peerj_json_files/PeerJ_Json_12.json

{
    "v1_Abstract": "Copepods as feed promote better growth and development in marine fish larvae than rotifers. However, unlike rotifers, copepods contain several minerals such as iodine (I), at potentially toxic levels. Iodine is an essential trace element and both under and over supply of I can inhibit the production of the I containing thyroid hormones. It is unknown whether marine fish larvae require copepod levels of I or if mechanisms are present that prevent I toxicity. In this study, larval Atlantic cod (Gadus morhua) were fed rotifers enriched to intermediate (26 mg I kg dry weight; MI group) or copepod (129 mg I kg DW; HI group) I levels and compared to cod larvae fed control rotifers (0.6 mg I kg DW). Larval I concentrations were increased by 3 (MI) and 7 (HI) fold compared to controls during the rotifer feeding period. No differences in growth were observed, but the HI diet increased thyroid follicle colloid to epithelium ratios, and effected the essential element concentrations of larvae compared to the other groups. The thyroid follicle morphology in the HI larvae is typical of colloid goitre, a condition resulting from excessive I intake, even though whole body I levels were below those found previously in copepod fed cod larvae. This is the first observation of dietary induced I toxicity in fish, and suggests I toxicity may be determined to a greater extent by bioavailability and nutrient interactions than by total body I concentrations in fish larvae. Rotifers with 0.6 mg I kg DW appeared sufficient to prevent gross signs of I deficiency in cod larvae reared with continuous water exchange, while modelling of cod larvae versus rotifer I levels suggests that optimum I levels in rotifers for cod larvae is 3.5 mg I kg DW.",
    "v1_text": "acknowledgements : This work was financed by the Norwegian Research Council (project no. 185006/S40). Thank you to technical staff at IMR Austevoll and NIFES for fish husbandry, sampling help and skilled analytical assistance, especially Stig Ove Utskot and Berit Solli. Thank you to Karin Pittman for allowing access to equipment for thyroid follicle morphology quantification. Contributions of the authors. S. P., K. H., A. N. and T.H designed the project, T. H. conducted animal trials, S. P., K. H. and T. H. sampled. S. P., O.S. and S.H. analysed samples. S. P. statistically analysed data. All authors contributed to manuscript writing and revision. Conflicts of interest. None. 16 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 Pre Prin ts Pre Prin ts 2 cod larvae rearing : The experiment was performed at the Institute of Marine Research (IMR), Austevoll Research Station, Norway. This study was carried out within the Norwegian animal welfare act guidelines (code 750.000) at an approved facility. As this trial was assumed to be a nutrition trial based on all available studies up to the date of the trial, no specific permit was required under the guidelines. Naturally spawned and fertilised Atlantic cod eggs were obtained from in house second generation brood stock. Prior to incubation, eggs were disinfected with 200 mg L-1 glutaraldehyde for 9 min at 6\u00b0C and eggs were incubated with a standard protocol as described in Penglase et al. (2010). Upon 100% hatching (16 dpf, 99 day degrees), larval density in the incubators was measured via tube sampling and ranged from 2000-3000 larvae L-1. Three days post 100% hatching (dph), larvae (65 \u00b1 2 \u00b5g DW fish-1, n = 2 where n is a pool of 428 or 520 fish, and 4.9 \u00b1 0.2 mm fish-1, n = 30 (mean \u00b1 SD), measured 5 dph) were transferred into the experimental tanks. Larvae were stocked at an estimated density of 50 000 larvae (120 larvae L -1) in each of the nine 500 L (400 L water volume) experimental tanks (n=3), using volumes of larvae taken from incubators based on the initial larval density measurements. The larval tanks, including colour, material, water inlets, filter size, cleaning procedures and algal additions were as described previously (Penglase et al., 2010). Water inflow to each tank (temperature 8.0\u00b0C, salinity 34.8 \u00b1 0.2\u2030, 20-\u00b5m sand/lamella filtered, degassed, from 160 m depth) started at 0.8 L min-1 at larval transfer and increased over time to reach 4 L min -1 by 39 dph. The water temperature in larval tanks at transfer was 8.0\u00b0C (3 dph), and gradually increased and then maintained at 11.5\u00b0C from 27 dph. Oxygen saturation (75\u2013102%) and temperature were measured once daily in the outlet pipe of each tank. Dim light was provided continuously. Rotifers (Brachionus plicatilis. \u2018Cayman\u2019, adult lorica length 184 \u00b1 10 \u00b5m, width 134 \u00b1 11 \u00b5m) were batch cultured in 500 L tanks and washed as previously described (Penglase et al., 2011) (section 2.2.5 and 2.2.6) with the exception that algae paste (Chlorella sp., Docosa, SV12, Japan) was used as the culture feed. After washing, rotifers were enriched with either a control or treatment diet. The control enrichment was 250 mg Ori-green (Skretting, Norway) million-1 rotifers. The treatment enrichment (I+rotifers) was as per controls, but in addition 60 mg L-1 of sodium iodide (NaI; VWR, Belgium art. no. 27915.297) was added to the water at the start of rotifer enrichment. Ori-green was prepared to manufacturer\u2019s directions, while NaI 5 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 Pre Prin ts Pre Prin ts was dissolved in cold tap water, prior to addition to rotifer enrichment tanks. Rotifers were enriched for 2 h at densities between 1000-2000 mL-1 in water (as for rotifer culturing) with continual aeration and oxygenation (oxygen saturation was kept above 80%). After 1.5 h of this enrichment, an antibacterial (Pyceze, Novartis, Switzerland) was added to both control and treatment enrichment tanks at a rate of 0.2 ml L-1. Pyceze was used to lower rotifer bacterial numbers and thus control for any antibacterial effect of I enrichment. After enrichment, rotifers were washed, concentrated to 2000-4500 rotifers mL-1, transferred to storage tanks with aeration, and cooled rapidly (<10 min) to 8.5\u00b0C. To maintain I concentrations in the I+rotifers, 60 mg I L-1 (as NaI) was added to the treatment rotifer holding tank. Rotifers samples for element analysis were collected from rotifer storage tanks on 4 separate days during the larvae feeding trial. Rotifers were collected on 62 \u00b5m mesh, washed for 5 min with 12\u00b0C saltwater, placed in 25 mL containers and stored at -20\u00b0C. The feeding trial started at 4 dph, using rotifers as prepared in section 2.2. Larvae were fed control or I+rotifers (HI+rotifers) or a mixture of both (80:20, control:I+, MI+rotifers). Each tank received increasing quantities of rotifers with time, starting from 3.5 million rotifers tank-1 day-1 at 4 dph increasing to 6 million rotifers tank-1 day-1 by 39 dph. The same quantity of rotifers was fed to each tank, and larvae were assumed to be fed to satiation. The rotifers were fed daily to larvae in two batch feedings of equal rotifer quantity at 10:00 and 15:00. Rotifers were poured gently into larval tanks in a circular motion to ensure even rotifer distribution and minimal larvae disturbance. Control larvae and HI+larvae were fed only control or treatment rotifers respectively. The MI+larvae were fed I+ and control rotifers at 10:00, and only control rotifers at 15:00 resulting in the overall feeding ration consisting of 80:20 control: I+rotifers. For later analysis of skeletal deformities, fish were reared on identical diets from 40 to 124 dph. Larvae were co-fed Artemia (OK performance cysts, INVE, Belgium) and rotifers from 40-44 dph. Both Artemia and rotifers were enriched with Ori-green as per directions. Fish were fed only Artemia from 45-68 dph, co-fed Artemia and formulated diet (AgloNorse-EX1, Trofi, Troms\u00f8, Norway) from 69-91 dph. Only formulated feed was fed from 92 dph (EX1; 92-94 dph, EX1 and 2; 95-103 dph, EX2; 104-115 dph, EX3; 116-124 dph). Formulated feed was administered continuously for 24 hrs day-1 by belt feeders. Flow rate was increased from 4 L min-1 at 30 dph to 8 L min-1 at 108 dph, while water current speed in tanks was minimized by letting water enter through a 32 mm diameter inlet tube. Along with the increased water 6 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 Pre Prin ts Pre Prin ts flow rate, oxygen saturation (64-96%) was maintained by removal of fingerling cod when required. Larvae were sampled for weight and length at 5, 9, 19, 30, and 124 dph, element and thyroid hormone analysis at 5, 9, 19 and 30 dph, thyroid follicle morphology at 19, 30 and 37 dph and for analysis of skeletal deformities at 124 dph. All fish were euthanised with an overdose of tricaine methane sulfonate (MS 222) upon sampling. Larvae sampled for weight, minerals and thyroid hormones were collected on mesh (62 \u00b5m), briefly rinsed with ddH20, and patted dry from underneath with paper towel. Larvae were then placed in pre weighed tubes and frozen immediately in liquid nitrogen and stored at -80\u00b0C until analysis. All tubes were then reweighed to determine sample wet weights. Tubes sampled for weight determination were thawed and larvae were counted (n=20-100) to determine wet weight per larvae. Dry weight was determined from dry matter, which in turn was determined from tubes weighed before and after lyophilising. The standard length of the larvae was measured according to Hamre et al. (2008a) on 10 larvae tank-1. Larval densities in tanks were measured at 30 dph as described by Penglase et al. (2010). Larvae for thyroid follicle (3 fish tank-1) were selectively sampled to be similar in length, thus representing similar levels of morphological development (S\u00e6le and Pittman, 2010). Larvae were placed in individual tubes containing 1 ml of 4% paraformaldehyde in PBS buffer at pH 7.2. Samples were left overnight and then transferred to separate tubes containing 70% ethanol, where they remained at 4\u00b0C until embedding. For analysis of skeletal deformities, cod juveniles (124 dph, n=50 per tank) were measured for length and weight, frozen flat and subsequently stored in individual labelled plastic bags at -20\u00b0C until analysis. Survival in one HI+ tank decreased to zero prior to this sampling, so data represents the mean \u00b1 SD n=2 for the HI+fish at 124 dph. Samples for analysis of total I were digested under alkaline conditions using tetra methyl ammonium hydroxide ((CH3)4NOH, Tamapure-AA, Tama chemicals, Japan) and then analysed with ICP-MS (Agilent 7500 series, USA) as described by Julshamn, Dahl and Eckhoff (2001) with cod muscle (BCR-422, Belgium) used as the standard reference material. Samples for the analysis of other elements were prepared by wet digestion with nitric acid (65 7 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 Pre Prin ts Pre Prin ts % HNO3 Suprapur\u00ae, Merck, Germany) and hydrogen peroxide (30 % H2O2, Merck, Germany), in a microwave (Ethos 1600, Milestone, USA) as described by Julshamn et al. (2004). The samples were then analysed with ICP-MS along with blanks and standard reference material as described previously (Julshamn et al., 2004), with modifications to the mass of Mn (Mass 55) and Pb (Mass 208) measured. The standard reference materials used (NIST-SRM 1566, Oyster tissue, USA; TORT-2, NRC, lobster hepatopancreas, Canada) had similar concentrations of minerals as the samples analysed. Thyroid hormones extraction from larvae was carried out according to Einarsdottir et al. (2006) with some modifications. Approximately 500 mg WW of larvae (440 to 630 mg) was homogenised (Precellys 24, Bertin technologies, France) in 1 ml of ice-cold methanol (Sigma-Aldrich, Germany), and then stored over night at -20oC. Samples were then centrifuged (30 min, 3000 rpm, 4 \u00baC) and the supernatant removed from the pellet. This extraction procedure was then repeated on the pellet twice more. Nitrogen was used to dry the supernatant of methanol. Lipids were removed from samples by modified Folch extraction. To eliminate any lipid in samples, the dried extracts were dissolved in barbital buffer (0.1 M pH 8.6), methanol and chloroform (1:1:2). The aqueous phase containing T4 and T3 was transferred to a fresh tube, evaporated with nitrogen and frozen at -20o C until use. To estimate extraction efficiency, \u22481000 cpm of [125I]-rT3 (NEX109, Perkin Elmer, USA) were added to the homogenates after homogenisation. The extraction efficiency ranged between 71 to 81%. The T4 and T3 content were determined by radioimmunoassay using an external standard curve according to Einarsdottir et al. (2006), and further corrected for the extraction efficiency of each sample. Larval T3 contents were also reanalysed by a DELFIA\u00ae T3 Kit (PerkinElmer, Turku, Finland) according to manufacturer\u2019s instructions. Larvae were dehydrated in an increasing gradient of ethanol and embedded in Technovit 7100 as per directions (Heraeus Kulzer, Wehrheim, Germany). The resin blocks were then sectioned into 5 \u03bcm thick slices, and every second section was placed on a slide and stained with toluidine blue. The follicle number within the pharyngeal region was counted and the area of epithelium and colloid were measured for each larvae (2 larvae tank -1 at 19 and 30 dph, 1 larvae tank-1 at 37 dph) at 200\u00d7 magnification using a microscope and computer assisted program CAST-grid version 2 (Olympus, Albertslund, Denmark) according to Saele et al. (2003). The colloid and epithelium volume were calculated using area and width of sections, 8 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 Pre Prin ts Pre Prin ts and skipped sections were assumed to have the same volume as that measured on the preceding section. Larval sections were also scanned for evidence of thyroid follicles in the kidneys, as has been observed for some other fish species such as common carp (Cyprinus carpio) (Geven et al., 2007), but none were observed. The radiographic imaging and analysis of skeletal deformities was performed as described previously (Penglase et al., 2010) with skeletal deformities and degree of deformities classified according to Baeverfjord et al. (undated). Briefly, radiographic images were visually examined for any skeletal pathology, and deviations from normal were recorded. Deviations were classified in axial deviations, vertebral deviations and head deformities, and further classified into sub categories and degrees of severity. Larval survival was adjusted using a linear individual probability timeline for each tank to calculate the probable survival of sampled larvae had they remained in the tanks until density measurements at 30 dph, using the following equation; Estimated survival of sampled larvae at time point Y = 100 \u2013 (((100-S)/T1)*(T1-T2))/100)*X) where S is the measured survival % at 30 dph, T1 equals the time period in days from the start (5 dph) and end (30 dph) survival measurements (25 d), T2 equals the number of days post 100% survival (trial start) and X equals the number of larvae sampled at time point Y. This equation was used to produce two numbers, one for the sampling at 9 (T2 = 4) and one at 19 (T2 = 14) dph, and along with the number of larvae sampled on day 30, were added to the larvae measured in tanks from density measurements taken after sampling at 30 dph. Specific growth rates (SGR) of cod larvae were calculated with the following equation SGR = (e^((lnW1 \u2212 lnW0)/(t2-t1))-1)\u00d7100 where W0 and W1 are the initial and final dry weights (tank means) respectively, and t2 \u2013 t1 is the time interval in days between age t1 and t2 (Ricker, 1958). Fulton\u2019s condition factor (FC) was calculated using FC = Weight (g)*100/Length(cm)3. The I concentration ratio (CR) between larvae and feed was CR = Larval I content (mg kg-1 DW)/rotifer I content (mg kg-1 DW). Statistica software (Statsoft Inc., 2008, Tulsa, USA, Ver.9) was used for statistical analysis of data except when GraphPad Prism (GraphPad Software, San Diego, CA, USA, Ver. 5) was 9 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 Pre Prin ts Pre Prin ts used to model fit the I concentration of cod larvae versus rotifers, and on data from 124 dph, as the loss of one replicate in the HI larvae group prevented ANOVA analysis between all three groups. Data analysed with Statistica were checked for homogeneity of variances using Levene\u2019s test before having significance tested with one-way ANOVA followed with Fisher\u2019s least significant difference (LSD) homogeneity post-hoc test at each time point. Data with significantly different variance between treatments according to Levene\u2019s test (p<0.05) was log transformed before analysis. As the density of cod larvae has a large effect on growth (Koedijk et al., 2010), growth data during the larval stage was analysed with ANCOVA with the final larval density in tanks included as a continuous predictor. Data from 124 dph were analysed using regression, and tested against the null hypothesis that rotifer I content had no effect on outcome. Differences among means were considered significant at p<0.05. 10 277 278 279 280 281 282 283 284 285 286 287 Pre Prin ts Pre Prin ts 3 Results 3 cod larvae growth : No statistically significant differences in cod larvae length (p > 0.08) or dry weight (p > 0.25) occurred between treatments, although high variation between tanks may have masked effects (Fig. 1). On average, cod larvae grew from 4.9 \u00b1 0.2 mm to 6.8 \u00b1 0.5 mm in length, and 0.065 \u00b1 0.002 mg to 0.27 \u00b1 0.07 mg fish-1 in dry weight from 5 to 30 dph (Fig. 1), representing a specific growth rate of 6.3% day-1 during this period. There were no statistical differences (p>0.39) in the cod larval survival adjusted for sampled larvae (see section 2.6) between treatments at 30 dph which were 28 \u00b1 2, 39 \u00b1 20 and 30 \u00b1 15 % for controls, MI and HI groups respectively, while the average for all groups was 32 \u00b1 13 %. Survivals based solely on densities in tanks at 30 dph without taking into account sampled larvae were 12 \u00b1 2, 19 \u00b1 12 and 12 \u00b1 10 % for controls, MI and HI groups respectively. Control rotifers contained 0.60 \u00b1 0.33 mg I kg-1 DW while HI+rotifers contained 129 \u00b1 101 mg I kg-1 DW (Table 1). Whole body I levels in cod larvae were significantly different between groups (p<0.01, Fig. 2). Cod larvae (5 dph) had a starting concentration of 4.0 \u00b1 0.3 mg I kg-1 DW. Between 9 and 30 dph average I concentrations were 1.6 \u00b1 0.3 mg I kg-1 DW for control larvae, while MI larvae had 3 fold higher levels (4.9 \u00b1 2.4 mg I kg-1 DW), and HI larvae 7 fold higher levels (11.0 \u00b1 3.3 mg I kg-1 DW) than controls. Other element concentrations were also affected by treatment in both rotifers and cod larvae. HI+rotifers contained less Fe and Mn than controls (Table 1), while HI larvae contained more Mn, Fe and Cu than controls and MI larvae at one or more time points (Fig. 2b-c, e). Both HI and MI larvae contained higher levels of Co than controls (Fig. 2d), while larval Zn and Se concentrations were unaffected by treatment (Fig. 2f-g). Rotifer macro mineral concentrations were unaffected by treatment (Table 1), but increased levels of Ca and Mg, and lower levels of P and K were observed in HI larvae in comparison to controls and/or MI larvae during the rotifer feeding period (Fig. 3). The rate of increase in cod larvae I concentrations decreased as dietary I levels (rotifer I concentration) increased, and thus the I concentration ratio between cod larvae and rotifers displayed a negative trend (Fig 4; p<0.01). The age of the cod larvae did not effect their I concentration ratio (p = 0.96). The model predicts that the ratio of I in cod larvae versus rotifers equals 1 when rotifers have 3.5 mg I kg-1 DW (Fig. 4). 11 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 Pre Prin ts Pre Prin ts There were no differences in thyroid hormone levels or ratios between treatments (Fig. 5). Data was normalised to aid interpretation, as T3 results were higher than obtained previously (Penglase et al., 2010) and the high result was validated by the analysis of T3 with a second method (see methods). The total volume of thyroid follicle was 1.3 fold lower and the total epithelium volume was 1.4 fold lower per fish in HI versus MI larvae, but not controls, at 30 dph (Fig. 6a, c). The thyroid follicle colloid to epithelium ratio was higher in HI larvae than controls at 19 (1.7 fold) and 37 (1.8 fold) dph, while MI larvae did not differ from controls (Fig. 6d). No statistically significant differences were observed between groups in colloid volume or total number of thyroid follicles (Fig. 6b, e). Images of thyroid follicle sections from control and HI larvae at 37 dph are shown in figure 7. There were no significant differences in the weights (average; 2.50 \u00b1 0.19 g), lengths (6.14 \u00b1 0.16 cm) or condition factors (1.05 \u00b1 0.03) between groups at 124 dph (Table 2, n=400). Neck axis angle became closer to normal (180 \u00b1 3 degrees) with increasing I levels in rotifers, but there were no differences in any of the other skeletal deformity measurements (Table 2). 12 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 Pre Prin ts Pre Prin ts Discussion The hypothesis of this study was that commercially reared cod larvae fed rotifers would benefit from increased dietary I. The bases for this hypothesis were, one; rotifer I concentrations are often at the lower end or below juvenile/adult fish requirements (NRC, 2011), two; rotifers have 6 - 600 fold lower concentrations of I than copepods (Hamre et al., 2008b), the natural feed of cod larvae (Thompson and Harrop, 1991), three; cod larvae have better growth and development when fed copepods versus rotifers (Busch et al., 2010; Koedijk et al., 2010), four; I levels in copepod fed cod larvae are higher than rotifer fed cod larvae (Busch et al., 2010), and five; increased growth and/or survival has been observed in larval stages of several marine fish species fed or reared in environments with increased levels of bioavailable I (Hamre et al., 2008a; Witt et al., 2009; Ribeiro et al., 2011; Ribeiro et al., 2012). However, in contrast to the hypothesis, the increased thyroid follicle colloid to epithelium (C/E) ratio observed in this study indicates that I toxicity occurred in cod larvae fed rotifers with 129 mg I kg-1 DW. This observation purely in relation to the I level is not unexpected. A high C/E ratio in thyroid follicles is a classic symptom of I induced toxicity termed I (Baker, 2004) or colloid goitre, and occurs in mice at dietary I concentrations 10 fold higher than requirements with increasing severity developing with increasing I ingestion rates (Yang et al., 2006). What is interesting is that despite copepods containing 50 \u2013 350 mg I kg-1 DW (Solbakken et al., 2002; Hamre et al., 2008b), cod larvae have either similar or lower thyroid follicle C/E ratios when fed copepods compared to rotifers (Gr\u00f8tan, 2005). Furthermore, I concentrations observed in cod larvae fed natural zooplankton (29 mg I kg-1 DW at 27 dph; (Busch et al., 2010)) were 2.2 fold higher than the highest level observed in the current study (HI larvae, 30 dph; 13 \u00b1 4 mg I kg-1 DW). Thus it appears that high I concentrations in copepods do not induce morphological changes in thyroid follicles consistent with I toxicity, but do appear to be effectively transferred from copepods to fish larvae upon consumption. It is possible that copepods do not induce I toxicity in fish larvae due to nutrient interactions. For example, I toxicity can be prevented by the simultaneous presence of the bromine anion (Br-) in animals ranging from chicks (Gallus gallus) (Baker, Parr and Augspurger, 2003) to Artemia (S. Penglase et al., unpublished data). The exact mechanism for this Br-/I- interaction is still unknown, but it has been demonstrated that Br- decreases iodide accumulation in the thyroid follicles and increases I excretion via the kidneys in rats (Pavelka, 2004). Although the bromide concentrations in whole copepods and rotifers are unknown, we speculate that copepods have relatively high levels of bromide reflecting the high levels found in other 13 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 Pre Prin ts Pre Prin ts marine organisms, and this bromide helps prevent I toxicity in fish larvae. In the marine ecosystem, bromine is naturally found at similar high concentrations as I in seaweed (Romaris-Hortas, Moreda-Pineiro and Bermejo-Barrera, 2009), adult fish (Arafa et al., 2000; Wan et al., 2010), and as part of the hard chitin structures of crustaceans such as crabs (Cribb et al., 2009; Schofield et al., 2009) and copepods (Perry, Grime and Watt, 1988). Thyroid hormone levels and ratios were similar between cod larvae groups, and this is probably due to the compensatory changes observed in the thyroid follicles. For example pathological changes of over 70% in thyroid gland morphology have been observed in dogs (Canis lupus familiaris) with little change in circulating TH levels (Graham, Refsal and Nachreiner, 2007), and significant changes in fish thyroid follicle morphology with little change in thyroid hormone levels have also been reported for fish (Hawkyard et al., 2011; Morris et al., 2011). Few differences were found between the control and MI larvae groups, with the exception of whole body I concentrations. The increased whole body I content of cod larvae demonstrates the effective transfer of I from the rotifers to the cod larvae. The current study differs to previous studies exploring the uptake of I in fish larvae, as it attempted to ensure the ingestion of graded levels of I by maintaining the I concentration in the prey organism up until the point of feeding. Srivastava et al. (2012) found that I leaches rapidly from rotifers after enrichment with sodium iodide. Previous studies have found no difference in the I concentration of cod larvae fed control or I supplemented rotifers (Hamre et al., 2008a); S. Penglase et al., unpublished data), and this is probably a consequence of I leaching from rotifers in the minimum 1.5 to 2 h period between rotifer enrichment and feeding of the rotifers to cod larvae in these studies. In the current study, cod larvae iodine level increases were proportionally smaller for each increase in dietary I levels; control fish were 2.7 fold higher, while MI were 5 fold lower and HI larvae were 12 fold lower in I than their respective diets. Body stores of minerals are a good indicator of status (Baker, 1986), and the decreasing level of I retention in cod larvae relative to feed I levels indicates that requirements were met at levels lower than those fed to MI larvae. Modelling of the ratio between cod larvae and rotifer I concentrations predicts that based on a ratio of 1:1, rotifer I concentrations of 3.5 mg kg-1 DW meet cod larvae requirements. Both food and water contribute to the I status of adult, juvenile (Lall, 2002) and larval fish (Witt et al., 2009; Ribeiro et al., 2011). Alongside the I content in the continuously 14 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 Pre Prin ts Pre Prin ts exchanging seawater (88 \u00b5g I L-1, Moren, Sloth and Hamre, 2008), the control rotifers in the current study with 0.6 mg I kg-1 DW appeared to prevent any gross signs of I deficiency in cod larvae. This is at the lower end of the 0.6 \u2013 1.1 mg I kg-1 DW recommended by the national research council (NRC, 2011) as the I requirements of juvenile/adult fish. The reason that symptoms of severe I deficiency such as classic goitre have been observed in other fish studies is probably due to water parameters. Clear signs of I deficiency (goitre, decreased growth and/or decreased survival) occurred in fish larvae reared in either recirculation systems (Ribeiro et al., 2011; Ribeiro et al., 2012) or well water (Witt et al., 2009). Nitrate (NO3-) is goitrogenic as it competitively blocks iodide uptake by the sodium iodide symporter (Tonacchera et al., 2004), and NO3- at levels commonly found in recirculation systems causes goitre in sharks (Crow et al., 1998; Morris et al., 2011). Furthermore, ozone (O3) used as a disinfectant in recirculation systems readily oxidises bioavailable I, iodide (I-) to iodate (IO3-) (Sherrill, Whitaker and Wong, 2004). Dissolve iodate is presumed to have low bioavailablity for fish (Sherrill, Whitaker and Wong, 2004), and higher levels of iodate compared to iodide were correlated to poor growth and survival in well water reared pacific threadfin larvae (Witt et al., 2009). Thus in recirculation systems, the presence of high levels of goitrogens (NO3-) and low levels of dissolved bioavailable I (I-) may increase the dietary I requirements of fish larvae over those reared with continuous water exchange where nitrate and its precursors are continuously removed and iodide continuously replaced, such as in the current study. Along with thyroid follicle morphology, dietary I also influenced the mineral composition of cod larvae. While most of the tested mineral concentrations in MI larvae were similar to controls, HI larvae had 10 to 25% higher levels of Ca, Mg, Mn, Fe, Co and Cu and around 10% lower levels of P and K than controls at one or more time points within the rotifer feeding period. For most of the minerals, differences in levels were observed by the first sampling point (9 dph; 4 days of feeding on rotifers). The differences cannot be explained by the feed; the HI rotifers had \u2248 10% less Mn and Fe, and no statistical differences were observed in Ca, Mg, K, P, Cu or Co concentrations. Hamre et al. (2008a) found that cod larvae fed increased levels of both I and Se had an 8% increase in whole body copper levels, similar to this study. Nguyen et al. (2008) found increased or decreased copper levels (20%) in red sea bream (Pagrus major) larvae depending on whether they were fed rotifers enriched with Mn alone or alongside Zn. While it is known that I deficiency can alter mineral distribution and homeostasis of Cu, Mn, Fe and Zn (Giray et al., 2003), to our knowledge this 15 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 Pre Prin ts Pre Prin ts is the first data demonstrating that I oversupply can also effect mineral homeostasis. Although there were few differences in growth or skeletal deformities observed between treatments at 124 dph, there was a small but statistically significant improvement in the neck axis angle in the HI compared to the control and MI cod groups (Table 2), and this may be linked to the differences in cod mineral concentrations in the larval stage. Conclusion Iodine enriched rotifers increased the levels of I in cod larvae, although as I levels in rotifers increased the increases in cod larvae I levels became proportionally smaller. Few differences occurred between cod larvae reared on control diets with 0.6 mg I kg -1 DW and those reared on diets with 26 mg I kg-1 DW, while the I concentration ratio between cod larvae and rotifers suggests cod larvae have an I requirement of 3.5 mg I kg -1 rotifers DW. Rotifers with copepod levels of I (129 mg I kg-1 DW) changed cod larvae whole body concentration of many essential minerals and induced changes in thyroid follicles morphology consistent with colloid goitre. The data presents one of the first observations of dietary induced I toxicity in fish, and suggests that I toxicity in fish larvae may be determined to a greater extent by I bioavailability and nutrient interactions than by body burdens of I. l ength (data set l; mm fish -1 , left y axis) and dry weight (data set w; mg fish -1 , right y axis) of : cod larvae fed control (\u25a1), MI ( \u25cb ) or HI ( \u25cf ) rotifers, from 5 to 30 dph. At 5 dph, data are mean \u00b1 SD of 2 analytical parallels for dry weight and mean \u00b1 SD (n=30) for length. Data at all other dph are mean \u00b1 SD (n=3) where n represents the average of 10 larvae tank -1 measured for length, and a group of 47 to 520 larvae group weighed then counted to determine individual mass. Pre Prin ts Pre Prin ts Figure 2 Essential micro element concentration in whole cod larvae Essential micro element concentrations (mg kg -1 DW) in whole cod larvae fed either control (\u25a1), MI ( \u25cb ) or HI ( \u25cf ) rotifers, from 5 to 30 dph. Letters denote statistically significant differences in mineral concentrations between treatments at a given day (one-way ANOVA; p<0.05). Data are mean \u00b1 SD (n=3), except at 5 dph where data are mean \u00b1 SD of analytical parallels. Pre Prin ts Pre Prin ts Figure 3 Essential micro element concentration in whole cod larvae Pre Prin ts Pre Prin ts Figure 4 Essential micro element concentration in whole cod larvae Pre Prin ts Pre Prin ts Figure 5 Essential micro element concentration in whole cod larvae Pre Prin ts Pre Prin ts Figure 6 Essential micro element concentration in whole cod larvae Pre Prin ts Pre Prin ts Figure 7 Essential micro element concentration in whole cod larvae Pre Prin ts Pre Prin ts Figure 8 Essential micro element concentration in whole cod larvae Pre Prin ts Pre Prin ts Figure 9 Essential macro element concentrations in whole cod larvae Essential macro mineral concentrations (g kg -1 DW) in whole cod larvae fed either control (\u25a1), MI ( \u25cb ) or HI ( \u25cf ) rotifers, from 5 to 30 dph. Letters denote statistically significant differences in mineral concentrations between treatments at a given day (one-way ANOVA, p<0.05). Data are mean \u00b1 SD (n=3), except at 5 dph where data are from a single analysis. Pre Prin ts Pre Prin ts Figure 10 Essential macro element concentrations in whole cod larvae Pre Prin ts Pre Prin ts Figure 11 Essential macro element concentrations in whole cod larvae Pre Prin ts Pre Prin ts Figure 12 Essential macro element concentrations in whole cod larvae Pre Prin ts Pre Prin ts Figure 13 Cod larvae iodine concentration in relation to their feed Ratio of iodine concentration (mg kg -1 DW) in cod larvae versus their diet (rotifers iodine levels (mg kg -1 DW)). X axis is log transformed. Line represents best fit model (Morrison Ki, R 2 = 0.94). Data are mean \u00b1 SD (n=9). Pre Prin ts Pre Prin ts Figure 14 Cod larvae thyroid hormone levels and ratios Normalised mean thyroid hormone levels (NML) in cod larvae fed either control (\u25a1), MI ( \u25cb ) or HI ( \u25cf ) rotifers. Graph A is tri-iodothyronine (T 3 ), Graph B is thyroxine (T 4 ), while graph C is the ratio between the NML of T 3 /T 4 . Data are mean \u00b1 SD (n=3) for all data points except controls at 30 dph which has an outlier removed in graph B and C (n=2). Pre Prin ts Pre Prin ts Figure 15 Cod larvae thyroid hormone levels and ratios Pre Prin ts Pre Prin ts Figure 16 Cod larvae thyroid hormone levels and ratios Pre Prin ts Pre Prin ts Figure 17 Cod larvae thyroid follicle morphology Thyroid follicle morphology in cod larvae fed either control (\u25a1), MI ( \u25cb ) or HI ( \u25cf ) rotifers. Graph A shows the total number of follicles per fish, Graph B is the total thyroid follicle volume per fish, Graphs C and D show the volume of colloid or epithelium per fish, Graph E shows the ratio between the colloid and epithelium volumes. Letters denote statistically significant differences between treatments per time point (one-way ANOVA, p<0.05). Data are mean \u00b1 SD (n=3) where n consists of the average measurements from two fish per tank at 19 and 30 dph, and one fish per tank at 37 dph. Pre Prin ts Pre Prin ts Figure 18 Cod larvae thyroid follicle morphology Pre Prin ts Pre Prin ts Figure 19 Cod larvae thyroid follicle morphology Pre Prin ts Pre Prin ts Figure 20 Cod larvae thyroid follicle morphology Pre Prin ts Pre Prin ts Figure 21 Cod larvae thyroid follicle morphology Pre Prin ts Pre Prin ts Figure 22 Thyroid follicle sections from cod larvae Thyroid follicle section from c od larvae (37 dph) fed either control (A) or HI ( B ) rotifers. Sections are stained with toulidine blue. C; thyroid follicle colloid, E; example of thyroid follicle epithelium. Scale bars are 100 \u00b5m. Pre Prin ts Pre Prin ts Figure 23 Thyroid follicle sections from cod larvae Pre Prin ts Pre Prin ts",
    "v2_text": "acknowledgements : Thank you to technical staff at IMR Austevoll and NIFES for fish husbandry, sampling help and skilled analytical assistance, especially Stig Ove Utskot and Berit Solli. Thank you to Karin Pittman for allowing access to equipment for thyroid follicle morphology quantification. Contributions of the authors. S. P., K. H., A. N. and T.H. designed the project, T. H. conducted animal trials, S. P., K. H. and T. H. sampled. S. P., O.S. and S.H. analysed samples. S. P. statistically analysed data. All authors contributed to manuscript writing and revision. 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 Pre Prin ts Pre Prin ts 2 cod larvae rearing : The experiment was performed at the Institute of Marine Research (IMR), Austevoll Research Station, Norway. This study was carried out within the Norwegian animal welfare act guidelines (code 750.000) at an approved facility. As this trial was assumed to be a nutrition trial based on all available studies up to the date of the trial, no specific permit was required under the guidelines. Naturally spawned and fertilised Atlantic cod eggs were obtained from in house second generation brood stock. Prior to incubation, eggs were disinfected with 200 mg L-1 glutaraldehyde for 9 min at 6\u00b0C and eggs were incubated with a standard protocol as described in Penglase et al. (2010). Upon 100% hatching (16 dpf, 99 day degrees), larval density in the incubators was measured via tube sampling and ranged from 2000-3000 larvae L-1. Three days post 100% hatching (dph), larvae (65 \u00b1 2 \u00b5g DW fish-1, n = 2 where n is a pool of 428 or 520 fish, and 4.9 \u00b1 0.2 mm fish-1, n = 30 (mean \u00b1 SD), measured 5 dph) were transferred into the experimental tanks. Larvae were stocked at an estimated density of 50 000 larvae (120 larvae L -1) in each of the nine 500 L (400 L water volume) experimental tanks (n=3), using volumes of larvae taken from incubators based on the initial larval density measurements. The larval tanks, including colour, material, water inlets, filter size, cleaning procedures and algal additions were as described previously (Penglase et al., 2010). Water inflow to each tank (temperature 8.0\u00b0C, salinity 34.8 \u00b1 0.2 \u2030, 20-\u00b5m sand/lamella filtered, degassed, from 160 m depth) started at 0.8 L min-1 at larval transfer and increased over time to reach 4 L min -1 by 39 dph. The water temperature in larval tanks at transfer was 8.0\u00b0C (3 dph), and gradually increased and then maintained at 11.5\u00b0C from 27 dph. Oxygen saturation (75\u2013 102%) and temperature were measured once daily in the outlet pipe of each tank. Dim light was provided continuously. Rotifers (Brachionus plicatilis. \u2018Cayman\u2019, adult lorica length 184 \u00b1 10 \u00b5m, width 134 \u00b1 11 \u00b5m) were batch cultured in 500 L tanks and washed as previously described (Penglase et al., 2011) (section 2.2.5 and 2.2.6) with the exception that algae paste (Chlorella sp., Docosa, SV12, Japan) was used as the culture feed. After washing, rotifers were enriched with either a control or treatment diet. The control enrichment was 250 mg Ori-green (Skretting, Norway) million-1 rotifers. The treatment enrichment (I+rotifers) was as per controls, but in addition 60 mg L-1 of sodium iodide (NaI; VWR, Belgium art. no. 27915.297) was added to the water at the start of rotifer enrichment. Ori-green was prepared to manufacturer\u2019s directions, while NaI 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 Pre Prin ts Pre Prin ts was dissolved in cold tap water, prior to addition to rotifer enrichment tanks. Rotifers were enriched for 2 h at densities between 1000-2000 mL-1 in water (as for rotifer culturing) with continual aeration and oxygenation (oxygen saturation was kept above 80%). After 1.5 h of this enrichment, an antibacterial (Pyceze, Novartis, Switzerland) was added to both control and treatment enrichment tanks at a rate of 0.2 ml L-1. Pyceze was used to lower rotifer bacterial numbers and thus control for any antibacterial effect of I enrichment. After enrichment, rotifers were washed, concentrated to 2000-4500 rotifers mL-1, transferred to storage tanks with aeration, and cooled rapidly (<10 min) to 8.5\u00b0C. To maintain I concentrations in the I+rotifers, 60 mg I L-1 (as NaI) was added to the treatment rotifer holding tank. Rotifers samples for element analysis were collected from rotifer storage tanks on 4 separate days during the larvae feeding trial. Rotifers were collected on 62 \u00b5m mesh, washed for 5 min with 12\u00b0C saltwater, placed in 25 mL containers and stored at -20\u00b0C. The feeding trial started at 4 dph, using rotifers as prepared in section 2.2. Larvae were fed control or I+rotifers (HI+rotifers) or a mixture of both (80:20, control:I+, MI+rotifers). Each tank received increasing quantities of rotifers with time, starting from 3.5 million rotifers tank-1 day-1 at 4 dph increasing to 6 million rotifers tank-1 day-1 by 39 dph. The same quantity of rotifers was fed to each tank, and larvae were assumed to be fed to satiation. The rotifers were fed daily to larvae in two batch feedings of equal rotifer quantity at 10:00 and 15:00. Rotifers were poured gently into larval tanks in a circular motion to ensure even rotifer distribution and minimal larvae disturbance. Control larvae and HI+larvae were fed only control or treatment rotifers respectively. The MI+larvae were fed I+ and control rotifers at 10:00, and only control rotifers at 15:00 resulting in the overall feeding ration consisting of 80:20 control: I+rotifers. For later analysis of skeletal deformities, fish were reared on identical diets from 40 to 124 dph. Larvae were co-fed Artemia (OK performance cysts, INVE, Belgium) and rotifers from 40-44 dph. Both Artemia and rotifers were enriched with Ori-green as per directions. Fish were fed only Artemia from 45-68 dph, co-fed Artemia and formulated diet (AgloNorse-EX1, Trofi, Troms\u00f8, Norway) from 69-91 dph. Only formulated feed was fed from 92 dph (EX1; 92-94 dph, EX1 and 2; 95-103 dph, EX2; 104-115 dph, EX3; 116-124 dph). Formulated feed was administered continuously for 24 hrs day-1 by belt feeders. Flow rate was increased from 4.0 L min-1 at 30 dph to 8 L min-1 at 108 dph, while water current speed in tanks was minimized by letting water enter through a 32 mm diameter inlet tube. Along with the 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 Pre Prin ts Pre Prin ts increased water flow rate, oxygen saturation (64-96%) was maintained by removal of fingerling cod when required. Larvae were sampled for weight and length at 5, 9, 19, 30, and 124 dph, element and thyroid hormone analysis at 5, 9, 19 and 30 dph, thyroid follicle morphology at 19, 30 and 37 dph and for analysis of skeletal deformities at 124 dph. All fish were euthanised with an overdose of tricaine methane sulfonate (MS 222) upon sampling. Larvae sampled for weight, minerals and thyroid hormones were collected on mesh (62 \u00b5m), briefly rinsed with ddH20, and patted dry from underneath with paper towel. Larvae were then placed in pre weighed tubes and frozen immediately in liquid nitrogen and stored at -80\u00b0C until analysis. All tubes were then reweighed to determine sample wet weights. Tubes sampled for weight determination were thawed and larvae were counted (n=20-100) to determine wet weight per larvae. Dry weight was determined from dry matter, which in turn was determined from tubes weighed before and after lyophilising. The standard length of the larvae was measured according to Hamre et al. (2008a) on 10 larvae tank-1. Larval densities in tanks were measured at 30 dph as described by Penglase et al. (2010). Larvae for thyroid follicle (3 fish tank-1) were selectively sampled to be similar in length, thus representing similar levels of morphological development (S\u00e6le and Pittman, 2010). Larvae were placed in individual tubes containing 1 ml of 4% paraformaldehyde in PBS buffer at pH 7.2. Samples were left overnight and then transferred to separate tubes containing 70% ethanol, where they remained at 4\u00b0C until embedding. For analysis of skeletal deformities, cod juveniles (124 dph, n=50 per tank) were measured for length and weight, frozen flat and subsequently stored in individual labelled plastic bags at -20\u00b0C until analysis. Survival in one HI+ tank decreased to zero prior to this sampling, so data represents the mean \u00b1 SD n=2 for the HI+fish at 124 dph. Samples for analysis of total I were digested under alkaline conditions using tetra methyl ammonium hydroxide ((CH3)4NOH, Tamapure-AA, Tama chemicals, Japan) and then analysed with ICP-MS (Agilent 7500 series, USA) as described by Julshamn, Dahl and Eckhoff (2001) with cod muscle (BCR-422, Belgium) used as the standard reference material. Samples for the analysis of other elements were prepared by wet digestion with nitric acid (65 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 Pre Prin ts Pre Prin ts % HNO3 Suprapur\u00ae, Merck, Germany) and hydrogen peroxide (30 % H2O2, Merck, Germany), in a microwave (Ethos 1600, Milestone, USA) as described by Julshamn et al. (2004). The samples were then analysed with ICP-MS along with blanks and standard reference material as described previously (Julshamn et al., 2004), with modifications to the mass of Mn (Mass 55) and Pb (Mass 208) measured. The standard reference materials used (NIST-SRM 1566, Oyster tissue, USA; TORT-2, NRC, lobster hepatopancreas, Canada) had similar concentrations of minerals as the samples analysed. Thyroid hormones extraction from larvae was carried out according to Einarsdottir et al. (2006) with some modifications. Approximately 500 mg WW of larvae (440 to 630 mg) was homogenised (Precellys 24, Bertin technologies, France) in 1 ml of ice-cold methanol (Sigma-Aldrich, Germany), and then stored over night at -20oC. Samples were then centrifuged (30 min, 3000 rpm, 4 \u00baC) and the supernatant removed from the pellet. This extraction procedure was then repeated on the pellet twice more. Nitrogen was used to dry the supernatant of methanol. Lipids were removed from samples by modified Folch extraction. To eliminate any lipid in samples, the dried extracts were dissolved in barbital buffer (0.1 M pH 8.6), methanol and chloroform (1:1:2). The aqueous phase containing T4 and T3 was transferred to a fresh tube, evaporated with nitrogen and frozen at -20o C until use. To estimate extraction efficiency, \u22481000 cpm of [125I]-rT3 (NEX109, Perkin Elmer, USA) were added to the homogenates after homogenisation. The extraction efficiency ranged between 71 to 81%. The T4 and T3 content were determined by radioimmunoassay using an external standard curve according to Einarsdottir et al. (2006), and further corrected for the extraction efficiency of each sample. Larval T3 contents were also reanalysed by a DELFIA\u00ae T3 Kit (PerkinElmer, Turku, Finland) according to manufacturer\u2019s instructions. Larvae were dehydrated in an increasing gradient of ethanol and embedded in Technovit 7100 as per directions (Heraeus Kulzer, Wehrheim, Germany). The resin blocks were then sectioned into 5 \u03bcm thick slices, and every second section was placed on a slide and stained with toluidine blue. The follicle number was counted and the area of epithelium and colloid were measured for each larvae (2 larvae tank-1 at 19 and 30 dph, 1 larvae tank-1 at 37 dph) at 200\u00d7 magnification using a microscope and computer assisted program CAST-grid version 2 (Olympus, Albertslund, Denmark) according to Saele et al. (2003). The colloid and 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 Pre Prin ts Pre Prin ts epithelium volume were calculated using area and width of sections, and skipped sections were assumed to have the same volume as that measured on the preceding section. The radiographic imaging and analysis of skeletal deformities was performed as described previously (Penglase et al., 2010). Larval survival was adjusted for sampled fish using a linear individual probability timeline for each tank to calculate the probable survival of sampled larvae had they not been sampled at each sampling point using the following equation; Estimated survival of sampled larvae at time point Y = 100 \u2013 (((100-S)/T1)*(T1-T2))/100)*X) where S is the measured survival % at 30 dph, T1 equals the time period in days from the start (5 dph) and end (30 dph) survival measurements (= 25 d), T2 equals the number of days post 100% survival (trial start) and X equals the number of larvae sampled at time point Y. This equation was used to produce two numbers, one for the sampling at 9 (T2 = 4) and one at 19 (T2 = 14) dph, and along with the number of larvae sampled on day 30, were added to the larvae measured in tanks from density measurements taken after sampling at 30 dph. Specific growth rates (SGR) of cod larvae were calculated with the following equation SGR = (e^((lnW1 \u2212 lnW0)/(t2-t1))-1)\u00d7100 where W0 and W1 are the initial and final dry weights (tank means) respectively, and t2 \u2013 t1 is the time interval in days between age t1 and t2 (Ricker, 1958). Fulton\u2019s condition factor (FC) was calculated using FC = Weight (g)*100/Length(cm)3. The I concentration ratio (CR) between larvae and feed was CR = Larval I content (mg kg-1 DW)/rotifer I content (mg kg-1 DW). Statistica software (Statsoft Inc., 2008, Tulsa, USA, Ver.9) was used for statistical analysis of data except when GraphPad Prism (GraphPad Software, San Diego, CA, USA, Ver. 5) was used to model fit the I concentration of cod larvae versus rotifers, and on data from 124 dph, as the loss of one replicate in the HI larvae group prevented ANOVA analysis between all three groups. Data analysed with Statistica were checked for homogeneity of variances using Levene\u2019s test before having significance tested with one-way ANOVA followed with Fisher\u2019s least significant difference (LSD) homogeneity post-hoc test at each time point. Data with significantly different variance between treatments according to Levene\u2019s test (p<0.05) was 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 Pre Prin ts Pre Prin ts log transformed before analysis. As the density of cod larvae has a large effect on growth (Koedijk et al., 2010), growth data during the larval stage was analysed with ANCOVA with the final larval density in tanks included as a continuous predictor. Data from 124 dph were analysed using regression, and tested against the null hypothesis that rotifer I content had no effect on outcome. Differences among means were considered significant at p<0.05. 254 255 256 257 258 Pre Prin ts Pre Prin ts 3 Results 3 cod larvae growth : No statistically significant differences in cod larvae length (p > 0.08) or dry weight (p > 0.25) occurred between treatments, although high variation between tanks may have masked effects (Fig. 1). On average, cod larvae grew from 4.9 \u00b1 0.2 mm to 6.8 \u00b1 0.5 mm in length, and 0.065 \u00b1 0.002 mg to 0.27 \u00b1 0.07 mg fish-1 in dry weight from 5 to 30 dph (Fig. 1), representing a specific growth rate of 6.3% day-1 during this period. There were no statistical differences (p>0.39) in the cod larval survival adjusted for sampled larvae (see section 2.6) between treatments at 30 dph which were 28 \u00b1 2, 39 \u00b1 20 and 30 \u00b1 15 % for controls, MI and HI groups respectively, while the average for all groups was 32 \u00b1 13 %. Survivals based solely on densities in tanks at 30 dph without taking into account sampled larvae were 12 \u00b1 2, 19 \u00b1 12 and 12 \u00b1 10 % for controls, MI and HI groups respectively. Control rotifers contained 0.60 \u00b1 0.33 mg I kg-1 DW while HI+rotifers contained 129 \u00b1 101 mg I kg-1 DW (Table 1). Whole body I levels in cod larvae were significantly different between groups (p<0.01, Fig. 2). Cod larvae (5 dph) had a starting concentration of 4.0 \u00b1 0.3 mg I kg-1 DW. Between 9 and 30 dph average I concentrations were 1.6 \u00b1 0.3 mg I kg-1 DW for control larvae, while MI larvae had 3 fold higher levels (4.9 \u00b1 2.4 mg I kg-1 DW), and HI larvae 7 fold higher levels (11.0 \u00b1 3.3 mg I kg-1 DW) than controls. Other element concentrations were also affected by treatment in both rotifers and cod larvae. HI+rotifers contained less Fe and Mn than controls (Table 1), while HI larvae contained more Mn, Fe and Cu than controls and MI larvae at one or more time points (Fig. 2b,c,e). Both HI and MI larvae contained higher levels of Co than controls (Fig. 2d), while larval Zn and Se concentrations were unaffected by treatment (Fig. 2f,g). Rotifer macro mineral concentrations were unaffected by treatment (Table 1), but increased levels of Ca and Mg, and lower levels of P and K were observed in HI larvae in comparison to controls and/or MI larvae during the rotifer feeding period (Fig. 3). The rate of increase in cod larvae I concentrations decreased as dietary I levels (rotifer I concentration) increased, and thus the I concentration ratio between cod larvae and rotifers displayed a negative trend (Fig 4; p<0.01). The age of the cod larvae did not affect their I concentration ratio (p = 0.96). The model predicts that the ratio of I in cod larvae versus rotifers equals 1 when rotifers have 3.5 mg I kg-1 DW (Fig. 4). 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 Pre Prin ts Pre Prin ts There were no differences in thyroid hormone levels or ratios between treatments (Fig. 5). Data was normalised to aid interpretation, as T3 results were higher than obtained previously (Penglase et al., 2010) and the high result was validated by the analysis of T3 with a second method (see methods). The total volume of thyroid follicle was 1.3 fold lower and the total epithelium volume was 1.4 fold lower per fish in HI versus MI larvae, but not controls, at 30 dph (Fig. 6a,c). The thyroid follicle colloid to epithelium ratio was higher in HI larvae than controls at 19 (1.7 fold) and 37 (1.8 fold) dph, while MI larvae did not differ from controls (Fig. 6d). No statistically significant differences were observed between groups in colloid volume or total number of thyroid follicles (Fig. 6b,e). Images of thyroid follicle sections from control and HI larvae at 37 dph are shown in figure 7. There were no significant differences in the weights (average; 2.50 \u00b1 0.19 g), lengths (6.14 \u00b1 0.16 cm) or condition factors (1.05 \u00b1 0.03) between groups at 124 dph (Table 2, n=400). Neck axis angle became closer to normal (180 \u00b1 3 degrees) with increasing I levels in rotifers, but there were no differences in any of the other skeletal deformity measurements (Table 2). 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 Pre Prin ts Pre Prin ts Discussion The hypothesis of this study was that commercially reared cod larvae fed rotifers would benefit from increased dietary I. The bases for this hypothesis were, one; rotifer I concentrations are often at the lower end or below juvenile/adult fish requirements (NRC, 2011), two; rotifers have 6 - 600 fold lower concentrations of I than copepods (Hamre et al., 2008b), the natural feed of cod larvae (Thompson and Harrop, 1991), three; cod larvae have better growth and development when fed copepods versus rotifers (Busch et al., 2010; Koedijk et al., 2010), four; I levels in copepod fed cod larvae are higher than rotifer fed cod larvae (Busch et al., 2010), and five; increased growth and/or survival has been observed in larval stages of several marine fish species fed or reared in environments with increased levels of bioavailable I (Hamre et al., 2008a; Witt et al., 2009; Ribeiro et al., 2011; Ribeiro et al., 2012). However, in contrast to the hypothesis, the increased thyroid follicle colloid to epithelium (C/E) ratio observed in this study indicates that I toxicity occurred in cod larvae fed rotifers with 129 mg I kg-1 DW. This observation purely in relation to the I level is not unexpected. A high C/E ratio in thyroid follicles is a classic symptom of I induced toxicity termed I (Baker, 2004) or colloid goitre, and occurs in mice at dietary I concentrations 10 fold higher than requirements with increasing severity developing with increasing I ingestion rates (Yang et al., 2006). What is interesting is that despite copepods containing 50 \u2013 350 mg I kg-1 DW (Solbakken et al., 2002; Hamre et al., 2008b), cod larvae have either similar or lower thyroid follicle C/E ratios when fed copepods compared to rotifers (Gr\u00f8tan, 2005). Furthermore, I concentrations observed in cod larvae fed natural zooplankton (29 mg I kg-1 DW at 27 dph; (Busch et al., 2010)) were 2.2 fold higher than the highest level observed in the current study (HI larvae, 30 dph; 13 \u00b1 4 mg I kg-1 DW). Thus it appears that high I concentrations in copepods do not induce morphological changes in thyroid follicles consistent with I toxicity, but do appear to be effectively transferred from copepods to fish larvae upon consumption. It is possible that copepods do not induce I toxicity in fish larvae due to nutrient interactions. For example, I toxicity can be prevented by the simultaneous presence of the bromine anion (Br-) in animals ranging from chicks (Gallus gallus) (Baker, Parr and Augspurger, 2003) to Artemia (S. Penglase et al., unpublished data). The exact mechanism for this Br-/I- interaction is still unknown, but it has been demonstrated that Br- decreases iodide accumulation in the thyroid follicles and increases I excretion via the kidneys in rats (Pavelka, 2004). Although the bromide concentrations in whole copepods and rotifers are unknown, we speculate that copepods have relatively high levels of bromide reflecting the high levels found in other 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 Pre Prin ts Pre Prin ts marine organisms, and this bromide helps prevent I toxicity in fish larvae. In the marine ecosystem, bromine is naturally found at similar high concentrations as I in seaweed (Romaris-Hortas, Moreda-Pineiro and Bermejo-Barrera, 2009), adult fish (Arafa et al., 2000; Wan et al., 2010), and as part of the hard chitin structures of crustaceans such as crabs (Cribb et al., 2009; Schofield et al., 2009) and copepods (Perry, Grime and Watt, 1988). Thyroid hormone levels and ratios were similar between cod larvae groups, and this is probably due to the compensatory changes observed in the thyroid follicles. For example pathological changes of over 70% in thyroid gland morphology have been observed in dogs (Canis lupus familiaris) with little change in circulating TH levels (Graham, Refsal and Nachreiner, 2007), and significant changes in fish thyroid follicle morphology with little change in thyroid hormone levels have also been reported for fish (Hawkyard et al., 2011; Morris et al., 2011). Few differences were found between the control and MI larvae groups, with the exception of whole body I concentrations. The increased whole body I content of cod larvae demonstrates the effective transfer of I from the rotifers to the cod larvae. The current study differs to previous studies exploring the uptake of I in fish larvae, as it attempted to ensure the ingestion of graded levels of I by maintaining the I concentration in the prey organism up until the point of feeding. Srivastava et al. (2012) found that I leaches rapidly from rotifers after enrichment with sodium iodide. Previous studies have found no difference in the I concentration of cod larvae fed control or I supplemented rotifers (Hamre et al., 2008a; S. Penglase et al., unpublished data), and this is probably a consequence of I leaching from rotifers in the minimum 1.5 to 2 h period between rotifer enrichment and feeding of the rotifers to cod larvae in these studies. In the current study, cod larvae iodine level increases were proportionally smaller for each increase in dietary I levels; control fish were 2.7 fold higher, while MI were 5 fold lower and HI larvae were 12 fold lower in I than their respective diets. Body stores of minerals are a good indicator of status (Baker, 1986), and the decreasing level of I retention in cod larvae relative to feed I levels indicates that requirements were met at levels lower than those fed to MI larvae. Modelling of the ratio between cod larvae and rotifer I concentrations predicts that based on a ratio of 1:1, rotifer I concentrations of 3.5 mg kg-1 DW meet cod larvae requirements. Both food and water contribute to the I status of adult, juvenile (Lall, 2002) and larval fish (Witt et al., 2009; Ribeiro et al., 2011). Alongside the I content in the continuously 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 Pre Prin ts Pre Prin ts exchanging seawater (88 \u00b5g I L-1, Moren, Sloth and Hamre, 2008), the control rotifers in the current study with 0.6 mg I kg-1 DW appeared to prevent any gross signs of I deficiency in cod larvae. This is at the lower end of the 0.6 \u2013 1.1 mg I kg-1 DW recommended by the national research council (NRC, 2011) as the I requirements of juvenile/adult fish. The reason that symptoms of severe I deficiency such as classic goitre have been observed in other fish studies is probably due to water parameters. Clear signs of I deficiency occurred in fish larvae reared in either recirculation systems (Ribeiro et al., 2011; Ribeiro et al., 2012) or well water (Witt et al., 2009). Nitrate (NO3-) is goitrogenic as it competitively blocks iodide uptake by the sodium iodide symporter (Tonacchera et al., 2004), and NO3- at levels commonly found in recirculation systems causes goitre in sharks (Crow et al., 1998; Morris et al., 2011). Furthermore, ozone (O3) used as a disinfectant in recirculation systems readily oxidises bioavailable I, iodide (I-) to iodate (IO3-) (Sherrill, Whitaker and Wong, 2004). Dissolve iodate is presumed to have low bioavailablity for fish (Sherrill, Whitaker and Wong, 2004), and higher levels of iodate compared to iodide were correlated to poor growth and survival in well water reared pacific threadfin larvae (Witt et al., 2009). Thus in recirculation systems, the presence of high levels of goitrogens (NO3-) and low levels of dissolved bioavailable I (I-) may increase the dietary I requirements of fish larvae over those reared with continuous water exchange where nitrate and its precursors are continuously removed and iodide continuously replaced, such as in the current study. Along with thyroid follicle morphology, dietary I also influenced the mineral composition of cod larvae. While most of the tested mineral concentrations in MI larvae were similar to controls, HI larvae had 10 to 25% higher levels of Ca, Mg, Mn, Fe, Co and Cu and around 10% lower levels of P and K than controls at one or more time points within the rotifer feeding period. For most of the minerals, differences in levels were observed by the first sampling point (9 dph; 4 days of feeding on rotifers). The differences cannot be explained by the feed; the HI rotifers had \u2248 10% less Mn and Fe, and no statistical differences were observed in Ca, Mg, K, P, Cu or Co concentrations. Hamre et al. (2008a) found that cod larvae fed increased levels of both I and Se had an 8% increase in whole body copper levels, similar to this study. Nguyen et al. (2008) found increased or decreased copper levels (20%) in red sea bream (Pagrus major) larvae depending on whether they were fed rotifers enriched with manganese alone or alongside zinc. While it is known that I deficiency can alter mineral distribution and homeostasis of Cu, Mn, Fe and Zn (Giray et al., 2003), to our knowledge this is the first data demonstrating that I oversupply can also effect mineral homeostasis. Although 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 Pre Prin ts Pre Prin ts there were few differences in growth or skeletal deformities observed between treatments at 124 dph, there was a small but statistically significant improvement in the neck axis angle in the HI compared to the control and MI cod groups (Table 2), and this may be linked to the differences in cod mineral concentrations in the larval stage. Conclusion Iodine enriched rotifers increased the levels of I in cod larvae, although as I levels in rotifers increased the increases in cod larvae I levels became proportionally smaller. Few differences occurred between cod larvae reared on control diets with 0.6 mg I kg -1 DW and those reared on diets with 26 mg I kg-1 DW, while the I concentration ratio between cod larvae and rotifers suggests cod larvae have an I requirement of 3.5 mg I kg -1 rotifers DW. Rotifers with copepod levels of I (129 mg I kg-1 DW) changed cod larvae whole body concentration of many essential minerals and induced changes in thyroid follicles morphology consistent with colloid goitre. The data presents one of the first observations of dietary induced I toxicity in fish, and suggests that I toxicity in fish larvae may be determined to a greater extent by I bioavailability and nutrient interactions than by body burdens of I.",
    "url": "https://peerj.com/articles/22/reviews/",
    "review_1": "David Solomon \u00b7 Jan 8, 2013 \u00b7 Academic Editor\nACCEPT\nThank you for addressing the feedback from the reviewers. I look forward to seeing your manuscript in print.",
    "review_2": "David Solomon \u00b7 Dec 21, 2012 \u00b7 Academic Editor\nMINOR REVISIONS\nThank you for submitting this impressive study for publication in PeerJ. I feel with a few revisions it will be an excellent addition to the literature.\n\nI apologize for the delay in providing you with feedback. I had difficulty finding reviewers who had the necessary expertise necessary and were able to review the manuscript.\n\nThe two reviewers provide a wealth of useful feedback I hope you will find helpful in revising your manuscript. There are a few what I feel to be key points that I would appreciate if you could address in a revision of the paper. Along with the four items below, please consider carefully the other feedback provided by the reviewers using your own judgment on what to implement in the revision.\n\n1. Although your study is fairly theoretical, it would seem it would have some important implications for educational practice with second language learners. As noted by both reviewers, the paper would be significantly enhanced if the discussion provided some specific suggestions on how what has been learned from your study could be applied in educational settings or in better preparing second language learners for entering these settings.\n\n2. While the research questions and hypotheses are discussed 133-155 as noted by the second reviewer, they are not stated as clearly as they could be. Just as suggestion, along with discussing them it might be helpful to state them specifically 1, 2,3 etc.\n\n3. As noted by the second reviewer, it is unclear how the 113 subjects for the second part of the study were selected. Please clarify how they were selected from the 582 students who participated in the first phase of the study.\n\n4. Please check the document carefully for typographical and grammatical errors.",
    "review_3": "Sonia Crandall \u00b7 Dec 21, 2012\nBasic reporting\nThe purpose of the current study is to explore whether students\u2019 verbal working memory influences their academic achievement. The theoretical framework for this study focused on the challenges faced by students in an environment where they are learning in a second language. The authors make a compelling case for the study.\nExperimental design\nThe authors designed an elaborate study using both self-report and observed measures. The design is novel and interesting with a comprehensive statistical analysis plan that addresses multiple confounding variables. This human subjects approved study has been rigorously conducted.\nValidity of the findings\nThe findings are well justified. The authors provide interesting data displays that contribute to overall understanding of the results explained within the text. Study limitations are adequately addressed. Conclusions are sound, clearly reported and are connected to the original question.\nAdditional comments\nOverall this is an excellently conducted research study. However, the educational significance is missing. What are medical educators supposed to do with these results? Nearly all, if not all, medical schools have L2 learners and would benefit from the authors\u2019 recommendations for supporting students\u2019 learning. Educational research must support practice.\n\nNeeded revisions:\nThere are numerous acronyms to keep in one\u2019s mind while reading the paper. Is it possible to include an acronym table that can be referred to easily?\n\nAbstract: Please explain what is meant by tertiary medical students. The readership is international and many may not be familiar with this term.\n\nTypos:\nLine 295: The word data implies plural; thus, the verb should match: was should be were.\n\nLine 398: Cronbach is spelled incorrectly.\nCite this review as\nCrandall S (2013) Peer Review #1 of \"Poorer verbal working memory for a second language selectively impacts academic achievement in university medical students (v0.1)\". PeerJ https://doi.org/10.7287/peerj.22v0.1/reviews/1",
    "review_4": "Reviewer 2 \u00b7 Dec 16, 2012\nBasic reporting\nIn the abstract, information like independent variable(s), dependent variable(s), expected results and actual results all should be included. However, the authors didn\u2019t include briefly stated results in the abstract. In addition, as an expected factor which may influence the results of the study, the age of English language acquisition was left behind in the abstract too.\n\nThe paper also emphasizes the role of age of language acquisition in speech perception, mentioning several differences in the ability to process speech between monolinguals and late bilinguals. It would be better if the authors show expected and actual results about the effects of age of second language acquisition in the abstract of the paper.\nIn the introduction, the article should make more clarification about what the theoretical and empirical significance of the current study is and how the study can contribute to the understanding of the impact of verbal working memory on academic attainment, particularly for English as second language users.\n\nThe hypothesis/hypotheses of the study should be more clearly defined. It seemed in the abstract that the authors hypothesized poorer working memory under high noisy contexts significantly reduced the academic performance for L2. But the paper neither clearly stated the hypothesis/hypotheses or research question(s) in the discussion, nor did it explain why and how other constructs such as English proficiency (e.g., AoAoE, PEP) related to the hypothesis/hypotheses or research question(s).\n\nBesides, the paper discussed in great details about why English proficiency would not be a confounding variable in the relationship between vWM and academic performance because all international students have to take standard tests in order to get the admission. However, this is a fair weak argument since passing stringent measures of English proficiency prior to enrolment, such as the IELTS or the TOEFL, would not guarantee that international students would performance as well as their local counterparts in academic achievements. In addition, in later examination, the paper extended to consider English proficiency as an indicator of academic achievements in the research design. It could be very confusing for readers to identify the role of English proficiency in this article.\n\nMoreover, the evidence exhibited in the article does not sufficiently support the relationship between the \u201cability to process speech and to recall academic material.\u201d Only one study (e.g., Ljung et al., 2010) from line 118 to 124 was cited for illustration. Given that this relationship is crucial for developing hypothesis/hypotheses, more relevant research from the extant literature would be better to enhance the argument.\nExperimental design\n1. There\u2019s no reliability report for the Perceived Stress Scale and the Index of learning Styles Questionnaire used in the research design.\n\n2. Stress and learning styles of the medical undergraduate students were measured as two potential factors which may affect their academic achievements. However, the authors failed to justify the reason why these two factors may or may not cause an impact. Except for two studies mentioned in line 165 to 169, there were insufficient literature reviews and explications to support this point.\n\n3. Participants: Line 182 to 189 showed that 103 students were recruited into the complete research project. However, the authors didn\u2019t clearly mention where they recruited the 54 participants for the year second study. Were they from the 103 participants of the first year study? Or were they from the 582 students who returned the surveys and different from the 103 students? Why there was a half loss of the sample? Would the sample loss cause any impact on the expected effect size?\n\n4. The criterion that distinguishes \u201clocal\u201d and \u201cinternational\u201d cannot rule out the possibility that an increasing number of students in \u201cmigrant families\u201d who hold the permanent residency but not a L1 student or cannot speak English fluently and idiomatically. This may constitute a confound effect in the comparison between L1 and L2.\n\n5. The authors didn\u2019t rationalize and explain clearly the investigation of students\u2019 academic achievements for both first and second year of the course. It is not clarified how they collected the second year\u2019s data.\n\n6. In terms of the limitations of the study, the within-subject experimental design may result in participants\u2019 fatigue particularly when each participant in the study was required to listen and verbally repeat 60 sentences in total under three noise backgrounds (high, moderate and low). The author explained that high noise group would not be the first condition in order to putting the participants in a difficult situation at the very beginning. However, there is still a possibility that the participants who should have performed well scored low in the last group because of fatigue.\n\n7. The use of everyday clinically-used sentences may pose a threat to ecological validity. Given that students are taught academic contents, which are different semantically with everyday language (e.g., academic lecture use more long sentences and professional vocabulary), in undergraduate medical courses, they may not be able to fully emerge in experimental settings.\nValidity of the findings\n1. From line 367 to 374, when conducting the multiple regressions, the authors used five items significantly correlated to SNR50 that pertained to English proficiency and/or usage, such as AoAoE, PEP, MSE. However, there were insufficient justifications and supportive literature review to explain why the authors did this subdividing and how did these five items pertained to English proficiency and usage.\n2. In line 395, the authors did the principle component analysis for the five items pertaining to English proficiency and usage to rule out the threat of multicollinearity. What\u2019s the VIF and tolerance for the new scale renamed \u201cEnglish Language Skills\u201d (ELS)? Have the multicollinearity problem been solved successfully?\n3. The authors indicated that the new ELS was an approximation of the students\u2019 overall English proficiency. How did they draw out this conclusion? Is there any empirical evidence or related literature to support this point?\n4. In the discussion section, the authors classified the background noise into 2 groups: energetic and informational, which a) did not have literature to support the categorization; b) were ambiguous in the way that how the categorization fit into the hypothesis and the design of the experiment. According to the article, the author mentioned the use of headphone to play noise in line 232 and \u201cbabble noise\u201d in line 219, but it is not clear what category do these belong to.\nAdditional comments\nThe article includes quite a few grammatical and stylistic mistakes. For example, when listing previous literatures, \u201cet al.\u201d has been used as \u201cet al\u201d without the period in a few places. In line 489, it is not necessary to list all three authors and put on \u201cet al.\u201d at the end. When mentioning \u201clocal participants\u201d and \u201cinternational participants\u201d, the capitalization of the letter \"L\" in the word \"local\" should be consistent in the paper. For example, in line 137, \"L\" is not capitalized, while it is in line 457. The letter \"I\" in the word \"international\" is another case. In line 516, it is better to explain what are \u201cPSE; the SNR50\u201d used for (are they examples or illustrations). Also, like in the line of 235, the first line of the paragraph should be indented. From the line of 479 to 485, the format of the text needs to be fixed.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Poorer verbal working memory for a second language selectively impacts academic achievement in university medical students (v0.1)\". PeerJ https://doi.org/10.7287/peerj.22v0.1/reviews/2",
    "pdf_1": "https://peerj.com/articles/22v0.2/submission",
    "pdf_2": "https://peerj.com/articles/22v0.1/submission",
    "all_reviews": "Review 1: David Solomon \u00b7 Jan 8, 2013 \u00b7 Academic Editor\nACCEPT\nThank you for addressing the feedback from the reviewers. I look forward to seeing your manuscript in print.\nReview 2: David Solomon \u00b7 Dec 21, 2012 \u00b7 Academic Editor\nMINOR REVISIONS\nThank you for submitting this impressive study for publication in PeerJ. I feel with a few revisions it will be an excellent addition to the literature.\n\nI apologize for the delay in providing you with feedback. I had difficulty finding reviewers who had the necessary expertise necessary and were able to review the manuscript.\n\nThe two reviewers provide a wealth of useful feedback I hope you will find helpful in revising your manuscript. There are a few what I feel to be key points that I would appreciate if you could address in a revision of the paper. Along with the four items below, please consider carefully the other feedback provided by the reviewers using your own judgment on what to implement in the revision.\n\n1. Although your study is fairly theoretical, it would seem it would have some important implications for educational practice with second language learners. As noted by both reviewers, the paper would be significantly enhanced if the discussion provided some specific suggestions on how what has been learned from your study could be applied in educational settings or in better preparing second language learners for entering these settings.\n\n2. While the research questions and hypotheses are discussed 133-155 as noted by the second reviewer, they are not stated as clearly as they could be. Just as suggestion, along with discussing them it might be helpful to state them specifically 1, 2,3 etc.\n\n3. As noted by the second reviewer, it is unclear how the 113 subjects for the second part of the study were selected. Please clarify how they were selected from the 582 students who participated in the first phase of the study.\n\n4. Please check the document carefully for typographical and grammatical errors.\nReview 3: Sonia Crandall \u00b7 Dec 21, 2012\nBasic reporting\nThe purpose of the current study is to explore whether students\u2019 verbal working memory influences their academic achievement. The theoretical framework for this study focused on the challenges faced by students in an environment where they are learning in a second language. The authors make a compelling case for the study.\nExperimental design\nThe authors designed an elaborate study using both self-report and observed measures. The design is novel and interesting with a comprehensive statistical analysis plan that addresses multiple confounding variables. This human subjects approved study has been rigorously conducted.\nValidity of the findings\nThe findings are well justified. The authors provide interesting data displays that contribute to overall understanding of the results explained within the text. Study limitations are adequately addressed. Conclusions are sound, clearly reported and are connected to the original question.\nAdditional comments\nOverall this is an excellently conducted research study. However, the educational significance is missing. What are medical educators supposed to do with these results? Nearly all, if not all, medical schools have L2 learners and would benefit from the authors\u2019 recommendations for supporting students\u2019 learning. Educational research must support practice.\n\nNeeded revisions:\nThere are numerous acronyms to keep in one\u2019s mind while reading the paper. Is it possible to include an acronym table that can be referred to easily?\n\nAbstract: Please explain what is meant by tertiary medical students. The readership is international and many may not be familiar with this term.\n\nTypos:\nLine 295: The word data implies plural; thus, the verb should match: was should be were.\n\nLine 398: Cronbach is spelled incorrectly.\nCite this review as\nCrandall S (2013) Peer Review #1 of \"Poorer verbal working memory for a second language selectively impacts academic achievement in university medical students (v0.1)\". PeerJ https://doi.org/10.7287/peerj.22v0.1/reviews/1\nReview 4: Reviewer 2 \u00b7 Dec 16, 2012\nBasic reporting\nIn the abstract, information like independent variable(s), dependent variable(s), expected results and actual results all should be included. However, the authors didn\u2019t include briefly stated results in the abstract. In addition, as an expected factor which may influence the results of the study, the age of English language acquisition was left behind in the abstract too.\n\nThe paper also emphasizes the role of age of language acquisition in speech perception, mentioning several differences in the ability to process speech between monolinguals and late bilinguals. It would be better if the authors show expected and actual results about the effects of age of second language acquisition in the abstract of the paper.\nIn the introduction, the article should make more clarification about what the theoretical and empirical significance of the current study is and how the study can contribute to the understanding of the impact of verbal working memory on academic attainment, particularly for English as second language users.\n\nThe hypothesis/hypotheses of the study should be more clearly defined. It seemed in the abstract that the authors hypothesized poorer working memory under high noisy contexts significantly reduced the academic performance for L2. But the paper neither clearly stated the hypothesis/hypotheses or research question(s) in the discussion, nor did it explain why and how other constructs such as English proficiency (e.g., AoAoE, PEP) related to the hypothesis/hypotheses or research question(s).\n\nBesides, the paper discussed in great details about why English proficiency would not be a confounding variable in the relationship between vWM and academic performance because all international students have to take standard tests in order to get the admission. However, this is a fair weak argument since passing stringent measures of English proficiency prior to enrolment, such as the IELTS or the TOEFL, would not guarantee that international students would performance as well as their local counterparts in academic achievements. In addition, in later examination, the paper extended to consider English proficiency as an indicator of academic achievements in the research design. It could be very confusing for readers to identify the role of English proficiency in this article.\n\nMoreover, the evidence exhibited in the article does not sufficiently support the relationship between the \u201cability to process speech and to recall academic material.\u201d Only one study (e.g., Ljung et al., 2010) from line 118 to 124 was cited for illustration. Given that this relationship is crucial for developing hypothesis/hypotheses, more relevant research from the extant literature would be better to enhance the argument.\nExperimental design\n1. There\u2019s no reliability report for the Perceived Stress Scale and the Index of learning Styles Questionnaire used in the research design.\n\n2. Stress and learning styles of the medical undergraduate students were measured as two potential factors which may affect their academic achievements. However, the authors failed to justify the reason why these two factors may or may not cause an impact. Except for two studies mentioned in line 165 to 169, there were insufficient literature reviews and explications to support this point.\n\n3. Participants: Line 182 to 189 showed that 103 students were recruited into the complete research project. However, the authors didn\u2019t clearly mention where they recruited the 54 participants for the year second study. Were they from the 103 participants of the first year study? Or were they from the 582 students who returned the surveys and different from the 103 students? Why there was a half loss of the sample? Would the sample loss cause any impact on the expected effect size?\n\n4. The criterion that distinguishes \u201clocal\u201d and \u201cinternational\u201d cannot rule out the possibility that an increasing number of students in \u201cmigrant families\u201d who hold the permanent residency but not a L1 student or cannot speak English fluently and idiomatically. This may constitute a confound effect in the comparison between L1 and L2.\n\n5. The authors didn\u2019t rationalize and explain clearly the investigation of students\u2019 academic achievements for both first and second year of the course. It is not clarified how they collected the second year\u2019s data.\n\n6. In terms of the limitations of the study, the within-subject experimental design may result in participants\u2019 fatigue particularly when each participant in the study was required to listen and verbally repeat 60 sentences in total under three noise backgrounds (high, moderate and low). The author explained that high noise group would not be the first condition in order to putting the participants in a difficult situation at the very beginning. However, there is still a possibility that the participants who should have performed well scored low in the last group because of fatigue.\n\n7. The use of everyday clinically-used sentences may pose a threat to ecological validity. Given that students are taught academic contents, which are different semantically with everyday language (e.g., academic lecture use more long sentences and professional vocabulary), in undergraduate medical courses, they may not be able to fully emerge in experimental settings.\nValidity of the findings\n1. From line 367 to 374, when conducting the multiple regressions, the authors used five items significantly correlated to SNR50 that pertained to English proficiency and/or usage, such as AoAoE, PEP, MSE. However, there were insufficient justifications and supportive literature review to explain why the authors did this subdividing and how did these five items pertained to English proficiency and usage.\n2. In line 395, the authors did the principle component analysis for the five items pertaining to English proficiency and usage to rule out the threat of multicollinearity. What\u2019s the VIF and tolerance for the new scale renamed \u201cEnglish Language Skills\u201d (ELS)? Have the multicollinearity problem been solved successfully?\n3. The authors indicated that the new ELS was an approximation of the students\u2019 overall English proficiency. How did they draw out this conclusion? Is there any empirical evidence or related literature to support this point?\n4. In the discussion section, the authors classified the background noise into 2 groups: energetic and informational, which a) did not have literature to support the categorization; b) were ambiguous in the way that how the categorization fit into the hypothesis and the design of the experiment. According to the article, the author mentioned the use of headphone to play noise in line 232 and \u201cbabble noise\u201d in line 219, but it is not clear what category do these belong to.\nAdditional comments\nThe article includes quite a few grammatical and stylistic mistakes. For example, when listing previous literatures, \u201cet al.\u201d has been used as \u201cet al\u201d without the period in a few places. In line 489, it is not necessary to list all three authors and put on \u201cet al.\u201d at the end. When mentioning \u201clocal participants\u201d and \u201cinternational participants\u201d, the capitalization of the letter \"L\" in the word \"local\" should be consistent in the paper. For example, in line 137, \"L\" is not capitalized, while it is in line 457. The letter \"I\" in the word \"international\" is another case. In line 516, it is better to explain what are \u201cPSE; the SNR50\u201d used for (are they examples or illustrations). Also, like in the line of 235, the first line of the paragraph should be indented. From the line of 479 to 485, the format of the text needs to be fixed.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Poorer verbal working memory for a second language selectively impacts academic achievement in university medical students (v0.1)\". PeerJ https://doi.org/10.7287/peerj.22v0.1/reviews/2\nReview 5: nan\nReview 6: \n",
    "LLM_extracted_review": "['Review 1: Thank you for addressing the feedback from the reviewers. I look forward to seeing your manuscript in print.\\n\\nReview 2: \\n1. The paper would be significantly enhanced if the discussion provided specific suggestions on how the study\\'s findings could be applied in educational settings for second language learners.\\n2. The research questions and hypotheses are not stated as clearly as they could be; it might be helpful to list them specifically.\\n3. It is unclear how the 113 subjects for the second part of the study were selected; clarification is needed.\\n4. The document should be checked for typographical and grammatical errors.\\n\\nReview 3: Overall, this is an excellently conducted research study. However, the educational significance is missing; recommendations for supporting students\u2019 learning should be included. \\n- An acronym table is needed for clarity.\\n- The term \"tertiary medical students\" should be explained in the abstract for an international readership.\\n- There are typographical errors that need correction.\\n\\nReview 4: \\n1. The abstract should include independent and dependent variables, expected results, and actual results.\\n2. The theoretical and empirical significance of the study should be clarified in the introduction.\\n3. The hypothesis/hypotheses should be more clearly defined.\\n4. The argument regarding English proficiency as a confounding variable is weak and needs more justification.\\n5. The recruitment process for participants is unclear, and the sample loss should be addressed.\\n6. The investigation of academic achievements for both first and second year students needs clarification.\\n7. The within-subject experimental design may lead to participant fatigue, affecting results.\\n8. The use of everyday language may threaten ecological validity in the context of academic learning.\\n\\nValidity of the findings:\\n1. Insufficient justification for the subdivision of items related to English proficiency.\\n2. Clarification is needed on the multicollinearity analysis and its results.\\n3. The conclusion regarding the new English Language Skills scale needs empirical support.\\n4. The categorization of background noise lacks literature support and clarity.\\n\\nAdditional comments: The article contains grammatical and stylistic mistakes that need correction.\\n\\nReview 5: nan\\n\\nReview 6: ']"
}