respectively. Predictors with p

260 values > 0.05 were excluded from further analysis. Linear regressions were conducted using

261 the distance-based linear modelling (DISTLM) with distance based redundancy analysis

262 (dbRDA) routine of Primer 6 (Primer-E, Plymouth) with PERMANOVA+ extension

263 (v.6.1.18) software. The BEST model selection routine with AICc selection criterion (a

264 modified version of Akaike's Information Criterion) and BIC selection criterion (Schwarz’s

265 Bayesian Information Criterion), based on 9999 permutations, were used to select the five

266 best fitting models. As analyses run using BIC produced similar models to AICc, only the

267 AICc models are presented.

268 3. Results

269 3.1. Effects of diet on survival of A. latezonatus

270 At 7 dph, survival of larval A. latezonatus fed PsC (82%) and PsE (76%) diets was

271 higher than larvae fed SsE (53%) and Un (47%) diets (One-way ANOVA, F3, 16 = 6.36, p = 272 0.0052, post-hoc pair-wise test, PsC = PsE > SsE = Un, Fig. 1). Survival was not different

273 between larvae fed PsC or PsE diets, and there was no difference in the survival of larvae fed

274 SsE or Un diets (Fig. 1).

275 At 14 dph, larval A. latezonatus fed the PsC diet had higher survival (64.6%) than any

276 other diet (F3, 15 = 20.60, p < 0.0001, PsC > PsE = SsE > Un, Fig. 1). Survival of larvae fed

277 PsE (38.6%) and SsE (15.3%) diets was not different to each other, and higher than the

278 survival of larvae fed the Un diet (0.8%) (Fig. 1).

279 3.2. Effects of diet on growth and morphology of A. latezonatus

280 At 7 dph, the eye diameter of A. latezonatus larvae fed the PsE diet (0.78 mm) was

281 larger than those fed SsE (0.76 mm) and Un (0.75 mm) diets (F3, 16 = 4.83, p = 0.0131, PsE =

282 PsC > PsC = SsE = Un, Fig. 2A). The eye diameter of A. latezonatus fed the PsC diet (0.77

283 mm) was not different to all other diets (Fig. 2A).

284 At 7 dph, the body depth of A. latezonatus larvae fed PsC (1.00 mm) and PsE (1.01

285 mm) diets were not different to each other and greater than larvae fed the SsE (0.90 mm) diet

286 (F3, 16 = 3.73, p = 0.0339, PsC = PsE = Un > Un = SsE, Fig. 2B). The body depth of larvae

287 fed the Un diet (0.94 mm) was not different to all other diets (Fig. 2B).

288 At 7 dph, the standard length of larval A. latezonatus fed PsC (5.49 mm) and PsE

289 (5.49 mm) diets were longer than larvae fed SsE (5.16 mm) and Un (5.26 mm) diets (F3, 16 =

290 8.64, p = 0.0019, PsC = PsE > SsE = Un, Fig. 2C). Standard length was not different between

291 larvae fed PsC or PsE diets, and there was no difference in the standard length of larvae fed

292 the SsE or Un diets (Fig. 2C).

293 At 14 dph, there was no differences in eye diameter (F3, 12 = 0.38, p = 0.7703), body

294 depth (F3, 12 = 0.28, p = 0.8328), or standard length (F3, 12 = 0.26, p = 0.8362) of A.

295 latezonatus among any diets (Fig. 2). 296 3.3. Lipid profiles of live feed enhancements

297 P. sulcata had the highest levels of total polyunsaturated fatty acids (PUFA, 560.8 mg

298 g lipid-1), and EPA (111.0 mg g lipid-1), while Selco S.presso had the highest n-3 LC-PUFA

299 (256.5 mg g lipid-1) and total lipid content (852.9 mg g sample dry weight (DW)-1, Table 2).

300 Selco S.presso had the highest level of DHA (225.3 mg g lipid-1) followed by P. sulcata (37.4

301 mg g lipid-1, Table 2). Nannochloropsis paste did not contain DHA. Nannochloropsis paste

302 contained the highest levels of ARA (4.0 mg g lipid-1, Table 2). P. sulcata and Selco S.presso

303 had similar levels of DPAn-6 (19.35 and 18.92 mg g lipid-1, respectively), while

304 Nannochloropsis paste had amost no DPAn-6 (0.15 mg g lipid-1, Table 2).

305 P. sulcata had the highest proportion of polar lipids (924.2 mg g lipid-1)

306 predominantly in the form of acetone mobile polar lipids (AMPL, 565.96 mg g lipid-1),

307 followed by phosphatidylethanolamine (PE, 175.35 mg g lipid-1, Table 3). Selco S.presso did

308 not contain PE, but did contain phosphatidylcholine (PC, 119.35 mg g lipid-1), and was

309 predominantly composed of neutral lipids (841.2 mg g lipid-1, Table 3) Nannochloropsis was

310 predominantly composed of polar lipids (752.3 mg g lipid-1), mainly in the form of AMPL

311 (378.88 mg g lipid-1) and PE (238.40 mg g lipid-1, Table 3).

312 3.4. Characteristics of differently enhanced live feeds

313 3.4.1. Morphology

314 The volume of rotifers in the PsC diet (20.5 × 106 µm3) was larger than any other

315 treatment, and rotifers in the PsE diet (16.2 × 106 µm3) were larger than rotifers in both SsE

6 3 6 3 316 12.1 × 10 µm ) and Un diets (11.5 × 10 µm ) (F3, 196 = 35.02, p < 0.001, PsC > PsE > SsE =

317 Un, Fig. 3A).

318 The lorica of rotifers in the PsC diet (223 µm) was longer than any other treatment

319 (F3, 196 = 15.354, p < 0.001, PsC > PsE = Un > Un = SsE, Fig. 3B). Rotifers in the PsE diet 320 (206 µm) had longer lorica than those in the SsE diet (187 µm), while the lorica length of

321 rotifers in the Un diet (198 µm) was not different to those in PsE and SsE diets (Fig. 3B).

322 There was no difference in the length of Artemia in PsC/PsE (666 ± 10 µm), SsE (641

323 ± 10 µm) and Un (656 ± 9 µm) diets (F2, 147 = 1.534, p = 0.219).

324 3.4.2. Lipid profiles

325 Rotifers and Artemia enriched with Selco S.presso had the highest total lipid content

326 (165.6 and 191.3 mg g sample DW-1, respectively), and concentrations of total n-3 LC-PUFA

327 (239.4 and 124.6 mg g lipid-1, respectively) and DHA (141.6 and 71.1 mg g lipid-1,

328 respectively, Table 2). EPA levels were highest within rotifers cultured on P. sulcata (63.6

329 mg g lipid-1) and within Artemia enriched with Selco S.presso (29.8 mg g lipid-1, Table 2).

330 ARA levels were highest within rotifers that were unenriched (12.3 mg g lipid-1) and lowest

331 within rotifers cultured on P. sulcata (4.5 mg g lipid-1), but were similar within Artemia

332 across all diets (3.6–4.1 mg g lipid-1, Table 2).

333 Rotifers and Artemia enriched with Selco S.presso had the highest proportion of

334 neutral lipids (704.4 and 722.9 mg g lipid-1 respectively, Table 3). The highest proportions of

335 polar lipids were found in rotifers cultured on P. sulcata (698.3 mg g lipid-1) and were

336 predominantly in the form of PE (175.57 mg g lipid-1) and PC (192.36 mg g lipid-1, Table 3).

337 Both P. sulcata enriched and unenriched Artemia were predominantly composed of neutral

338 lipids (620.1 and 582.8 mg g lipid-1, respectively, Table 3). The most abundant polar lipid

339 class in P. sulcata enriched and unenriched Artemia was PC (146.91 and 158.13 mg g lipid-1,

340 respectively, Table 3).

341 3.5. Effect of diet on lipid profiles of A. latezonatus

342 3.5.1. Fatty acid composition 343 At 7 dph, A. latezonatus larvae fed the SsE diet had higher levels of DHA than larvae

344 fed all other diets (F3, 15 = 9.49, p = 0.0021, SsE > PsE = PsC = Un, Table 4). 7 dph A.

345 latezonatus larvae fed the SsE diet also had higher levels of linoleic acid (LNA, 18:2n-6) than

346 larvae fed the PsC and PsE diets, while larvae fed the Un diet had similar levels of LNA to

347 larvae fed the SsE and PsE diets (F3, 15 = 6.32, p = 0.006, SsE = Un > Un = PsE > PsE = PsC,

348 Table 4). There was no difference in the ARA or EPA content of 7 dph A. latezonatus larvae

349 among any diets (ARA, F3, 15 = 0.88, p = 0.4751; EPA, F3, 15 = 2.20, p = 0.13, Table 4). 7 dph

350 A. latezonatus fed the PsC and PsE diets had significantly higher amounts of stearidonic acid

351 (SDA, 18:4n-3) than larvae fed the SsE and Un diets (F3, 15 = 43.76, p = 0.0001, PsC = PsE >

352 SsE = Un, Table 4). The DPAn-6 content of 7 dph A. latezonatus larvae was not different

353 among PsC, PsE and SsE diets, but significantly higher than those fed the Un diet (F3, 15 =

354 10.02, p = 0.0006, PsC = PsE = SsE > Un, Table 4)

355 At 14 dph, A. latezonatus larvae fed the SsE diet had higher levels of DHA than

356 larvae fed the PsC and PsE diets where larvae had DHA levels that were statistically similar

357 (F2, 12 = 4.50, p = 0.03, SsE > PsC = PsE, Table 4). There was no difference in the ARA or

358 EPA content of 14 dph A. latezonatus larvae among the diets (ARA, F2, 12 = 2.23, p = 0.1511;

359 EPA, F2, 12 = 1.54, p = 0.257, Table 4).

360 Differences among diets for the remaining fatty acids and total lipid content for 7 and

361 14 dph A. latezonatus larvae are summarised in Table 4.

362 3.5.2. Fatty acid ratios

363 At 7 and 14 dph, A. latezonatus fed the SsE diet had higher DHA/EPA ratios than any

364 other treatment, followed by fish fed the PsC diet which had higher DHA/EPA ratios than

365 those fed PsE and Un diets (7 dph: F3, 15 = 43.45, p = 0.0001, SsE > PsC > PsE = Un; 14 dph:

366 F2, 12 = 6.62, p = 0.0084, SsE > PsC > PsE, Fig. 4A). 367 At 7 and 14 dph, there was no difference in the EPA/ARA ratios of A. latezonatus

368 larvae among any diets (7 dph: F3, 15 = 1.29, p = 0.3097, 14 dph: F2, 12 = 1.24, p = 0.3261, Fig.

369 4B).

370 At 7 and 14 dph, A. latezonatus fed the SsE diet had the highest DHA/ARA ratios,

371 followed by fish fed the PsC, PsE, and Un diets (7 dph: F3, 15 = 225.96, p = 0.0001, SsE >

372 PsC > PsE > Un; 14 dph: F2, 12 = 12.64, p = 0.0025, SsE > PsC > PsE, Fig. 4C).

373 At 7dph, A. latezonatus fed PsC and PsE diets had higher DPAn-6/DHA ratios than

374 those fed the SsE diet, which was also higher than fish fed the Un diet (F3, 15 = 217.76, p =

375 0.0001, PsC = PsE > SsE > Un, Fig. 4D). At 14 dph, A. latezonatus fed the PsC diet had a

376 higher DPAn-6/DHA ratio than those fed the SsE diet (F2, 12 = 4.11, p = 0.044, PsC = PsE >

377 PsE = SsE, Fig. 4D). The DPAn-6/DHA ratio of 14 dph A. latezonatus fed the PsE diet was

378 not different to any other treatment.

379 At 7 dph, A. latezonatus fed PsC, PsE, and Un diets had significantly higher (DPAn-6

380 + ARA)/(DHA + EPA) ratios than fish fed the SsE diet (F3, 15 = 35.92, p = 0.0001, PsC = PsE

381 = Un > SsE, Fig. 4E). At 14 dph A. latezonatus fed PsC and PsE diets had significantly

382 higher (DPAn-6 + ARA)/(DHA + EPA) ratios than fish fed the SsE diet (F2, 12 = 17.63, p =

383 0.0001, PsC = PsE > SsE, Fig. 4E)

384 At 7 dph, A. latezonatus larvae fed the SsE diet had a higher n-3 LC-PUFA/n-6 LC-

385 PUFA ratio than fish fed the Un diet., formed a heircarchy of overlapping signficance with

386 fish fed PsC and PsE diets, respectively (F3, 15 = 10.37, p = 0.001, SsE = PsC > PsC = PsE >

387 PsE = Un, Fig. 4F). At 14 dph, there was no difference in the n-3 LC-PUFA/n-6 LC-PUFA

388 ratio of A. latezonatus among any diets (F2, 12 = 2.62, p = 0.114, Fig. 4F).

389 3.5.3. Lipid classes

390 Diets had little influence on the proportion of lipid classes in 7 or 14 dph A.

391 latezonatus larvae (Table 5), except for 1,2 diacylglycerol (DAG). At 7 dph, larval A. 392 latezonatus fed the SsE and Un diets had higher levels of DAG than larvae fed the PsC and

393 PsE diets (F3, 15 = 3.04, p = 0.0202, SsE = Un > PsE = PsC, Table 5).

394 3.6. Relationships between growth and survival and fatty acid composition and lipid classes

395 of A. latezonatus

396 Survival, eye diameter, body depth at anus, and standard length of A. latezonatus at 7

397 dph was correlated with fatty acid composition (Table S4, Table 6). Models testing fatty acid

398 predictors accounted for 58–66% of the variation in survival, 38–51% of variation in eye

399 diameter, 41–54% of variation in body depth, and 48–52% of variation in standard length

400 (Table 6). The strongest models with the greatest AICc-weights (AICcwt) contained the same

401 fatty acid predictors, with other predictors contributing little to the models (Table 6). Survival

402 was correlated with the fatty acid predictors DPAn-6, SDA, and 20:2n-6 (Table 6). Eye

403 diameter was correlated with the fatty acid predictors DPAn-6/DHA and SDA. Body depth

404 was correlated with the fatty acid predictors (DPAn-6 + ARA)/(DHA + EPA), DPAn-6/DHA,

405 and 20:3n-3. Standard length was correlated with the fatty acid predictors SDA and (DPAn-6

406 + ARA)/(DHA + EPA) (Table 6).

407 Survival, eye diameter, and body depth at anus of A. latezonatus at 7 dph were

408 correlated with lipid classes (Table S6). Survival was correlated with 1,2 diacylglycerols (1,2

409 DAG) (R2 = 0.30, Table S6). Eye diameter was correlated with AMPL (R2 = 0.24). Marginal

410 tests indicated body depth was correlated with 1,2 DAG (R2 = 0.22) and AMPL (R2 = 0.23)

411 (Table S6), although the model combining these two predictors was weaker than models

412 where either predictor was used on its own (AICc: AMPL 160.10; 1,2 DAG 160.46;

413 combined 161.32, R2 = 0.29). Standard length of A. latezonatus at 7 dph was not correlated

414 with lipid classes (Table S6). 415 Survival of A. latezonatus to 14 dph was correlated with fatty acid composition (Table

416 S5), but not lipid classes (Table S7). Only one fatty acid predictor was significant in marginal

417 tests; 20:0, which accounted for 27% of the variation in survival to 14 dph (Table S5).

418 4. Discussion

419 A. latezonatus larvae fed live feeds enriched with, or cultured on, the microalgae P.

420 sulcata grew and survived better than larvae fed live feeds enriched with the commercial lipid

421 emulsion Selco S.presso. This may be explained by the higher proportions of phospholipids

422 in live feeds enhanced with P. sulcata. Survival and growth of A. latezonatus was positively

423 correlated with DPAn-6 and DPAn-6/DHA in body tissues, and was highest in fish fed P.

424 sulcata enhanced live feeds. A. latezonatus fed live feeds enriched with a commercial lipid

425 emulsion contained the highest levels of DHA, but this did not correlate with survival or

426 growth. These results highlight the potential of using microalgae as an alternative live feed

427 enhancement to commercial enrichments.

428 A. latezonatus larvae fed live feeds either cultured on or enriched with P. sulcata had

429 better survival and growth at 7 dph compared to larvae fed rotifers enriched with the

430 commercial lipid emulsion Selco S.presso or unenriched controls. Enhancing live feeds with

431 commercial enrichments can improve larval fish rearing, but some species of fish perform

432 poorly when fed these diets (Dhert et al., 2001; Gapasin and Duray, 2001; Payne et al., 2001;

433 Watanabe et al., 2016; Zeng et al., 2018). Ma and Qin (2014) found enriching live feeds with

434 a mixture of Nannochloropsis and Isochrysis was better for rearing larval Seriola lalandi than

435 some commercial products, although enriching with Selco S.presso was just as effective as

436 these algae. Selecting appropriate microalgae to feed to live feeds is important as nutritional

437 profiles vary among species (Jónasdóttir, 2019). Enhancing live feeds with microalgae such

438 as Nannochloropsis and Pavlova can result in poor larval fish performance compared to

439 commercial enrichments (Ferreira et al., 2009; Rehberg-Haas et al., 2015a). Our results 440 demonstrate the potential of the cryptomonad alga P. sulcata as an alternative to commercial

441 lipid emulsions for enhancing the nutritional quality of live feeds. This may be advantageous

442 in developing nations where small-scale ornamental fish aquaculture is rapidly expanding and

443 commercial lipid emulsions may be prohibitively expensive (Rhyne et al., 2012; Gisondo,

444 2014). Future studies should examine the efficacy of microalgae other than P. sulcata as live

445 feed enhancements to expand the range of species available for commercial use, and identify

446 strains that are best suited to local conditions with respect to ease of production.

447 At 14 dph the benefit of rearing rotifers through several generations on P. sulcata was

448 evident; A. latezonatus larvae initially fed rotifers cultured on P. sulcata had better survival

449 than larvae fed rotifers enriched for 12 hours on P. sulcata, and had the highest survival

450 overall. We found that more than 92% of lipids within P. sulcata were polar lipids.

451 Consequently, rotifers cultured on P. sulcata contained higher proportions of phospholipids

452 compared to any other live feed. These phospholipids were predominantly in the form of

453 phosphatidylethanolamine and phosphatidylcholine, which are major components of

454 biological membranes and crucial for the development of the nervous system in fish (Sargent

455 et al., 1999; Hamre et al., 2013). Lipids in Selco S.presso enriched live feeds were mainly

456 present as less biologically available neutral lipids, which is typical of live feeds enhanced

457 with commercial emulsions (Coutteau and Mourente, 1997). Marine fish larvae can convert

458 fatty acids in the form of phospholipids into biological structures more efficiently than

459 neutral lipids, and this may explain why diets high in phospholipids drive comparatively

460 better growth and survival for many species, including A. latezonatus in this study (Cahu et

461 al., 2003; Hamre et al., 2013; Li et al., 2018). Despite differences in survival among larvae

462 fed different diets at 14 dph, the size of the fish were the same across the treatments, likely

463 due to the mortality of smaller fish in all treatments (Folkvord 1991). These results

464 demonstrate that using appropriate microalgae to culture invertebrates can generate high 465 quality live feeds and eliminate the need for short term enrichments using commercial

466 products.

467 An interesting side-effect of enhancing rotifers using P. sulcata was that they became

468 larger. In particular, rotifers that were grown on P. sulcata were the longest and had the

469 greatest volume. The size of Brachionus rotifers can vary considerably when fed different

470 microalgae diets, although there is little information on the effect of cryptomonads (Korstad

471 et al., 1989; Xi et al., 2002). It is possible that consuming the larger P. sulcata enhanced

472 rotifers were more energetically efficient than rotifers in other diets, contributing to the

473 higher growth and survival of A. latezonatus fed these diets (Gill, 2003).

474 Despite differences in the relative proportions of lipid classes among the live feeds in

475 the four diets, the proportions of polar lipids and neutral lipids in A. latezonatus larvae were

476 similar across all diets. Other studies that have compared lipid classes among fish larvae

477 raised on different diets have also found the proportions of lipid classes were similar, and are

478 probably due to the need for a fixed lipid ratio in the construction of membranes (Olsen et al.,

479 2014; Folkvord et al., 2018). Neutral lipids may have been similar across diets in this study

480 because the A. latezonatus had not reached a size where body fat is accumulated.

481 Alternatively, it is possible all diets satisfied the amount of neutral lipids required by A.

482 latezonatus larvae (Rainuzzo et al., 1994b).

483 Omega-6 docosapentaenoic acid (DPAn-6) was positively correlated with survival of

484 7 dph A. latezonatus, and the proportion of DPAn-6 relative to DHA was positively

485 correlated with body length, depth, and eye diameter. Rotifers cultured on P. sulcata had the

486 highest levels of DPAn-6, and fish fed P. sulcata enhanced live feeds had the highest ratios of

487 DPAn-6 to DHA. These results are similar to Garcia et al. (2008a) who found Gadus morhua

488 larvae grew and survived better when fed diets with high DPAn-6/DHA ratios. DPAn-6

489 appears to be important in promoting growth and survival across several species of marine 490 fish and invertebrate larvae (Pernet et al., 2005; Garcia et al., 2008a; 2008b). Despite being

491 identified as an essential fatty acid for marine fish larvae, DPAn-6 remains under-studied

492 relative to other LC-PUFA (Parrish et al, 2007, Garcia et al., 2008c). The benefits of DPAn-6

493 in promoting growth and survival of larval fish might be linked to the competitive production

494 of immunoregulatory docosonoids by DPAn-6 and DHA, similar to eicosanoid production by

495 ARA and EPA (Garcia et al., 2008a). However, the exact mechanisms by which DPAn-6

496 benefits larval fish requires further investigation.

497 Higher levels of stearidonic acid (SDA) was strongly correlated with higher survival

498 and larger size in 7 dph A. latezonatus. SDA was most abundant in P. sulcata enhanced diets,

499 and only present in A. latezonatus fed rotifers enhanced with P. sulcata. SDA appears to have

500 important roles in the development and immune response of juvenile marine fish (Bell et al.,

501 2006; Villalta et al., 2008), but marine larvae are unable to elongate and further desaturate

502 SDA into more biologically important fatty acids, such as EPA (Glencross, 2009). It remains

503 unclear then as to why SDA levels were positively correlated with growth of larvae in this

504 study.

505 A. latezonatus larvae fed live feeds enriched with Selco S.presso had the highest

506 levels of DHA in their tissue. The ratios of DHA to EPA and DHA to ARA in these fish were

507 also higher than those from other diets, and were at ratios considered optimal for marine fish

508 (Sargent et al., 1997, McKinnon et al., 2003, Hamre et al., 2013). Despite this, DHA was not

509 correlated with survival and growth, and A. latezonatus had higher survival and growth in P.

510 sulcata enhanced treatments despite having half as much DHA in their bodies compared to

511 those fed Selco S.presso enriched diets. These results are unusual as many studies show a

512 positive correlation between DHA content and larval fish performance within the range of

513 DHA levels seen in this study (Watanabe, 1993; Izquierdo, 1996; Hamre et al., 2013; Pinto et

514 al, 2016). Our result may be due to the high investment of resources by female anemonefish 515 in their eggs, which hatch as large competent larvae relative to other species of fish (Olivotto

516 and Geffroy, 2017). As a result, larvae of anemonefish species such as Amphiprion ocellaris

517 can be grown without live feed enhancement, although provision of DHA and other PUFA in

518 their diet improves pigmentation and survival (Avella et al., 2007; Olivotto and Geffroy,

519 2017). It is important to note that some DHA in live feeds appears necessary for A.

520 latezonatus. Fish fed unenriched diets had little DHA in their bodies, grew poorly and almost

521 all died by 14 dph. It is possible that A. latezonatus require relatively little DHA to meet their

522 nutritional needs, after which other nutrients such as DPAn-6 become more important.

523 However, future consideration should be given to the form that these individual fatty acids

524 are delivered in (e.g. polar vs neutral) considering the likelihood that the developing digestive

525 systems of Amphiprion ocellaris have a limited capacity to adequately assimilate these lipid

526 classes to the same extent.

527 5. Conclusion

528 This study highlights the potential of P. sulcata and possibly other cryptomonad

529 microalgae as an effective live feed enrichment for larval marine fish. Culturing rotifers on P.

530 sulcata increases their phospholipid content, making them ideal live feeds for A. latezonatus

531 larvae and possibly other larval fish. Our results also highlight the need for further research

532 into n-6 LC-PUFA such as DPAn-6 to elucidate their importance in marine larval fish

533 ontogeny. It is possible that other macronutrients such as protein and vitamins are driving the

534 high growth and survival in fish fed P. sulcata enhanced live feeds (Øie et al., 1997;

535 Rønnestad et al., 2003; Hamre et al., 2008). Cryptomonads have high levels of protein

536 relative to other algae (Brown et al., 1997; Seixas et al., 2009), and commercial enrichments

537 such as Selco S.presso are mainly designed to increase lipid content. Lipids and fatty acids,

538 while important, receive a greater amount of research attention compared to other 539 macronutrients (Hamre et al., 2013). Future studies should investigate the effects of other

540 nutrients within microalgae such as P. sulcata on the larval rearing of marine fish.

541 Acknowledgements

542 This study was approved by the Southern Cross University Animal Care and Ethics

543 Committee (approval number 17/27). Financial support was provided by Mars

544 Symbioscience (grant number 51647) and the Australian Government Research Training

545 Program scholarship.

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674 The potential of tropical paracalanid copepods as live feeds in aquaculture.

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683 The use of the Mediterranean calanoid copepod Centropages typicus in Yellowtail 684 clownfish (Amphiprion clarkii) larviculture. Aquaculture 284, 211-216.

685 https://doi.org/10.1016/j.aquaculture.2008.07.057

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690 DHA content in dietary phospholipids affects DHA content in phospholipids of cod

691 larvae and larval performance. Aquaculture 428, 203-214.

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695 https://doi.org/10.4319/lo.2007.52.1.0476

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697 Australian dhufish (Glaucosoma hebraicum) and pink snapper (Pagrus auratus)

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716 8486(97)00121-x

717 Rehberg-Haas, S., Meyer, S., Tielmann, M., Lippemeier, S., Vadstein, O., Bakke, I.,

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722 different Pavlova sp. products for cultivation of Brachionus plicatilis. Aquaculture

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727 https://doi.org/10.1371/journal.pone.0035808

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752 fish larviculture. Aquaculture 200, 147-159. https://doi.org/10.1016/s0044-

753 8486(01)00698-6 754 Southgate, P.C., Lou, D.C., 1995. Improving the n− 3 HUFA composition of Artemia using

755 microcapsules containing marine oils. Aquaculture 134, 91-99.

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758 responses to supplementation with particular polyunsaturated fatty acids.

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773 larviculture of Atlantic red porgy Pagrus pagrus. Aquaculture Reports 3, 93-107.

774 https://doi.org/10.1016/j.aqrep.2016.01.003

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777 without water exchange during seed production of amberjack Seriola dumerili. Fish.

778 Sci. 75, 697-705. https://doi.org/10.1007/s12562-009-0084-2 779 Zeng, C., Shao, L., Ricketts, A., Moorhead, J., 2018. The importance of copepods as live feed

780 for larval rearing of the green mandarin fish Synchiropus splendidus. Aquaculture

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784 Fresh. Ecol. 17, 185-190. https://doi.org/10.1080/02705060.2002.9663886 785 List of tables and figures

786 Table 1. Description of the four diets (PsC, PsE, SsE, Un) fed to larval Amphiprion

787 latezonatus.

Diet Day 0–6 Day 7–14

Rotifers cultured on PsC Proteomonas sulcata Instar II Artemia for 10–17 days enriched on Rotifers enriched with Proteomonas sulcata PsE Proteomonas sulcata for 12 h for 12 h

Instar II Artemia Rotifers enriched with SsE enriched on Selco Selco S.presso for 12 h S.presso for 12 h

Unenriched instar II Un Unenriched rotifers Artemia 788

789

790

791

792

793

794 Fig. 1. The effect of four diets on survival of larval A. latezonatus to 14 days post hatch

795 (dph). A full description of the diets (PsC, PsE, SsE, Un) is provided in Table 1. Points

796 labelled with different letters are significantly different according to one-way ANOVA

797 followed by post-hoc pairwise test at 7 and 14 dph. Data are means ± S.E., n = 5. 798

799

800

801

802

803

804

805

806

807

808

809 Fig. 2. The effect of four diets on (A) eye diameter, (B) body depth at anus, and (C) standard

810 length of larval Amphiprion latezonatus at 7 and 14 days post hatch (dph). A full description

811 of the diets (PsC, PsE, SsE, Un) is provided in Table 1. Within each time point, bars labelled

812 with different letters are significantly different according to one-way ANOVA followed by

813 post-hoc pairwise test. Data are means ± S.E. n = 5 for each diet. Data was unavailable for the

814 Un diet at 14 dph due to high mortality in this treatment. 815

816

817

818

819

820

821

822

823 Fig. 3. The (A) volume and (B) lorica length of rotifers in four diets fed to larval Amphiprion

824 latezonatus. Diets were rotifers cultured on P. sulcata over several generations (PsC), rotifers

825 enriched with P. sulcata (PsE), rotifers enriched with Selco S.presso (SsE), and unenriched

826 rotifers (Un) (also see Table 1). Bars labelled with different letters are significantly different

827 according to one-way ANOVA followed by post-hoc pairwise test. Data are means ± SE, n =

828 50, each replicate represents an individual rotifer. 829 Table 2. Total fatty acid content and fatty acid composition of live feed enhancements

830 Proteomonas sulcata, Selco S.presso, and Nannochloropsis paste (Nanno 3600), and

831 differently enhanced live feeds (rotifers and Artemia) fed to Amphiprion latezonatus larvae:

832 Proteomonas sulcata cultured, P. sulcata enriched, Selco S.presso enriched, and unenriched.

833 SFA is saturated fatty acids, MUFA is monounsaturated fatty acids, PUFA is polyunsaturated

834 fatty acids, LC-PUFA is long chain polyunsaturated fatty acids. Fatty acids are in mg g lipid-

835 1, total lipid is mg g sample DW-1 and are from a single sample. Full table of all detected fatty

836 acids are listed in supplementary material (Table S1, S2).

Enhancements Rotifers Artemia Selco Nanno P. sulcata P. sulcata Selco Un- P. sulcata Selco Un- Fatty Acid P. sulcata S.presso 3600 cultured enriched enriched enriched enriched enriched enriched Σ SFA 140.64 89.56 29.27 132.17 106.00 131.36 121.01 121.27 141.20 118.78 Σ MUFA 33.25 92.11 31.88 31.78 73.57 138.03 98.10 184.79 184.74 183.80 Σ PUFA 560.82 333.77 42.58 379.87 285.24 407.22 192.79 374.89 411.76 324.37 18:3n-3 ALA 210.28 6.41 6.91 91.58 57.95 19.98 25.20 222.82 174.95 201.87 18:4n-3 SDA 11.26 0.12 0.04 51.06 27.32 2.99 1.53 50.36 31.18 38.37 20:4n-3 112.60 1.24 0.37 90.85 28.71 7.54 9.79 8.04 5.29 5.59 20:5n-3 EPA 110.95 22.05 20.06 63.56 57.35 46.70 51.70 23.87 29.76 13.32 22:6n-3 DHA 37.37 225.27 0 21.35 12.43 141.61 0 2.76 71.10 1.76 Σ n-3 PUFA 487.76 264.10 27.34 347.67 205.61 262.37 107.87 317.27 330.74 269.89 Σ n-3 LC-PUFA 164.89 256.46 20.06 205.03 120.33 239.40 81.14 44.09 124.61 29.65 18:2n-6 LNA 35.38 43.43 9.29 9.74 39.58 113.89 51.10 37.69 53.92 36.36 20:4n-6 ARA 1.77 2.97 4.04 4.53 8.88 6.76 12.31 4.10 3.55 3.90 22:5n-6 DPAn-6 19.35 18.92 0.15 10.72 7.18 9.55 2.08 1.12 8.10 0 Σ n-6 PUFA 64.12 67.78 14.95 24.99 64.26 140.24 78.87 49.29 72.49 45.75 Σ n-6 LC-PUFA 21.12 24.06 5.36 15.24 24.10 24.42 25.05 7.27 15.31 5.42 Σ Fatty Acids 734.71 515.44 103.73 543.82 464.81 676.61 411.90 680.95 737.70 626.95 Total Lipid 213.03 852.90 465.47 64.25 94.03 165.64 110.77 151.63 191.34 159.52 837 838 Table 3. Lipid classes of live feed enhancements Proteomonas sulcata, Selco S.presso, and

839 Nannochloropsis paste (Nanno 3600) and differently enhanced live feeds (rotifers and

840 Artemia) fed to Amphiprion latezonatus larvae. Polar lipids are sterols, acetone mobile polar

841 lipids (AMPL), phosphatidylethanolamines (PE), phosphatidylserine/inositols (PS/PI), and

842 phosphatidylcholines (PC). Neutral lipids are sterol esters, triacylglycerols (TAG), free fatty

843 acids (FFA), and 1,2 diacylglycerols (1,2 DAG). Values are mg g lipid-1 and are from a single

844 sample.

Enhancements Rotifers Artemia Selco Nanno P. sulcata P. sulcata Selco Un- P. sulcata Selco Un- Lipid Class P. sulcata S.presso 3600 cultured enriched enriched enriched enriched enriched enriched Sterol ester 28.86 227.13 11.07 27.94 65.40 17.60 84.93 262.97 299.35 264.15 TAG 23.15 424.65 107.24 196.37 235.66 660.28 307.15 352.94 412.89 315.02 FFA 14.61 76.47 91.47 9.30 54.50 26.52 10.31 4.22 10.65 3.65 1,2 DAG 0 112.97 0 0 0 0 0 0 0 0 Sterols 28.71 39.42 68.15 35.92 51.63 18.46 40.05 24.10 21.28 31.66 AMPL 565.96 0 378.88 167.59 212.69 90.87 134.19 98.83 80.91 119.04 PE 175.35 0 238.40 254.18 175.57 72.66 218.56 99.31 57.80 101.40 PS/PI 88.95 0 0 0 0 0 0 0 0 0 PC 65.23 119.35 66.84 240.62 192.36 104.42 195.27 146.91 103.53 158.13 Lyso-PC 9.16 0 37.95 68.07 12.19 9.19 9.53 10.73 13.58 6.95 Σ Polar Lipid 924.2 158.8 752.3 698.3 632.3 286.4 588.1 369.1 263.5 410.2 Σ Neutral Lipid 66.6 841.2 209.8 233.6 355.6 704.4 402.4 620.1 722.9 582.8 845 846 Table 4. The effect of four diets on the fatty acid composition of 7 and 14 days post hatch

847 (dph) Amphiprion latezonatus. A full description of the diets (PsC, PsE, SsE, Un) is provided

848 in Table 1. SFA is saturated fatty acids, MUFA is monounsaturated fatty acids, PUFA is

849 polyunsaturated fatty acids, LC-PUFA is long chain polyunsaturated fatty acids. Data are

850 means ± S.E. n = 5 for each diet except PsC at 7 dph where n = 4. Values with different

851 superscript are significantly different according to one-way ANOVA followed by post-hoc

852 pairwise test. Fatty acids are in mg g lipid-1, total lipid is mg g sample DW-1. Full table of all

853 detected fatty acids are listed in supplementary material (Table S3).

7 dph Amphiprion latezonatus 14 dph Amphiprion latezonatus Fatty Acid PsC Diet PsE Diet SsE Diet Un Diet PsC Diet PsE Diet SsE Diet 17:0 2.7 ± 0.4 2.5 ± 0.1 2.3 ± 0.1 2.8 ± 0.2 2.0 ± 0.1A 2.5 ± 0.1B 2.1 ± 0.2AB Σ SFA 65.5 ± 10.1 64.4 ± 4.0 63.8 ± 4.4 64.9 ± 5.3 55.1 ± 4.3 68.3 ± 4.6 58.5 ± 3.9 15:1n-5 0.8 ± 0.2 0.7 ± 0.0 0.6 ± 0.1 0.6 ± 0.1 0.5 ± 0.1AB 0.6 ± 0.1A 0.4 ± 0.0B Σ MUFA 29.3 ± 3.6 32.6 ± 4.4 35.3 ± 4.7 41.9 ± 7.3 51.5 ± 6.3 59.7 ± 8.6 46.7 ± 6.8 18:2n-4 0a 0.3 ± 0.0b 0.2 ± 0.1b 0.4 ± 0.1b 0.2 ± 0.0 0.4 ± 0.1 0.3 ± 0.0 Σ PUFA 66.2 ± 12.7 66.3 ± 3.8 72.0 ± 5.5 60.5 ± 4.7 74.3 ± 6.3 90.5 ± 12.2 75.5 ± 7.7 18:3n-3 ALA 7.9 ± 1.4 7.7 ± 0.7 7.2 ± 1.0 6.6 ± 0.7 26.7 ± 2.8 35.7 ± 4.9 24.0 ± 2.2 18:4n-3 SDA 1.1 ± 0.2a 1.4 ± 0.1a 0b 0b 4.2 ± 0.5 5.4 ± 1.1 3.1 ± 0.4 20:3n-3 1.0 ± 0.2a 0.8 ± 0.0a 0.3 ± 0.1b 0.4 ± 0.1b 1.8 ± 0.2 2.1 ± 0.4 1.2 ± 0.1 20:4n-3 4.9 ± 1.1a 3.0 ± 0.2b 0.7 ± 0.0c 0.9 ± 0.1c 2.2 ± 0.2A 2.5 ± 0.4A 1.3 ± 0.1B 20:5n-3 EPA 12.6 ± 2.4 14.0 ± 1.5 9.3 ± 0.6 13.2 ± 1.1 10.6 ± 0.8 12.5 ± 1.2 10.3 ± 0.8 21:5n-3 0.8 ± 0.2a 0.4 ± 0.1ab 0.3 ± 0.1b 0.3 ± 0.1b 0.5 ± 0.3 0.5 ± 0.3 0.4 ± 0.1 22:6n-3 DHA 15.0 ± 3.1a 13.3 ± 0.7a 24.8 ± 2.5b 11.6 ± 1.2a 6.8 ± 0.6A 6.3 ± 1.1A 12.3 ± 2.4B Σ n-3 PUFA 49.2 ± 9.8 46.0 ± 2.6 47.9 ± 3.4 38.4 ± 2.7 55.0 ± 4.9 67.2 ± 9.6 54.9 ± 5.4 Σ n-3 LC-PUFA 40.2 ± 8.2 36.9 ± 2.3 40.7 ± 3.8 31.8 ± 3.0 24.1 ± 1.9 26.1 ± 3.5 27.8 ± 3.2 18:2n-6 LNA 5.0 ± 0.8a 7.6 ± 0.8ab 11.6 ± 1.3c 9.3 ± 1.2bc 9.3 ± 1.0 10.9 ± 1.7 10.1 ± 1.5 20:4n-6 ARA 4.4 ± 0.9 4.8 ± 0.4 3.9 ± 0.5 5.2 ± 0.7 3.3 ± 0.3 3.9 ± 0.6 2.7 ± 0.2 22:5n-6 DPAn-6 1.8 ± 0.4a 1.5 ± 0.1a 1.3 ± 0.1a 0.4 ± 0.0b 0.9 ± 0.1 0.9 ± 0.2 0.6 ± 0.1 Σ n-6 PUFA 15.8 ± 2.7 18.7 ± 1.2 22.8 ± 2.0 20.4 ± 2.0 16.9 ± 1.3 19.9 ± 2.6 18.0 ± 2.1 Σ n-6 LC-PUFA 8.4 ± 1.7 8.9 ± 0.6 8.0 ± 1.0 8.4 ± 1.3 5.9 ± 0.5 6.6 ± 1.3 5.0 ± 0.9 Σ Fatty Acids 160.9 ± 25.8 163.4 ± 11.8 171.0 ± 14.1 167.3 ± 13.6 180.9 ± 15.8 218.5 ± 24.8 180.6 ± 17.2 Total Lipid 431.5 ± 56.6 514.1 ± 23.6 528.8 ± 30.9 558.6 ± 49.1 521.7 ± 39.9 463.6 ± 52.4 560.4 ± 12.0 854 855

856

857

858

859

860

861

862

863

864

865

866

867

868 Fig. 4. The effects of four diets on the ratio of key fatty acids in Amphiprion latezonatus

869 larvae at 7 and 14 days post hatch (dph). (A) docosahexanoic acid (DHA) to eicosapentanoic

870 acid (EPA), (B) EPA to arachidonic acid (ARA), (C) DHA to ARA, (D) omega-6

871 docosapentaenoic acid (DPAn-6) to DHA, (E) DPAn-6 plus ARA to DHA plus EPA, and (F)

872 omega-3 long chain-polyunsaturated fatty acids (n-3 LC-PUFA) to n-6 LC-PUFA. A full

873 description of the diets (PsC, PsE, SsE, Un) is provided in Table 1. Within each time point,

874 bars labelled with different letters are significantly different according to one-way ANOVA

875 followed by post-hoc pairwise test. Data are means ± S.E. n = 5 for each diet, except PsC at 7

876 dph where n = 4. 877 Table 5. Lipid classes of 7 and 14 days post hatch (dph) Amphiprion latezonatus larvae fed

878 four diets (PsC, PsE, SsE, Un; see Table 1 for full description). Polar lipids are sterols,

879 acetone mobile polar lipids (AMPL), phosphatidylethanolamines (PE),

880 phosphatidylserine/inositol (PS/PI), and phosphatidylcholines (PC). Neutral lipids are sterol

881 esters, triacylglycerols (TAG), free fatty acids (FFA), and 1,2 diacylglycerols (1,2 DAG).

882 Data are means ± S.E. n = 5 for each diet except PsC at 7 dph where n = 4. Values with

883 different superscript are significantly different according to one-way PERMANOVA

884 followed by post-hoc pairwise test. Individual lipid class values are mg g lipid-1.

7 dph Amphiprion latezonatus 14 dph Amphiprion latezonatus Lipid Class PsC Diet PsE Diet SsE Diet Un Diet PsC Diet PsE Diet SsE Diet Sterol ester 159.9 ± 60.9 118.9 ± 5.7 116.1 ± 8.4 128.6 ± 9.7 133.5 ± 21.0 93.0 ± 17.7 203.8 ± 66.5 TAG 41.2 ± 8.3 31.2 ± 6.7 31.7 ± 3.2 58.1 ± 24.6 202.2 ± 39.5 172.8 ± 33.6 108.4 ± 42.9 FFA 28.0 ± 2.3 33.0 ± 2.6 38.9 ± 2.8 36.0 ± 4.7 23.8 ± 2.8 24.6 ± 2.7 31.4 ± 6.6 1,2 DAG 38.2 ± 13.6a 52.6 ± 1.9a 65.3 ± 04.3b 60.5 ± 4.3b 30.8 ± 4.4 39.4 ± 6.1 41.3 ± 13.5 Sterols 96.4 ± 8.2 107.2 ± 4.5 104.6 ± 4.5 78.6 ± 19.9 91.9 ± 7.4 93.4 ± 7.7 82.9 ± 8.5 AMPL 216.6 ± 10.9 231.4 ± 16.2 260.8 ± 19.6 255.0 ± 22.4 201.0 ± 20.4 171.3 ± 6.4 229.1 ± 30.2 PE 130.4 ± 15.1 135.7 ± 7.9 120.4 ± 9.6 118.5 ± 10.7 103.4 ± 15.1 120.2 ± 3.3 99.3 ± 6.9 PS/PI 0 0 0 0 0 28.1 ± 28.1 0 PC 228.3 ± 22.2 244.3 ± 22.1 222.6 ± 22.4 224.8 ± 21.3 189.7 ± 16.6 214.1 ± 20.6 182.0 ± 14.4 Lyso-PC 61.0 ± 22.6 45.7 ± 11.5 39.4 ± 17.1 40.0 ± 17.3 23.8 ± 7.1 43.1 ± 8.5 21.7 ± 9.7 Σ Polar Lipid 671.7 ± 48.9 718.6 ± 19.3 708.6 ± 18.7 676.9 ± 29.2 585.9 ± 45.9 627.0 ± 14.5 593.3 ± 51.9 Σ Neutral Lipid 267.4 ± 70.3 235.7 ± 10.9 252.1 ± 11.7 283.2 ± 26.7 390.3 ± 50.1 329.9 ± 19.3 385.0 ± 43.0 885 886 Table 6. Model selection to estimate the relationship between fatty acid composition and

887 survival, eye diameter, body depth at anus, and standard length of Amphiprion latezonatus at

888 7 dph (days post hatch) when fed four diets (full description of the diets is provided in Table

889 1). In each case, ‘predictors’ refers to the combination of explanatory variables included in

890 the model. ΔAICc is the difference in AICc between the model and the best fitting model.

891 AICc-weight (AICcwt) is the probability that this model represents the best-fitting model

892 among those considered. ‘Sum of AICcwt’ refers to the sum of AICcwt scores for the given

893 model and all better-fitting models. For each dataset, the five best models are presented.

894 ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; DPAn-6,

895 docosapentaenoic acid, omega-6; PUFA, polyunsaturated fatty acids.

2 Dataset Predictors AICc ΔAICc AICcwt Sum of AICcwt R Survival 7 dph DPAn-6, 20:2n-6 103.49 0 0.31 0.31 0.62 18:4n-3, 20:2n-6 103.75 0.26 0.28 0.59 0.61 DPAn-6, 18:4n-3, 20:2n-6 104.83 1.34 0.16 0.75 0.66 DPAn-6, n-6 PUFA 105.25 1.76 0.13 0.88 0.58 20:2n-6, n-3 PUFA/n-6 PUFA 105.42 1.93 0.12 1.00 0.58 Eye diameter DPAn-6/DHA 102.81 0 0.34 0.34 0.44 DPAn-6/DHA, 22:1 (isomers) 103.24 0.43 0.28 0.62 0.51 18:4n-3, 22:1 (isomers) 104.32 1.51 0.16 0.78 0.48 18:4n-3 104.94 2.13 0.12 0.90 0.38 18:4n-3; DPAn-6/DHA 105.26 2.45 0.10 1.00 0.45 Body depth DPAn-6/DHA, (DPAn-6+ARA)/(DHA+EPA) 153.16 0 0.35 0.35 0.54 DPAn-6/DHA, DHA/ARA 153.84 0.68 0.25 0.59 0.53 20:3n-3, (DPAn-6+ARA)/(DHA+EPA) 154.88 1.72 0.15 0.74 0.50 20:3n-3 155.04 1.88 0.14 0.87 0.41 18:4n-3, (DPAn-6+ARA)/(DHA+EPA) 155.19 2.03 0.13 1.00 0.49 Standard length 18:4n-3 164.66 0 0.42 0.42 0.48 DPAn-6/DHA, (DPAn-6+ARA)/(DHA+EPA) 166.13 1.47 0.20 0.62 0.52 18:4n-3, 20:3n-3 166.97 2.31 0.13 0.75 0.50 18:4n-3, (DPAn-6+ARA)/(DHA+EPA) 167.07 2.41 0.13 0.88 0.50 18:4n-3, 20:4n-3 167.15 2.49 0.12 1.00 0.49 896 897 Supplementary Material

898 Table S1. Total fatty acid content and fatty acid composition of live feed enhancements

899 Proteomonas sulcata, Selco S.presso, and Nannochloropsis paste (Nanno 3600). SFA is

900 saturated fatty acids, MUFA is monounsaturated fatty acids, PUFA is polyunsaturated fatty

901 acids, LC-PUFA is long chain polyunsaturated fatty acids. Fatty acids are mg g lipid-1, total

902 lipid is mg g sample-1 (dry weight) and are from a single sample.

Fatty Acid Proteomonas sulcata Selco S.presso Nanno 3600 12:0 11.76 0.33 0.20 13:0 6.28 0.12 1.62 14:0 35.53 10.71 2.70 14:1n-5 5.96 0.26 0.97 15:0 1.20 1.08 0.25 15:1n-5 3.78 0.00 0.94 16:0 80.61 68.22 23.51 16:1n-7 19.84 4.56 19.85 16:2n-4 3.68 0.16 0 16:3n-4 3.88 1.10 0.29 16:4n-1 1.39 0.18 0 17:0 2.24 1.02 0.19 17:1n-7 0 0.38 3.12 18:0 3.02 7.19 0.79 18:1n-9 3.67 53.49 4.92 18:1n-9 (Trans) 0 1.23 0.16 18:1n-7 0 3.79 0.81 18:2n-6 35.38 43.43 9.29 18:2n-4 0.00 0.19 0 18:3n-6 7.61 0.29 0.30 18:3n-4 0 0.26 0 18:3n-3 210.28 6.41 6.91 18:4n-3 112.60 1.24 0.37 20:0 0 0.65 0 20:1 (isomers) 0 2.25 0.84 20:2n-6 0 0.34 0.17 20:3n-6 0 0.50 0 20:3n-3 0 0.23 0 20:4n-6 1.77 2.97 4.04 20:4n-3 16.57 1.75 0 20:5n-3 110.95 22.05 20.06 21:0 0 0.24 0 21:5n-3 0 6.20 0 22:1 (isomers) 0 0.65 0 22:2n-6 0 0.19 0.82 22:4n-6 0 1.14 0.18 22:5n-6 19.35 18.92 0.15 22:5n-3 0 0.96 0 22:6n-3 37.37 225.27 0 24:1n-9 0 25.50 0.28 Σ SFA 140.64 89.56 29.27 Σ MUFA 33.25 92.11 31.88 Σ PUFA 560.82 333.77 42.58 Σ n-3 PUFA 487.76 264.10 27.34 Σ n-3 LC PUFA 164.89 256.46 20.06 Σ n-6 PUFA 64.12 67.78 14.95 Σ n-6 LC PUFA 21.12 24.06 5.36 Σ Fatty Acids 734.71 515.44 103.73 Total Lipid 213.03 852.90 465.47 903 904 Table S2. Total fatty acid content and fatty acid composition of differently enhanced live

905 rotifers and Artemia; Proteomonas sulcata cultured, P. sulcata enriched, Selco S.presso

906 enriched, and unenriched (Nannochloropsis paste, Nanno 3600). SFA is saturated fatty acids,

907 MUFA is monounsaturated fatty acids, PUFA is polyunsaturated fatty acids, LC-PUFA is

908 long chain polyunsaturated fatty acids. Fatty acids are mg g lipid-1, total lipid is mg g sample-

909 1 (dry weight) and are from a single sample.

Rotifer Artemia P. sulcata P. sulcata Selco Un- P. sulcata Selco Un- Fatty Acid cultured enriched enriched enriched enriched enriched enriched 12:0 0 1.22 0.70 1.00 0 0 0 13:0 0 0.79 0 1.09 0 0 0 14:0 19.77 13.89 13.93 13.67 4.70 9.99 4.36 15:0 0 1.86 1.33 1.60 0.87 1.07 0.88 15:1n-5 0 0.49 0 0 0 0 0 16:0 87.21 73.77 99.22 80.91 76.73 94.18 74.38 16:1n-7 5.37 27.57 23.73 47.75 14.33 12.27 13.23 16:2n-4 0 8.01 0 0 1.01 0 0 16:3n-4 7.21 1.06 1.73 1.15 3.86 3.51 3.96 16:4n-1 0 0.48 0 0 1.29 1.13 0 17:0 4.65 0.63 4.83 10.11 3.34 3.99 3.18 17:1n-7 0.00 1.07 0.94 1.66 4.77 3.72 4.05 18:0 20.53 12.83 11.35 12.63 34.61 30.91 34.93 18:1n-9 4.47 8.69 83.40 13.31 125.20 133.77 124.86 18:1n-9 (Trans) 3.79 3.18 4.17 5.73 0.90 0 0 18:1n-7 8.43 18.25 10.40 14.59 35.25 31.11 37.13 18:2n-6 9.74 39.58 113.89 51.10 37.69 53.92 36.36 18:2n-4 0 4.23 2.88 4.90 0.48 2.54 2.95 18:3n-6 0 0.59 1.94 2.71 4.34 3.26 3.97 18:3n-4 0 1.59 0 0 1.68 1.36 1.81 18:3n-3 91.58 57.95 19.98 25.20 222.82 174.95 201.87 18:4n-3 51.06 27.32 2.99 1.53 50.36 31.18 38.37 20:0 0 1.00 0 0 1.02 1.05 1.05 20:1 (isomers) 9.73 12.53 13.32 13.10 4.34 3.87 4.54 20:2n-6 0 3.61 4.79 5.24 1.47 1.34 1.52 20:3n-6 0 3.58 2.63 4.16 0.58 0 0 20:3n-3 9.86 5.17 1.94 3.57 7.91 5.99 7.27 20:4n-6 4.53 8.88 6.76 12.31 4.10 3.55 3.90 20:4n-3 90.85 28.71 7.54 9.79 8.04 5.29 5.59 20:5n-3 63.56 57.35 46.70 51.70 23.87 29.76 13.32 21:5n-3 0 1.32 7.73 0 1.50 2.89 1.71 22:1 (isomers) 0 1.79 2.06 1.96 0 0 0 22:4n-6 0 0.86 0.70 1.27 0 2.31 0 22:5n-6 10.72 7.18 9.55 2.08 1.12 8.10 0 22:5n-3 19.41 15.35 33.87 16.09 0 9.57 0 22:6n-3 21.35 12.43 141.61 0 2.76 71.10 1.76 Σ SFA 132.17 106.00 131.36 121.01 121.27 141.20 118.78 Σ MUFA 31.78 73.57 138.03 98.10 184.79 184.74 183.80 Σ PUFA 379.87 285.24 407.22 192.79 374.89 411.76 324.37 Σ n-3 PUFA 347.67 205.61 262.37 107.87 317.27 330.74 269.89 Σ n-3 LC PUFA 205.03 120.33 239.40 81.14 44.09 124.61 29.65 Σ n-6 PUFA 24.99 64.26 140.24 78.87 49.29 72.49 45.75 Σ n-6 LC PUFA 15.24 24.10 24.42 25.05 7.27 15.31 5.42 Σ Fatty Acids 543.82 464.81 676.61 411.90 680.95 737.70 626.95 Total Lipid 64.25 94.03 165.64 110.77 151.63 191.34 159.52 910

911 912 Table S3. Fatty acid composition of 7 and 14 days post hatch (dph) Amphiprion latezonatus

913 larvae fed four diets (PsC, PsE, SsE, Un; see Table 1 for a full description). SFA is saturated

914 fatty acids, MUFA is monounsaturated fatty acids, PUFA is polyunsaturated fatty acids, LC-

915 PUFA is long chain polyunsaturated fatty acids. Data are means ± S.E. n = 5 for each diet

916 except PsC at 7 dph where n = 4. Values with different superscript are significantly different

917 according to one-way ANOVA followed by post-hoc pairwise test. Fatty acids are mg g lipid-

918 1, total lipid is mg g sample-1 (dry weight).

7 dph Amphiprion latezonatus 14 dph Amphiprion latezonatus Fatty Acid PsC Diet PsE Diet SsE Diet Un Diet PsC Diet PsE Diet SsE Diet 12:0 0.5 ± 0.1 0.5 ± 0.0 0.5 ± 0.0 0.5 ± 0.1 0.2 ± 0.0 0.4 ± 0.1 0.4 ± 0.1 13:0 0 0.3 ± 0.3 0 0.1 ± 0.1 0 0.1 ± 0.1 0 14:0 2.3 ± 0.4 2.5 ± 0.2 2.7 ± 0.3 2.2 ± 0.2 1.5 ± 0.1 2.0 ± 0.1 1.9 ± 0.3 14:1n-5 0.2 ± 0.1 0.4 ± 0.0 0.3 ± 0.1 0.1 ± 0.1 0.2 ± 0.0 0.2 ± 0.0 0.2 ± 0.1 15:0 0.9 ± 0.2 0.8 ± 0.1 0.9 ± 0.1 0.8 ± 0.1 0.5 ± 0.0 0.6 ± 0.0 0.6 ± 0.0 15:1n-5 0.8 ± 0.2 0.7 ± 0.0 0.6 ± 0.1 0.6 ± 0.1 0.5 ± 0.1AB 0.6 ± 0.1A 0.4 ± 0.0B 16:0 35.1 ± 5.5 35.5 ± 2.5 36.1 ± 2.8 36.1 ± 3.2 29.1 ± 2.5 35.6 ± 2.8 31.1 ± 2.4 16:1n-7 5.0 ± 0.9 7.5 ± 1.5 4.9 ± 0.7 7.8 ± 1.1 3.9 ± 0.5 4.3 ± 1.0 3.5 ± 0.6 16:2n-4 0 0 0 0.2 ± 0.2 0.1 ± 0.0 0.2 ± 0.0 0.1 ± 0.1 16:3n-4 1.0 ± 0.2 0.9 ± 0.1 0.9 ± 0.1 0.8 ± 0.1 1.2 ± 0.1 1.5 ± 0.1 1.2 ± 0.1 16:4n-1 0 0.2 ± 0.1 0 0.1 ± 0.1 0.5 ± 0.0 0.7 ± 0.1 0.5 ± 0.2 17:0 2.7 ± 0.4 2.5 ± 0.1 2.3 ± 0.1 2.8 ± 0.2 2.0 ± 0.1A 2.5 ± 0.1B 2.1 ± 0.2AB 17:1n-7 0 0.22 ± 0.2 0 0 0.5 ± 0.1 0.6 ± 0.2 0.4 ± 0.1 18:0 21.8 ± 3.3 20.5 ± 0.9 19.1 ± 1.3 20.3 ± 1.6 20.2 ± 1.6 25.0 ± 2.1 20.1 ± 1.0 18:1n-9 12.3 ± 1.9 11.9 ± 2.0 14.3 ± 1.3 20.2 ± 6.8 33.2 ± 4.5 37.8 ± 5.4 29.5 ± 4.7 18:1n-9 (Trans) 1.4 ± 0.2 1.6 ± 0.2 1.9 ± 0.2 1.6 ± 0.3 0.4 ± 0.0 0.4 ± 0.1 0.3 ± 0.1 18:1n-7 4.4 ± 0.8 5.2 ± 0.4 3.8 ± 0.4 5.5 ± 0.7 9.2 ± 0.9 11.5 ± 1.8 7.9 ± 0.9 18:2n-6 5.0 ± 0.8a 7.6 ± 0.8ab 11.6 ± 1.3c 9.3 ± 1.2bc 9.3 ± 1.0 10.9 ± 1.7 10.1 ± 1.5 18:2n-4 0a 0.3 ± 0.0b 0.2 ± 0.1b 0.4 ± 0.1b 0.2 ± 0.0 0.4 ± 0.1 0.3 ± 0.0 18:3n-6 2.4 ± 0.4 2.2 ± 0.4 3.2 ± 0.5 2.6 ± 0.4 1.8 ± 0.2 2.5 ± 0.5 2.9 ± 0.5 18:3n-4 0.2 ± 0.1 0.2 ±0.1 0.1 ± 0.1 0.2 ± 0.1 0.5 ± 0.0 0.6 ± 0.1 0.4 ± 0.0 18:3n-3 7.9 ± 1.4 7.7 ± 0.7 7.2 ± 1.0 6.6 ± 0.7 26.7 ± 2.8 35.7 ± 4.9 24.0 ± 2.2 18:4n-3 1.1 ± 0.2a 1.4 ± 0.1a 0b 0b 4.2 ± 0.5 5.4 ± 1.1 3.1 ± 0.4 20:0 1.5 ± 0.2 1.3 ± 0.1 1.5 ± 0.1 1.6 ± 0.1 1.2 ± 0.1 1.5 ± 0.1 1.5 ± 0.1 20:1 (isomers) 1.3 ± 0.2 1.8 ± 0.3 2.2 ± 0.3 2.3 ± 0.4 2.2 ± 0.4 2.4 ± 0.3 1.9 ± 0.2 20:2n-6 0.8 ± 0.2 1.1 ± 0.1 1.5 ± 0.2 1.3 ± 0.2 0.7 ± 0.0 0.8 ± 0.2 0.7 ± 0.1 20:3n-6 1.0 ± 0.2 1.0 ± 0.1 0.9 ± 0.1 1.0 ± 0.3 0.3 ± 0.0 0.4 ± 0.1 0.4 ± 0.1 20:3n-3 1.0 ± 0.2a 0.8 ± 0.0a 0.3 ± 0.1b 0.4 ± 0.1b 1.8 ± 0.2 2.1 ± 0.4 1.2 ± 0.1 20:4n-6 4.4 ± 0.9 4.8 ± 0.4 3.9 ± 0.5 5.2 ± 0.7 3.3 ± 0.3 3.9 ± 0.6 2.7 ± 0.2 20:4n-3 4.9 ± 1.1a 3.0 ± 0.2b 0.7 ± 0.0c 0.9 ± 0.1c 2.2 ± 0.2A 2.5 ± 0.4A 1.3 ± 0.1B 20:5n-3 12.6 ± 2.4 14.0 ± 1.5 9.3 ± 0.6 13.2 ± 1.1 10.6 ± 0.8 12.5 ± 1.2 10.3 ± 0.8 21:0 0.8 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 0.8 ± 0.0 0.6 ± 0.0 0.8 ± 0.1 0.8 ± 0.1 21:5n-3 0.8 ± 0.2a 0.4 ± 0.1ab 0.3 ± 0.1b 0.3 ± 0.1b 0.5 ± 0.3 0.5 ± 0.3 0.4 ± 0.1 22:1 (isomers) 0.1 ± 0.1 0.1 ± 0.1 0.1 ± 0.1 0.3 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 0.8 ± 0.5 22:2n-6 0 0 0 0 0.4 ± 0.1 0.5 ± 0.1 0.2 ± 0.2 22:4n-6 0.5 ± 0.1 0.5 ± 0.0 0.4 ± 0.0 0.6 ± 0.1 0.2 ± 0.0 0.2 ± 0.1 0.5 ± 0.4 22:5n-6 1.8 ± 0.4a 1.5 ± 0.1a 1.3 ± 0.1a 0.4 ± 0.0b 0.9 ± 0.1 0.9 ± 0.2 0.6 ± 0.1 22:5n-3 5.9 ± 1.3 5.5 ± 0.4 5.2 ± 0.8 5.5 ± 0.6 2.2 ± 0.2 2.2 ± 0.4 2.3 ± 0.3 22:6n-3 15.0 ± 3.1a 13.3 ± 0.7a 24.8 ± 2.5b 11.6 ± 1.2a 6.8 ± 0.6A 6.3 ± 1.1A 12.3 ± 2.4B 24:1n-9 3.7 ± 0.6 3.3 ± 0.8 7.2 ± 2.5 3.4 ± 0.9 1.4 ± 0.2 1.7 ± 0.2 2.0 ± 0.3 Σ SFA 65.5 ± 10.1 64.4 ± 4.0 63.8 ± 4.4 64.9 ± 5.3 55.1 ± 4.3 68.3 ± 4.6 58.5 ± 3.9 Σ MUFA 29.3 ± 3.6 32.6 ± 4.4 35.3 ± 4.7 41.9 ± 7.3 51.5 ± 6.3 59.7 ± 8.6 46.7 ± 6.8 Σ PUFA 66.2 ± 12.7 66.3 ± 3.8 72.0 ± 5.5 60.5 ± 4.7 74.3 ± 6.3 90.5 ± 12.2 75.5 ± 7.7 Σ n-3 PUFA 49.2 ± 9.8 46.0 ± 2.6 47.9 ± 3.4 38.4 ± 2.7 55.0 ± 4.9 67.2 ± 9.6 54.9 ± 5.4 Σ n-3 LC PUFA 40.2 ± 8.2 36.9 ± 2.3 40.7 ± 3.8 31.8 ± 3.0 24.1 ± 1.9 26.1 ± 3.5 27.8 ± 3.2 Σ n-6 PUFA 15.8 ± 2.7 18.7 ± 1.2 22.8 ± 2.0 20.4 ± 2.0 16.9 ± 1.3 19.9 ± 2.6 18.0 ± 2.1 Σ n-6 LC PUFA 8.4 ± 1.7 8.9 ± 0.6 8.0 ± 1.0 8.4 ± 1.3 5.9 ± 0.5 6.6 ± 1.3 5.0 ± 0.9 Σ Fatty Acids 160.9 ± 25.8 163.4 ± 11.8 171.0 ± 14.1 167.3 ± 13.6 180.9 ± 15.8 218.5 ± 24.8 180.6 ± 17.2 Total Lipid 431.5 ± 56.6 514.1 ± 23.6 528.8 ± 30.9 558.6 ± 49.1 521.7 ± 39.9 463.6 ± 52.4 560.4 ± 12.0 919 920 Table S4: Distance-based linear model (DISTLM) marginal tests for survival, eye diameter, body depth at anus, and standard length of

921 Amphiprion latezonatus at 7 dph (days post hatch) when fed four diets (Table 1), and selected fatty acids and ratios. Selection procedure: BEST,

922 selection criterion: AICc. ‘Prop.’ is the proportion of variation in the dataset explained by the variable. Variables with p < 0.05 are bold, and

923 were included in further analyses.

Survival Eye diameter Body depth Standard Length Variable SS (trace) F p Prop. SS (trace) F p Prop. SS (trace) F p Prop. SS (trace) F p Prop. 18:2n-6 2.33E3 7.34 0.014 0.30 75.14 0.22 0.650 0.01 8.65E3 1.85 0.181 0.10 2.66E4 3.24 0.092 0.16 18:3n-6 614.04 1.46 0.246 0.08 533.19 1.68 0.209 0.09 3.16E3 0.63 0.432 0.04 3.58E3 0.37 0.546 0.02 18:3n-3 156.44 0.35 0.566 0.02 26.07 0.07 0.790 0.00 5.41E3 1.11 0.312 0.06 1.65E4 1.88 0.182 0.10 20:4n-6 ARA 178.92 0.40 0.527 0.02 159.61 0.47 0.507 0.03 6.02E3 1.25 0.282 0.07 2.72E3 0.28 0.597 0.02 20:5n-3 EPA 83.97 0.19 0.677 0.01 470.18 1.46 0.244 0.08 1.67E4 3.98 0.062 0.19 2.04E4 2.37 0.141 0.12 22:5n-6 DPAn-6 1.96E3 5.75 0.031 0.25 1.38E3 5.16 0.040 0.23 1.22E4 2.72 0.116 0.14 3.46E4 4.46 0.048 0.21 22:6n-3 DHA 258.91 0.59 0.464 0.03 0.32 0.00 0.978 0.00 6.22E3 1.29 0.271 0.07 1.10E4 1.20 0.291 0.07 12:0 5.59 0.01 0.912 0.00 35.13 0.10 0.751 0.01 4.67E3 0.95 0.333 0.05 1.57E4 1.77 0.207 0.09 13:0 134.23 0.30 0.574 0.02 527.48 1.66 0.172 0.09 2.61E3 0.52 0.527 0.03 4.48E3 0.47 0.514 0.03 14:0 38.68 0.09 0.776 0.00 349.25 1.06 0.313 0.06 58.03 0.01 0.919 0.00 435.38 0.04 0.842 0.00 15:0 6.43 0.01 0.907 0.00 120.12 0.35 0.562 0.02 377.70 0.07 0.793 0.00 9.92 0.00 0.977 0.00 16:0 21.76 0.05 0.824 0.00 82.85 0.24 0.619 0.01 476.57 0.09 0.762 0.01 46.73 0.00 0.948 0.00 17:0 2.22 0.00 0.941 0.00 69.20 0.20 0.663 0.01 200.24 0.04 0.847 0.00 306.19 0.03 0.863 0.00 18:0 235.27 0.53 0.467 0.03 125.85 0.37 0.548 0.02 4.54E3 0.92 0.347 0.05 3.56E3 0.37 0.554 0.02 20:0 1.92 0.00 0.951 0.00 1.24E3 4.50 0.053 0.21 6.30E3 1.31 0.266 0.07 1.43E4 1.59 0.225 0.09 21:0 2.74 0.01 0.937 0.00 857.22 2.87 0.106 0.14 7.74E3 1.64 0.220 0.09 1.29E4 1.43 0.246 0.08 14:1n-5 578.88 1.37 0.263 0.07 104.29 0.30 0.593 0.02 2.01E3 0.40 0.532 0.02 7.99E3 0.86 0.367 0.05 15:1n-5 273.47 0.62 0.437 0.04 526.17 1.65 0.219 0.09 1.23E4 2.77 0.115 0.14 1.62E4 1.84 0.196 0.10 16:1n-7 251.09 0.57 0.456 0.03 372.36 1.14 0.307 0.06 6.08E3 1.26 0.276 0.07 2.92E3 0.30 0.578 0.02 17:1n-7 202.03 0.46 0.583 0.03 1.07E3 3.75 0.105 0.18 6.39E3 1.33 0.315 0.07 1.31E4 1.45 0.325 0.08 18:1n-7 28.15 0.06 0.808 0.00 156.27 0.46 0.501 0.03 7.59E3 1.60 0.224 0.09 5.27E3 0.56 0.464 0.03 18:1n-9 43.19 0.10 0.744 0.01 303.59 0.92 0.348 0.05 1.96E3 0.39 0.577 0.02 5.66E3 0.60 0.461 0.03 18:1n-9 t 806.33 1.98 0.181 0.10 11.77 0.03 0.862 0.00 1.20E3 0.23 0.626 0.01 4.25E3 0.45 0.516 0.03 20:1 (isomers) 1.28E3 3.37 0.084 0.17 15.78 0.05 0.833 0.00 1.90E3 0.38 0.542 0.02 9.45E3 1.02 0.327 0.06 22:1 (isomers) 5.94 0.01 0.910 0.00 1.40E3 5.22 0.037 0.23 1.22E4 2.74 0.116 0.14 3.60E4 4.70 0.045 0.22 24:1n-9 930.93 2.32 0.136 0.12 82.06 0.24 0.628 0.01 2.60E3 0.52 0.491 0.03 1.32E4 1.47 0.245 0.08 16:4n-1 256.09 0.58 0.457 0.03 403.75 1.24 0.277 0.07 1.33E4 3.02 0.100 0.15 2.46E4 2.94 0.107 0.15 18:4n-3 3.74E3 15.91 0.001 0.48 2.23E3 10.25 0.006 0.38 3.49E4 11.18 0.005 0.40 8.05E4 15.94 0.001 0.48 20:3n-3 2.20E3 6.76 0.021 0.28 1.13E3 3.97 0.061 0.19 3.63E4 11.93 0.001 0.41 6.43E4 10.70 0.004 0.39 20:4n-3 3.19E3 11.91 0.005 0.41 1.09E3 3.83 0.065 0.18 3.14E4 9.44 0.004 0.36 6.72E4 11.52 0.003 0.40 21:5n-3 776.59 1.90 0.188 0.10 207.96 0.62 0.434 0.04 1.38E4 3.17 0.099 0.16 2.33E4 2.77 0.112 0.14 22:5n-3 75.82 0.17 0.694 0.01 275.41 0.83 0.370 0.05 9.29E3 2.00 0.175 0.11 6.24E3 0.66 0.438 0.04 16:2n-4 441.48 1.03 0.260 0.06 351.74 1.07 0.370 0.06 621.84 0.12 0.737 0.01 5.80E3 0.61 0.469 0.03 16:3n-4 238.67 0.54 0.461 0.03 119.26 0.35 0.555 0.02 1.36E3 0.27 0.611 0.02 450.02 0.05 0.839 0.00 18:2n-4 1.14E3 2.92 0.105 0.15 294.33 0.89 0.362 0.05 2.82E3 0.56 0.460 0.03 1.07E4 1.17 0.295 0.06 18:3n-4 22.46 0.05 0.831 0.00 11.51 0.03 0.858 0.00 1.02E4 2.22 0.153 0.12 2.63E4 3.19 0.101 0.16 20:2n-6 2.77E3 9.47 0.007 0.36 55.07 0.16 0.697 0.01 4.38E3 0.89 0.357 0.05 2.02E4 2.35 0.146 0.12 20:3n-6 6.68 0.01 0.903 0.00 0.49 0.00 0.970 0.00 227.95 0.04 0.844 0.00 714.15 0.07 0.792 0.00 22:4n-6 331.17 0.76 0.391 0.04 2.52 0.01 0.927 0.00 4.25E3 0.86 0.371 0.05 702.63 0.07 0.784 0.00 Sum SFA 4.43 0.01 0.925 0.00 81.57 0.24 0.633 0.01 1.10E3 0.21 0.649 0.01 94.72 0.01 0.924 0.00 Sum MUFA 416.69 0.97 0.346 0.05 89.54 0.26 0.624 0.02 477.18 0.09 0.763 0.01 5.59E3 0.59 0.455 0.03 Sum PUFA 35.48 0.08 0.785 0.00 209.77 0.62 0.432 0.04 1.90E3 0.38 0.558 0.02 1.54E3 0.16 0.701 0.01 Sum TRANS 797.35 1.95 0.184 0.10 12.43 0.04 0.850 0.00 1.21E3 0.24 0.632 0.01 4.21E3 0.44 0.512 0.03 n-3 PUFA 113.02 0.25 0.614 0.01 432.94 1.34 0.262 0.07 5.52E3 1.14 0.305 0.06 8.93E3 0.96 0.343 0.05 n-3 LC-PUFA 20.94 0.05 0.839 0.00 336.98 1.02 0.333 0.06 2.79E3 0.56 0.461 0.03 3.20E3 0.33 0.577 0.02 n-6 PUFA 1.68E3 4.72 0.042 0.22 6.34 0.02 0.900 0.00 1.72E3 0.34 0.582 0.02 9.85E3 1.07 0.321 0.06 n-6 LC-PUFA 26.87 0.06 0.806 0.00 271.07 0.81 0.382 0.05 4.92E3 1.01 0.333 0.06 2.82E3 0.29 0.594 0.02 DHA/EPA 265.21 0.60 0.443 0.03 88.99 0.26 0.619 0.01 1.80E4 4.36 0.051 0.20 2.47E4 2.96 0.101 0.15 EPA/ARA 1.11E3 2.84 0.113 0.14 66.84 0.19 0.668 0.01 4.83E3 0.99 0.340 0.05 1.86E4 2.14 0.159 0.11 DHA/ARA 100.36 0.22 0.647 0.01 117.46 0.34 0.564 0.02 1.88E4 4.60 0.049 0.21 1.91E4 2.21 0.156 0.11 DPA/DHA 3.57E3 14.58 0.001 0.46 2.63E3 13.47 0.002 0.44 2.47E4 6.61 0.022 0.28 6.47E4 10.82 0.005 0.39 (DHA+EPA) 5.65 0.01 0.915 0.00 84.39 0.25 0.628 0.01 1.54E4 3.61 0.072 0.18 1.20E4 1.32 0.266 0.07 /ARA (ARA+DPAn-6) 478.82 1.12 0.305 0.06 627.50 2.01 0.170 0.11 3.15E4 9.48 0.006 0.36 3.65E4 4.77 0.044 0.22 /( DHA+EPA) n-3 PUFA 3.42E3 13.42 0.002 0.44 768.24 2.53 0.133 0.13 1.89E4 4.64 0.047 0.21 4.52E4 6.35 0.021 0.27 /n-6 PUFA n-3 LC-PUFA 117.77 0.26 0.610 0.02 18.54 0.05 0.821 0.00 4.73E3 0.96 0.338 0.05 732.79 0.08 0.795 0.00 /n-6 LC-PUFA 924 925 Table S5. Distance-based linear model (DISTLM) marginal tests for survival of Amphiprion

926 latezonatus at 14 dph (days post hatch) when fed four diets (Table 1), and selected fatty acids

927 and ratios. Selection procedure: BEST, selection criterion: AICc. ‘Prop.’ is the proportion of

928 variation in the dataset explained by the variable. Variables with p < 0.05 are bold.

Variable SS (trace) F p Prop. Variable SS (trace) F p Prop. 18:2n-6 751.59 1.23 0.282 0.09 20:3n-3 63.91 0.10 0.745 0.01 18:3n-6 1.77E3 3.31 0.096 0.20 20:4n-3 160.31 0.24 0.617 0.02 18:3n-3 151.42 0.23 0.625 0.02 21:5n-3 46.65 0.07 0.796 0.01 20:4n-6 ARA 1.98 0.00 0.957 0.00 22:5n-3 594.87 0.95 0.356 0.07 20:5n-3 EPA 460.99 0.73 0.397 0.05 16:2n-4 120.37 0.18 0.669 0.01 22:5n-6 DPAn-6 92.77 0.14 0.718 0.01 16:3n-4 683.47 1.11 0.305 0.08 22:6n-3 DHA 1.64E3 3.02 0.106 0.19 18:2n-4 117.68 0.18 0.740 0.01 12:0 1.29E3 2.26 0.158 0.15 18:3n-4 569.18 0.91 0.347 0.07 13:0 605.77 0.97 0.675 0.07 20:2n-6 665.98 1.08 0.342 0.08 14:0 1.10E3 1.88 0.196 0.13 20:3n-6 1.55E3 2.81 0.113 0.18 15:0 2.13E3 4.22 0.060 0.25 22:2n-6 72.74 0.11 0.744 0.01 16:0 1.21E3 2.10 0.173 0.14 22:4n-6 356.91 0.56 0.667 0.04 17:0 680.42 1.10 0.311 0.08 Sum SFA 1.17E3 2.02 0.178 0.13 18:0 586.18 0.94 0.352 0.07 Sum MUFA 159.43 0.24 0.622 0.02 20:0 2.38E3 4.89 0.049 0.27 Sum PUFA 582.69 0.93 0.350 0.07 21:0 695.52 1.13 0.318 0.08 Sum TRANS 80.89 0.12 0.732 0.01 14:1n-5 338.74 0.53 0.490 0.04 Sum n-3 PUFA 466.96 0.74 0.401 0.05 15:1n-5 1.80 0.00 0.963 0.00 n-3 LC-PUFA 1.21E3 2.10 0.173 0.14 16:1n-7 311.84 0.48 0.502 0.04 n-6 PUFA 1.01E3 1.70 0.208 0.12 17:1n-7 15.72 0.02 0.883 0.00 n-6 LC-PUFA 67.15 0.10 0.757 0.01 18:1n-7 34.60 0.05 0.817 0.00 DHA/EPA 1.01E3 1.71 0.210 0.12 18:1n-9 111.68 0.17 0.674 0.01 EPA/ARA 605.17 0.97 0.356 0.07 18:1n-9 (Trans) 80.89 0.12 0.724 0.01 DHA/ARA 1.59E3 2.89 0.111 0.18 20:1 (isomers) 61.75 0.09 0.757 0.01 DPAn-6/DHA 1.01E3 1.71 0.213 0.11 (DHA+EPA) 22:1 (isomers) 690.86 1.12 0.399 0.08 2.20E3 4.40 0.057 0.25 /ARA (ARA+DPAn-6) 24:1n-9 1.32E3 2.32 0.153 0.15 2.05E3 4.00 0.071 0.24 /( DHA+EPA) n-3 PUFA 16:4n-1 44.93 0.07 0.804 0.01 405.01 0.63 0.435 0.05 /n-6 PUFA

n-3 LC-PUFA 18:4n-3 13.87 0.02 0.888 0.00 372.47 0.58 0.458 0.04 /n-6 LC-PUFA

929 930 Table S6. Distance-based linear model (DISTLM) marginal tests for survival, eye diameter, body depth at anus, and standard length of

931 Amphiprion latezonatus at 7 dph (days post hatch) when fed four diets (Table 1), and lipid classes. Selection procedure: BEST, selection

932 criterion: AICc. ‘Prop.’ is the proportion of variation in the dataset explained by the variable.

Survival Eye diameter Body depth Standard length Variable SS (trace) F p Prop. SS (trace) F p Prop. SS (trace) F p Prop. SS (trace) F p Prop. Sterol ester 80.04 0.18 0.668 0.01 185.38 0.55 0.483 0.03 403.54 0.08 0.806 0.00 2.48E3 0.26 0.631 0.01 Triacylglycerol 6.05 0.01 0.908 0.00 38.07 0.11 0.740 0.01 60.92 0.01 0.918 0.00 383.22 0.04 0.860 0.00 Free fatty acids 566.56 1.34 0.262 0.07 834.12 2.78 0.115 0.14 1.80E4 4.36 0.053 0.20 2.75E4 3.36 0.086 0.17 1,2 diacylglycerol 2.34E3 7.35 0.022 0.30 232.89 0.69 0.421 0.04 1.92E4 4.75 0.038 0.22 3.01E4 3.76 0.073 0.18 Sterols 249.08 0.57 0.427 0.03 622.19 1.99 0.167 0.10 426.14 0.08 0.791 0.00 5.29E3 0.56 0.485 0.03 Acetone mobile polar lipids 380.87 0.88 0.368 0.05 1.43E3 5.40 0.031 0.24 2.05E4 5.17 0.037 0.23 2.98E4 3.71 0.078 0.18 Phosphatidylethanolamine 88.50 0.20 0.665 0.01 731.80 2.39 0.135 0.12 1.13E4 2.50 0.133 0.13 1.33E4 1.48 0.233 0.08 Phosphatidylcholine 13.63 0.03 0.864 0.00 222.91 0.66 0.423 0.04 6.61E3 1.38 0.248 0.08 6.36E3 0.68 0.424 0.04 Lyso-phosphatidylcholine 275.11 0.63 0.441 0.04 172.60 0.51 0.494 0.03 1.34E3 0.26 0.613 0.02 855.43 0.09 0.784 0.01 933 934 Table S7. Distance-based linear model (DISTLM) marginal tests for survival of Amphiprion

935 latezonatus at 14 dph (days post hatch) when fed four diets (Table 1), and lipid classes.

936 Selection procedure: BEST, selection criterion: AICc. ‘Prop.’ is the proportion of variation in

937 the dataset explained by the variable.

Survival 938 Variable SS (trace) F p Prop. Sterol ester 451.97 0.71 0.469 9390.05 Triacylglycerol 461.01 0.73 0.422 0.05 Free fatty acids 562.64 0.90 0.363 0.06 1,2 diacylglycerol 332.68 0.52 0.456 0.04 Sterols 115.52 0.17 0.664 0.01 Acetone mobile polar lipids 344.88 0.54 0.458 0.04 Phosphatidylethanolamine 146.52 0.22 0.666 0.02 Phosphatidylserine/inositol 605.77 0.97 0.655 0.07 Phosphatidylcholine 13.15 0.02 0.896 0.00 Lyso-phosphatidylcholine 536.56 0.85 0.352 0.06